**Early T-cell Precursor Acute Lymphoblastic Leukemia – A Characteristic Neoplasm Presenting the Phenotype of Common Hematopoietic Progenitors for both Myeloid and Lymphoid Lineages**

Hiroko Tsunemine and Takayuki Takahashi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60901

#### **Abstract**

Introduction: Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is a subtype of T-ALL and its clinical entity was established in recent years based on characteristic immunophenotyping and gene expression profiles. The cellular origin of ETP-ALL is supposed to be from common hematopoietic progenitors both for lymphoid and myeloid lineages because this leukemia phenotypically exhibits lymphoid, myeloid, and stem cell features. ETP-ALL comprises 5–15% of all T-ALL and is associated with a poor prognosis. The purpose of this chapter is to clarify the etiology, clinical picture, and therapeutic strategy of ETP-ALL showing two cases of this leukemia in our institution.

Cellular origin of ETP-ALL: The normal early T-cell precursors (ETPs) are considered to be a subset of early thymocytes which are originated from the bone marrow and subsequently reside in the thymus, retaining multilineage differentiation potential as the common lymphoid-myeloid hematopoietic progenitors. ETP-ALL is supposed to be a neoplastic counterpart of ETPs.

Immunophenotype and diagnosis of ETP-ALL: ETP-ALL is characterized by the lack of expression of T-lineage cell surface antigens (CD1a and CD8, weak or no expression of CD5) and expression of myeloid and hematopoietic stem cell markers such as CD13, CD33, CD34, and CD117. These characteristic immunophenotypic profiles have provided a scoring system or a criterion for the diagnosis of ETP-ALL, which distinguishes ETP-ALL from classical T-ALL.

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Clinical pictures: Clinical features are not substantially different between ETP-ALL and classical T-ALL, although ETP-ALL is associated with a higher rate of relapse and induction failure and a significantly worse overall survival. Two cases of ETP-ALL in our institution, which exhibited unique clinical pictures, that is, marked intestinal involvement and lymphoma-like systemic lymphadenopathy, respectively, will be discussed later in this chapter.

Gene profiles: Whole-genome sequencing studies on ETP-ALL have demonstrated several recurrent mutations involving genes coding cytokines, RAS signaling mediators (NRAS, KRAS, FLT3, IL7R, JAK3, LAK1, SH2B3, and BRAF), epigenetic controllers (EZH2, EED, SUZ12, SETD2, EP300 and DNMT3A), and hematopoietic transcriptional regulators (GATA3, ETV6, RUNX1, IKZF1, and EP300). These mutational spectrums are similar to those of acute myeloid leukemia.

Therapeutic strategies: These gene profiles suggest that treatment of ETP-ALL may benefit from a new chemotherapeutic approach, which is directed to the myeloid or stem cell natures of this leukemia, such as high-dose cytarabine, or epigenetic or molecular targeting therapy. Allogeneic stem cell transplantation (allo-SCT) may be a promising option for the treatment of ETP-ALL.

Conclusion: More precise and extensive cellular and molecular investigations are required to establish definite cellular origin and genetic or epigenetic nature of ETP-ALL. Accumulation of ETP-ALL cases and larger clinical trials will establish an effective therapeutic strategy for this high risk leukemia.

**Keywords:** Early T-cell precursor acute lymphoblastic leukemia

## **1. Introduction**

T-cell acute lymphoblastic leukemia (T-ALL) is a clonal malignant disorder of immature Tcells that accounts for 10–15% of childhood and 25% of adult ALL patients [1]. Despite the relatively high morbidity and mortality of T-ALL when compared to B-cell ALL, the prognosis of T-ALL has dramatically improved following the advancement of chemotherapy, and its long-term survival has become as high as 85% in both pediatric and adult T-ALL patients [2, 3]. However, a refractory subset of pediatric T-ALL associated with a poor prognosis has remained. In 2009, a study performed at St. Jude Children's Research Hospital identified a distinct subtype of pediatric T-ALL, which was designated as early T-cell precursor ALL (ETP-ALL) [4]. This new subtype of T-ALL was defined according to the characteristic gene expression profile and immunophenotypes of the leukemic cells and was found to be associ‐ ated with a high rate of remission induction failure or relapse when the patients were treated with conventional chemotherapy [4].

The purpose of this chapter is to clarify the recent advances in the biology, genetics, clinical characteristics, and therapeutic strategy of ETP-ALL and discuss two cases experienced at our institution.

## **2. Cellular origin of ETP-ALL**

Clinical pictures: Clinical features are not substantially different between ETP-ALL and classical T-ALL, although ETP-ALL is associated with a higher rate of relapse and induction failure and a significantly worse overall survival. Two cases of ETP-ALL in our institution, which exhibited unique clinical pictures, that is, marked intestinal involvement and lymphoma-like systemic lymphadenopathy, respectively, will be

Gene profiles: Whole-genome sequencing studies on ETP-ALL have demonstrated several recurrent mutations involving genes coding cytokines, RAS signaling mediators (NRAS, KRAS, FLT3, IL7R, JAK3, LAK1, SH2B3, and BRAF), epigenetic controllers (EZH2, EED, SUZ12, SETD2, EP300 and DNMT3A), and hematopoietic transcriptional regulators (GATA3, ETV6, RUNX1, IKZF1, and EP300). These

Therapeutic strategies: These gene profiles suggest that treatment of ETP-ALL may benefit from a new chemotherapeutic approach, which is directed to the myeloid or stem cell natures of this leukemia, such as high-dose cytarabine, or epigenetic or molecular targeting therapy. Allogeneic stem cell transplantation (allo-SCT) may be

Conclusion: More precise and extensive cellular and molecular investigations are required to establish definite cellular origin and genetic or epigenetic nature of ETP-ALL. Accumulation of ETP-ALL cases and larger clinical trials will establish an

T-cell acute lymphoblastic leukemia (T-ALL) is a clonal malignant disorder of immature Tcells that accounts for 10–15% of childhood and 25% of adult ALL patients [1]. Despite the relatively high morbidity and mortality of T-ALL when compared to B-cell ALL, the prognosis of T-ALL has dramatically improved following the advancement of chemotherapy, and its long-term survival has become as high as 85% in both pediatric and adult T-ALL patients [2, 3]. However, a refractory subset of pediatric T-ALL associated with a poor prognosis has remained. In 2009, a study performed at St. Jude Children's Research Hospital identified a distinct subtype of pediatric T-ALL, which was designated as early T-cell precursor ALL (ETP-ALL) [4]. This new subtype of T-ALL was defined according to the characteristic gene expression profile and immunophenotypes of the leukemic cells and was found to be associ‐ ated with a high rate of remission induction failure or relapse when the patients were treated

The purpose of this chapter is to clarify the recent advances in the biology, genetics, clinical characteristics, and therapeutic strategy of ETP-ALL and discuss two cases experienced at our

mutational spectrums are similar to those of acute myeloid leukemia.

a promising option for the treatment of ETP-ALL.

effective therapeutic strategy for this high risk leukemia.

**Keywords:** Early T-cell precursor acute lymphoblastic leukemia

discussed later in this chapter.

28 Leukemias - Updates and New Insights

**1. Introduction**

institution.

with conventional chemotherapy [4].

Normal early T-cell precursors (ETPs) are a subset of thymocytes, which have newly immi‐ grated from the bone marrow to the thymus, and they retain multilineage differentiation potential, suggesting their direct derivation from hematopoietic stem cells [5-7]. The initial stage of thymocyte development is characterized by the generation of cells that lack the expression of CD4 or CD8 antigen. Along with the differentiation of these double negative cells, a minimum of four distinct differentiation stages have been identified according to the differential expressions of CD44 and CD25, that is, DN1, DN2, DN3, and DN4 stages. The potential for myeloid, dendritic, and natural killer cell differentiation is retained at both the DN1 and early DN2 stages [6]. The ability to confer multilineage differentiation is lost at the DN3 stage, and provably, at the latter half of DN2 progression [8]. Therefore, it may be reasonable that the tumor-initiating cells in ETP-ALL could originate from DN1 and/or DN2 thymocytes (Figure 1). However, in recent years, a mouse model of T-ALL using a Sleeping-Beauty-based transposon system suggested that ETP-ALL may be derived from more mature T-cells [9]. Thus, the exact cellular origin of ETP-ALL remains to be elucidated.

**Figure 1.** Early T-cell development and supposed cellular origin of ETP-ALL.

## **3. Immunophenotyping and diagnosis of ETP-ALL**

Immunophenotyping of ETP-ALL cells is characterized by the lack of CD1a and CD8 expres‐ sions, weak CD5 expression (< 75% positive blasts), and the expression of one or more of the following myeloid or stem cell antigens on at least 25% of the leukemic cells: CD117, CD34, HLA-DR, CD13, CD33, CD11b, and/or CD65 [4]. Subsequently, a study proposed a scoring system based on the expression of commonly available eleven markers: CD5, CD8, CD13, CD33, CD34, HLA-DR, CD2, smCD3, CD4, CD10, and CD56 (Figure 2A) [10]. The specificity and sensitivity of this scoring system were 100% and 94%, respectively, when applied to the patients in the St. Jude cohort (Figure 2B) [10]. Recently, another study attempted to make a more simple diagnosis of ETP-ALL using the expression of CD5 and concluded that CD5 negative T-ALL could be diagnosed as ETP-ALL because CD5 negativity was associated with positive myeloid/stem cell antigens but not CD1a and CD8 expressions (Figure 3) [11]. Currently, precise immunophenotyping is the most important tool to make a diagnosis of ETP-ALL, and this analysis enables us to distinguish ETP-ALL from classical T-ALL.

**Figure 2.** A scoring system for immunophenotypical diagnosis of ETP-ALL. A: Scoring system based on the expression of 11 markers., B: Distribution of total score of 11-marker expression in ETP-ALL patients (right) and T-ALL (left) of the St Jude cohort. (Extract of Ref.10).

## **4. Clinical characteristics**

Following the early reports from the St. Jude Children's Research Hospital and the Associa‐ zione Italiana Ematologica Oncologica Pediatrica (AIEOP), comparative studies on the clinical characteristics between ETP-ALL and classical T-ALL were performed in six institutions: the Tokyo Children's Cancer Study Group [10], the Shanghai Children's Medical Center [12], the German Multicenter Study Group for adult ALL [13], Colombia University Medical Center [14], All India Institute of Medical Sciences [11], and the Medical Research Council UK-ALL 2003 trial [15] (Table 1). According to the results of these clinical studies, ETP-ALL was observed to comprise 5.5–16% of all T-ALL cases. The clinical characteristics were similar between ETP-ALL and classical T-ALL with regard to gender, hemoglobin concentration, and central nervous system involvement. However, ETP-ALL patients presented with a lower white blood cell (WBC) count [11, 12, 15], lower frequency of the mediastinal mass [13, 14], and higher age (10 years or older) [4, 11] at presentation when compared to those with classical T-ALL. Regarding the cytogenetic profile, Coustan-Smith et al. reported that ETP-ALL had Early T-cell Precursor Acute Lymphoblastic Leukemia – A Characteristic Neoplasm Presenting the Phenotype of… http://dx.doi.org/10.5772/60901 31

**Figure 3.** A flowchart for the diagnosis of ETP-ALL based on CD5 expression (Extract of Ref.11).

patients in the St. Jude cohort (Figure 2B) [10]. Recently, another study attempted to make a more simple diagnosis of ETP-ALL using the expression of CD5 and concluded that CD5 negative T-ALL could be diagnosed as ETP-ALL because CD5 negativity was associated with positive myeloid/stem cell antigens but not CD1a and CD8 expressions (Figure 3) [11]. Currently, precise immunophenotyping is the most important tool to make a diagnosis of ETP-

**Figure 2.** A scoring system for immunophenotypical diagnosis of ETP-ALL. A: Scoring system based on the expression of 11 markers., B: Distribution of total score of 11-marker expression in ETP-ALL patients (right) and T-ALL (left) of

Following the early reports from the St. Jude Children's Research Hospital and the Associa‐ zione Italiana Ematologica Oncologica Pediatrica (AIEOP), comparative studies on the clinical characteristics between ETP-ALL and classical T-ALL were performed in six institutions: the Tokyo Children's Cancer Study Group [10], the Shanghai Children's Medical Center [12], the German Multicenter Study Group for adult ALL [13], Colombia University Medical Center [14], All India Institute of Medical Sciences [11], and the Medical Research Council UK-ALL 2003 trial [15] (Table 1). According to the results of these clinical studies, ETP-ALL was observed to comprise 5.5–16% of all T-ALL cases. The clinical characteristics were similar between ETP-ALL and classical T-ALL with regard to gender, hemoglobin concentration, and central nervous system involvement. However, ETP-ALL patients presented with a lower white blood cell (WBC) count [11, 12, 15], lower frequency of the mediastinal mass [13, 14], and higher age (10 years or older) [4, 11] at presentation when compared to those with classical T-ALL. Regarding the cytogenetic profile, Coustan-Smith et al. reported that ETP-ALL had

the St Jude cohort. (Extract of Ref.10).

30 Leukemias - Updates and New Insights

**4. Clinical characteristics**

ALL, and this analysis enables us to distinguish ETP-ALL from classical T-ALL.

more 13q- and DNA copy number abnormalities than those in classical T-ALL [4]. Conversely, Allen et al. reported that the majority of patients with ETP-ALL exhibited a normal karyotype without recurrent cytogenetic abnormalities [14]. The monoclonal rearrangement of T-cell receptor genes was observed in 71% of the ETP-ALL cases, showing no significant difference between the two T-ALL subgroups [14].


**Table 1.** Comparative studies on the clinical characteristics between ETP-ALL and classical T-ALL.

As for the prognosis, ETP-ALL is associated with a higher rate of relapse and induction failure. ETP-ALL is additionally associated with a significantly worse overall survival with a 10-year event free survival (EFS) and overall survival (OS) rates of 22% and 19%, respectively, as compared with 69% EFS and 84% OS for all other subtypes of T-ALL, respectively, in the St. Jude cohort [4]. Similar results were obtained in the cohorts of four other institutions [4, 10-12]. More recently, however, two clinical studies showed no significant differences in the EFS and OS rates between the patients with ETP-ALL and classical T-ALL [14, 15]. Although the reason for this discrepancy is unclear, differences in the therapeutic protocol and patient cohort may have influenced the results of these clinical studies. However, an increased risk of relapse in the patients with ETP-ALL [4, 10-12, 15], especially children [4, 14], was a common result in all these previous studies. Thus, larger prospective studies are needed to determine the real prognosis of this T-ALL subtype.

## **5. Gene profiles**

The expression levels of oncogenic transcription factor genes were examined to establish genetic profiles of ETP-ALL in the St Jude Children's Research Hospital and AIEOP studies. Pediatric ETP-ALL had a higher expression of oncogenic transcription factors: *LMO1*, *LMO2*, *LYL1*, and *ERG* [4, 16]. *LMO1* and *LMO2* are binding partners with hematopoietic basic helixloop-helix transcription factors, such as *SCL/TAL1* or *LYL1*. These proteins interact together to form a transcription factor complex, and they are hypothesized to act through a common mechanism which leads to oncogenesis of T-ALL [17]. McCormack et al. demonstrated that *LYL1* is critical for the oncogenic function of *LMO2*, including the upregulation of a stem celllike gene signature, aberrant self-renewal of thymocytes, and subsequent generation of T-cell leukemia in *LMO2*-transgenic mice. Moreover, *LMO2* and *LYL1* are co-expressed in leukemic cells from the patients with ETP-ALL, and *LYL1* is indispensable for the growth of ETP-ALL cell lines [18]. Whole-genome sequencing studies showed that ETP-ALL had a high frequency of activating mutations in the genes involved in cytokine receptor and RAS signaling (e.g., *NRAS*, *KRAS*, *FLT3*, *IL-7R*, *JAK3*, *LAK1*, *SH2B3*, and *BRAF*) and inactivating mutations in the genes encoding key transcription factors involved in hematopoietic development (e.g., *GATA3*, *ETV6*, *RUNX1*, *IKZF1*, and *EP300*) and involved in epigenetic gene control (e.g., *EZH2*, *EED*, *SUZ12*, *SETD2*, and *EP300* genes) [16]. The gene mutations which affect cytokine receptor regulation and/or RAS signaling pathway are observed in two-thirds of ETP-ALL cases but only in 19% of non-ETP T-ALL cases [16]. These mutational gene spectrums in ETP-ALL are similar to those in acute myeloid leukemia (AML), but not in T- or B-lineage ALLs. Further‐ more, the global transcriptional profile of ETP-ALL is similar to that of normal hematopoietic stem cells, AML stem cells, and murine ETP. The activating mutations in the interleukin-7 receptor (IL-7R) gene were reported to be sufficient to generate ETP-ALL in mice, and this murine ETP-ALL model showed the blockage of thymocyte differentiation at the DN2 stage, at which the developmental potentials for both myeloid and T-cell lineages coexists [19]. These findings suggesting ETP-ALL is a neoplasm at the stage of less mature hematopoietic progen‐ itor or stem cells may account for the capacity of ETP-ALL to exert myeloid differentiation.

Gene expression profiling was also investigated in adult ETP-ALL patients. Whole-exome sequencing in adult ETP-ALL cells demonstrated a distinct mutation spectrum from that of pediatric ETP-ALL, particularly in affecting genes involved in epigenetic regulation with higher frequencies of *DNMT3A* and *FAT3* mutations [20]. *DNMT3A*, one of the genes for DNAmethyl-transferase, is a frequent mutational target in AML [21], whereas *FAT3* mutations have been frequently reported in ovarian carcinomas but not AML [22]. The incidence of *DNMT3A* mutations showed a clear age relationship [20]. Adult ETP-ALL patients also had mutations in the genes known to be involved in leukemogenesis, including *ETV6*, *NOTCH1*, *JAK1*, and *NF1*. In addition, more than 60% of the adult patients with ETP-ALL harbored at least a single genetic lesion in *DNMT3A*, *FLT3*, or *NOTCH1* [20]. Furthermore, adult ETP-ALL showed higher expression levels of *BAALC*, *IGFBP7*, *WT-1*, and *MN1* than those in classical T-ALL [4, 13, 18]. As described above, the high expression of *BAALC* and *ERG* were predictive for unfavorable outcomes in adult T-ALL [23, 24]. *IGFBP7* is a stem cell-associated gene, which is functionally highly related to *BAALC* and overexpressed in early T-ALL [25]. The *WT-1* gene is commonly overexpressed in AML [26], and its overexpression is associated with a poor prognosis in thymic T-ALL patients [27]. The overexpression of the *MN1* gene is additionally associated with a shorter survival in the patients with AML without karyotypic abnormalities [28, 29]. The *FLT3* mutations which are frequently observed in AML were found in 37.5% of the adult ETP-ALL but only in 1–3% of the classical T-ALL patients (37.5%) [13], although these *FLT3* mutations in ETP-ALL more frequently generated tyrosine kinase domain (TDK) abnormalities rather than internal tandem duplication (ITD) mutations, which are frequently observed in AML. In relation to the above-mentioned observations, mice that received a transplant of FLT3-ITD-transduced bone marrow cells developed myeloproliferative diseases, while those that received a transplant of FLT3-TDK-transduced bone marrow cells developed lymphoid disorders [30]. Collectively, it may be reasonable to separate ETP-ALL from classical T-ALL due to the distinct genetic profiles between ETP-ALL and other T-ALL subtypes, and the characteristic gene profile of ETP-ALL may provide new therapeutic strategies for this leukemia.

## **6. Therapeutic strategies**

As for the prognosis, ETP-ALL is associated with a higher rate of relapse and induction failure. ETP-ALL is additionally associated with a significantly worse overall survival with a 10-year event free survival (EFS) and overall survival (OS) rates of 22% and 19%, respectively, as compared with 69% EFS and 84% OS for all other subtypes of T-ALL, respectively, in the St. Jude cohort [4]. Similar results were obtained in the cohorts of four other institutions [4, 10-12]. More recently, however, two clinical studies showed no significant differences in the EFS and OS rates between the patients with ETP-ALL and classical T-ALL [14, 15]. Although the reason for this discrepancy is unclear, differences in the therapeutic protocol and patient cohort may have influenced the results of these clinical studies. However, an increased risk of relapse in the patients with ETP-ALL [4, 10-12, 15], especially children [4, 14], was a common result in all these previous studies. Thus, larger prospective studies are needed to determine the real

The expression levels of oncogenic transcription factor genes were examined to establish genetic profiles of ETP-ALL in the St Jude Children's Research Hospital and AIEOP studies. Pediatric ETP-ALL had a higher expression of oncogenic transcription factors: *LMO1*, *LMO2*, *LYL1*, and *ERG* [4, 16]. *LMO1* and *LMO2* are binding partners with hematopoietic basic helixloop-helix transcription factors, such as *SCL/TAL1* or *LYL1*. These proteins interact together to form a transcription factor complex, and they are hypothesized to act through a common mechanism which leads to oncogenesis of T-ALL [17]. McCormack et al. demonstrated that *LYL1* is critical for the oncogenic function of *LMO2*, including the upregulation of a stem celllike gene signature, aberrant self-renewal of thymocytes, and subsequent generation of T-cell leukemia in *LMO2*-transgenic mice. Moreover, *LMO2* and *LYL1* are co-expressed in leukemic cells from the patients with ETP-ALL, and *LYL1* is indispensable for the growth of ETP-ALL cell lines [18]. Whole-genome sequencing studies showed that ETP-ALL had a high frequency of activating mutations in the genes involved in cytokine receptor and RAS signaling (e.g., *NRAS*, *KRAS*, *FLT3*, *IL-7R*, *JAK3*, *LAK1*, *SH2B3*, and *BRAF*) and inactivating mutations in the genes encoding key transcription factors involved in hematopoietic development (e.g., *GATA3*, *ETV6*, *RUNX1*, *IKZF1*, and *EP300*) and involved in epigenetic gene control (e.g., *EZH2*, *EED*, *SUZ12*, *SETD2*, and *EP300* genes) [16]. The gene mutations which affect cytokine receptor regulation and/or RAS signaling pathway are observed in two-thirds of ETP-ALL cases but only in 19% of non-ETP T-ALL cases [16]. These mutational gene spectrums in ETP-ALL are similar to those in acute myeloid leukemia (AML), but not in T- or B-lineage ALLs. Further‐ more, the global transcriptional profile of ETP-ALL is similar to that of normal hematopoietic stem cells, AML stem cells, and murine ETP. The activating mutations in the interleukin-7 receptor (IL-7R) gene were reported to be sufficient to generate ETP-ALL in mice, and this murine ETP-ALL model showed the blockage of thymocyte differentiation at the DN2 stage, at which the developmental potentials for both myeloid and T-cell lineages coexists [19]. These findings suggesting ETP-ALL is a neoplasm at the stage of less mature hematopoietic progen‐ itor or stem cells may account for the capacity of ETP-ALL to exert myeloid differentiation.

prognosis of this T-ALL subtype.

32 Leukemias - Updates and New Insights

**5. Gene profiles**

Coustan-Smith et al. previously reported that the patients with ETP-ALL showed a poor initial response to standard intensive chemotherapies and unfavorable outcomes [4]. Subsequently, six clinical studies showed that ETP-ALL was associated with a very high risk for relapse, whereas two additional studies showed no significant differences in both the EFS and OS rates between the patients with ETP-ALL and classical T-ALL [14, 15]. In the TLLSGL99-15 study, three of four relapsed ETP-ALL patients were successfully treated with allogenic hemato‐ poietic stem cell transplantation (allo-SCT), indicating that allo-SCT could be an effective therapeutic option for ETP-ALL [10]. Prior to this report, the Berlin-Frankfurt-Munster group showed that allo-SCT was superior to chemotherapy alone for high-risk childhood T-ALL [31]. The UKALL 2003 study, which showed better outcomes of ETP-ALL, suggested the beneficial effects of a more intensive chemotherapeutic regimen and the employment of dexamethasone and pegylated asparaginase [15]. High-dose cytarabine combined with epigenetic treatment may be a promising option for ETP-ALL according to the results of whole-genome sequencing, which showed that the mutational spectrum of ETP-ALL was similar to that of AML and that the transcriptional profile was similar to that of normal hematopoietic stem cells and granu‐ locyte-macrophage progenitor cells [4], although these hypotheses need to be proven in future investigations. Additionally, other potential targets have been suggested according to the genetic alterations in the transcription factors. Stat4 phosphorylation was observed in *IL-7R* mutant-induced ETP-ALL cell lines, and consequently, ruxolitinib which is a selective JAK1 and JAK2 inhibitor, was shown to inhibit the proliferation of cells from the ETP-ALL cell lines and prolong the survival of mice xeno-transplanted with the *IL-7R* mutated ETP-ALL cells [19]. Tyrosine kinase inhibitors may be effective when *FLT3* mutations harboring ETP-ALL are molecularly targeted [13]. The antiapoptotic B-cell lymphoma-2 (BCL-2) protein is another attractive molecular target. BCL-2 is highly expressed in ETP-ALL and gradually decreases its expression along with the differentiation toward mature T-cells. ABT-199, an orally bioavail‐ able BCL-2 specific inhibitor, was demonstrated to induce apoptosis of ETP-ALL cells from patients with this subtype of leukemia and dramatically reduced the tumor burden in the bone marrow, spleen, and peripheral blood in mice transplanted with ETP-ALL patient-derived xenografts [32, 33]. In addition, a WT-1 peptide cancer vaccine may be a therapeutic option for relapsed patients or those with minimal residual disease in *WT-1*-overexpressed ETP-ALL, because this approach has demonstrated objective clinical responses in other hematological neoplasms and solid tumors [34].

## **7. Case study**

For a better understanding of ETP-ALL, we herein present two cases of ETP-ALL in our institution, which exhibited unique clinical pictures.

Case 1: A 24-year-old man developed epigastralgia and low-grade fever four months before the admission to our hospital. On gastrofiberscopy performed in a hospital, multiple nonulcerative mucosal nodules were observed. A biopsy specimen from the nodule histologically showed diffuse infiltration of small lymphocytes, which were positive for CD3, CD7, CD8, and CD56 but negative for TIA-1, Epstein-Barr virus-encoded small RNAs-in situ hybridization (EBER-ISH), and a suspected pathological diagnosis was lymphomatoid gastroenteropathy. Three months later, the patient was admitted to our hospital due to the exacerbation of abdominal distress. On this admission, he presented with multiple ulcerative nodules in the gastric mucosa (Figure 4), marked wall thickening of the small intestine, hepatosplenomegaly (Figure 5) and multiple nodular lesions in the bilateral lungs. A histological examination of the biopsied gastric mucosal nodule showed dense infiltration with small immature lympho‐ cytes (Figure 6). The WBC count elevated to 3.83×109 /L with 55% immature lymphocytes (Figure 7). Flow cytometry indicated that these cells were positive for cyCD3, CD7, CD8, CD13, and CD56 (partially), but negative for CD2, smCD3, CD34, TdT, B-cell antigens, and cytoplas‐ mic myeloperoxidase (MPO). A multiplex PCR analysis for TCRγ chain and immunoglobulin heavy chain genes yielded negative results regarding the monoclonal gene rearrangements. A cytogenetic examination of the bone marrow cells, including abundant leukemic cells, gave a normal karyotype of 46, XY. He was subsequently diagnosed with ETP-ALL according to these immunophenotypes of abnormal lymphocytes, which fulfilled the criteria of the TCCSG L99-15 study scoring system but not the St. Jude Criteria due to the CD8 positivity. Although the leukemia was resistant to CHOP (cyclophosphamide, adriamycin, vincristine, and prednisolone) and SMILE (dexamethasone, methotrexate, ifosfamide, L-asparaginase, and etoposide) [35] regimens, a complete remission (CR) was obtained with the MEC regimen (mitoxantrone, etoposide, and cytarabine) followed by nelarabine. He underwent unrelated allogeneic bone marrow transplantation and is currently maintaining CR. Importantly, a marked intestinal involvement at presentation has not been reported in ETP-ALL.

and pegylated asparaginase [15]. High-dose cytarabine combined with epigenetic treatment may be a promising option for ETP-ALL according to the results of whole-genome sequencing, which showed that the mutational spectrum of ETP-ALL was similar to that of AML and that the transcriptional profile was similar to that of normal hematopoietic stem cells and granu‐ locyte-macrophage progenitor cells [4], although these hypotheses need to be proven in future investigations. Additionally, other potential targets have been suggested according to the genetic alterations in the transcription factors. Stat4 phosphorylation was observed in *IL-7R* mutant-induced ETP-ALL cell lines, and consequently, ruxolitinib which is a selective JAK1 and JAK2 inhibitor, was shown to inhibit the proliferation of cells from the ETP-ALL cell lines and prolong the survival of mice xeno-transplanted with the *IL-7R* mutated ETP-ALL cells [19]. Tyrosine kinase inhibitors may be effective when *FLT3* mutations harboring ETP-ALL are molecularly targeted [13]. The antiapoptotic B-cell lymphoma-2 (BCL-2) protein is another attractive molecular target. BCL-2 is highly expressed in ETP-ALL and gradually decreases its expression along with the differentiation toward mature T-cells. ABT-199, an orally bioavail‐ able BCL-2 specific inhibitor, was demonstrated to induce apoptosis of ETP-ALL cells from patients with this subtype of leukemia and dramatically reduced the tumor burden in the bone marrow, spleen, and peripheral blood in mice transplanted with ETP-ALL patient-derived xenografts [32, 33]. In addition, a WT-1 peptide cancer vaccine may be a therapeutic option for relapsed patients or those with minimal residual disease in *WT-1*-overexpressed ETP-ALL, because this approach has demonstrated objective clinical responses in other hematological

For a better understanding of ETP-ALL, we herein present two cases of ETP-ALL in our

Case 1: A 24-year-old man developed epigastralgia and low-grade fever four months before the admission to our hospital. On gastrofiberscopy performed in a hospital, multiple nonulcerative mucosal nodules were observed. A biopsy specimen from the nodule histologically showed diffuse infiltration of small lymphocytes, which were positive for CD3, CD7, CD8, and CD56 but negative for TIA-1, Epstein-Barr virus-encoded small RNAs-in situ hybridization (EBER-ISH), and a suspected pathological diagnosis was lymphomatoid gastroenteropathy. Three months later, the patient was admitted to our hospital due to the exacerbation of abdominal distress. On this admission, he presented with multiple ulcerative nodules in the gastric mucosa (Figure 4), marked wall thickening of the small intestine, hepatosplenomegaly (Figure 5) and multiple nodular lesions in the bilateral lungs. A histological examination of the biopsied gastric mucosal nodule showed dense infiltration with small immature lympho‐

(Figure 7). Flow cytometry indicated that these cells were positive for cyCD3, CD7, CD8, CD13, and CD56 (partially), but negative for CD2, smCD3, CD34, TdT, B-cell antigens, and cytoplas‐ mic myeloperoxidase (MPO). A multiplex PCR analysis for TCRγ chain and immunoglobulin heavy chain genes yielded negative results regarding the monoclonal gene rearrangements. A

/L with 55% immature lymphocytes

neoplasms and solid tumors [34].

34 Leukemias - Updates and New Insights

institution, which exhibited unique clinical pictures.

cytes (Figure 6). The WBC count elevated to 3.83×109

**7. Case study**

**Figure 4.** Gastrofiberscopy of Case 1 on admission to our hospital. Multiple ulcerative nodules were visible on the gas‐ tric mucosa.

Case 2: An 83-year-old female who presented with generalized lymphadenopathy was referred to our hospital. She was tentatively diagnosed with peripheral T-cell lymphomaunspecified according to the findings from a biopsy specimen from a cervical lymph node, which histologically showed diffuse infiltration of CD3-positive lymphocytes and a proliferation of Langerhans cells without dysplastic features. The lymphadenopathy disappeared after CHOP chemotherapy; however, blast cells (Figure 8A) appeared in the peripheral blood and rapidly increased in number without recurrence of the lymphadenop‐ athy after the fourth round of CHOP chemotherapy. The blast cells expressed cyCD3, CD7, CD56, CD33, and CD34, but not CD2, smCD3, CD4, and CD8. PCR of the TCRγ chain gene

**Figure 5.** Contrast CT scanning of the abdomen (coronal image) in Case 1. Marked hepatosplenomegaly and wall thickening of the small intestine (arrows) were observed.

**Figure 6.** Histology of the biopsied gastric mucosal nodule in Case 1. Diffuse and dense infiltration of immature lym‐ phocytes is shown (H-E staining, 100×).

demonstrated a monoclonally rearranged faint band. These blast cells were negative for MPO staining; however, some of the cells were weakly positive for both α-naphthyl butyrate (Figure 8B) and naphthol AS-D chloroacetate esterase staining (Figure 8C), suggesting their ability to differentiate toward monocytes and granulocytes. A chromosomal analysis revealed an abnormal karyotype of 46, XX, t(12;20)(q13;q11.2) in seven of the 20 bone marrow cells analyzed. A final diagnosis of ETP-ALL was made according to these immunophenotypes, which fulfilled both the TCCSG L99-15 study scoring system and St. Jude criteria. Her leukemia was resistant to any chemotherapeutic protocols for lympho‐ ma, ALL, and AML, and she ultimately died due to disease progression.

Early T-cell Precursor Acute Lymphoblastic Leukemia – A Characteristic Neoplasm Presenting the Phenotype of… http://dx.doi.org/10.5772/60901 37

> increased in number blast cells expressed gene demonstrated a some of the cells were staining (Figure 8C), analysis revealed an A final diagnosis of study scoring system ALL, and AML, and

nuclei were irregular in

she ultimately died due to disease progression. progression.**Figure 7.** Abnormal lymphocytes in the peripheral blood in Case 1 (Wright-Giemsa staining, 1,000×).

Figure 8. Abnormal lymphocytes in the peripheral staining; C: naphthol AS-D chloroacetate esterase peripheral blood in Case 2. A: Wright-Giemsa staining, 1,000×); B: a-naphthyl esterase staining. Esterase staining was performed using a kit for double naphthyl butyrate esterase double esterase staining **Figure 8.** Abnormal lymphocytes in the peripheral blood in Case 2. A: Wright-Giemsa staining, 1,000×); B: a-naphthyl butyrate esterase staining; C: naphthol AS-D chloroacetate esterase staining. Esterase staining was performed using a kit for double esterase staining (Muto Pure Chemicals, Tokyo, Japan).

(Muto Pure Chemicals, Tokyo, Japan). In both cases, it was difficult to make a precise analysis was crucially important to determine phenotypic presentation reflecting an oncogenic in early normal hematopoiesis. In addition, precise diagnosis with a histopathological strategy, and the determine the final diagnosis. Both cases are very interesting oncogenic development at the level of granulocyte-macrophage addition, morphologically, leukemic cells in these two cases had a immunophenotypic interesting in terms of the macrophage-T-cell progenitors a slightly condensed In both cases, it was difficult to make a precise diagnosis with a histopathological strategy, and the immunophenotypic analysis was crucially important to determine the final diagnosis. Both cases are very interesting in terms of the phenotypic presentation reflecting an oncogenic development at the level of granulocyte-macrophage-T-cell progenitors in early normal hematopoiesis. In addition, morphologically, leukemic cells in these two cases had a slightly condensed chromatin network of the nucleus when compared with that of classical ALL blasts and these nuclei were irregular in shape.

compared with that of classical ALL blasts and these nuclei

#### 8. Conclusion **8. Conclusion**

Disclosure

shape.

chromatin network of the nucleus when compared

The authors declare that there are no conflicts

demonstrated a monoclonally rearranged faint band. These blast cells were negative for MPO staining; however, some of the cells were weakly positive for both α-naphthyl butyrate (Figure 8B) and naphthol AS-D chloroacetate esterase staining (Figure 8C), suggesting their ability to differentiate toward monocytes and granulocytes. A chromosomal analysis revealed an abnormal karyotype of 46, XX, t(12;20)(q13;q11.2) in seven of the 20 bone marrow cells analyzed. A final diagnosis of ETP-ALL was made according to these immunophenotypes, which fulfilled both the TCCSG L99-15 study scoring system and St. Jude criteria. Her leukemia was resistant to any chemotherapeutic protocols for lympho‐

**Figure 6.** Histology of the biopsied gastric mucosal nodule in Case 1. Diffuse and dense infiltration of immature lym‐

**Figure 5.** Contrast CT scanning of the abdomen (coronal image) in Case 1. Marked hepatosplenomegaly and wall

thickening of the small intestine (arrows) were observed.

36 Leukemias - Updates and New Insights

phocytes is shown (H-E staining, 100×).

ma, ALL, and AML, and she ultimately died due to disease progression.

More precise and extensive cellular and molecular genetic or epigenetic nature of ETP-ALL. effective therapeutic strategies for this highmolecular investigations are required to establish the definite An accumulation of ETP-ALL cases and larger clinical -risk leukemia. definite cellular origin and trials will establish More precise and extensive cellular and molecular investigations are required to establish the definite cellular origin and genetic or epigenetic nature of ETP-ALL. An accumulation of ETP-ALL cases and larger clinical trials will establish effective therapeutic strategies for this highrisk leukemia.

conflicts of interest with any individuals or companies.

## **Author details**

Hiroko Tsunemine and Takayuki Takahashi\*

\*Address all correspondence to: takahashi.takayuki@shinkohp.or.jp

Department of Hematology, Shinko Hospital, Kobe, Japan

The authors declare that there are no conflicts of interest with any individuals or companies.

## **References**


[11] Chopra A, Bakhshi S, Pramanik SK, Pandey RM, Singh S, Gajendra S, et al. Immuno‐ phenotypic analysis of T-acute lymphoblastic leukemia. A CD5-based ETP-ALL per‐ spective of non-ETP T-ALL. Eur J Haematol. 2014;92:211-8.

**Author details**

38 Leukemias - Updates and New Insights

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## **Leukemia and Retroviral Disease**

Lorena Loarca, Neil Sullivan, Vanessa Pirrone and Brian Wigdahl

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61254

#### **Abstract**

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[28] Heuser M, Beutel G, Krauter J, Dohner K, von Neuhoff N, Schlegelberger B, et al. High meningioma 1 (MN1) expression as a predictor for poor outcome in acute mye‐

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human acute leukemias. Leukemia. 1995;9:1060-7.

Two human retroviruses, identified as the human T-cell leukemia virus type 1 (HTLV-1) and human immunodeficiency virus type 1 (HIV-1), have been shown to affect millions of people worldwide. In the context of coinfection, the impact of their interactions with respect to HTLV-1-induced adult T-cell leukemia and neurologic disease as well as HIV-1 disease progression has been an understudied area of in‐ vestigation. HTLV-1/HIV-1 coinfections occur frequently, particularly in large met‐ ropolitan areas of the Americas, Africa, Europe, and Japan. The retroviruses HTLV-1 and HIV-1 share some similarities with regard to their genetic structure, general mechanisms of replication, modes of transmission, and cellular tropism; however, there are also significant differences in the details of these properties as well, and they also differ significantly with respect to the etiology of their pathogen‐ ic and disease outcomes. Both viruses impair the host immune system with HIV-1 demonstrated to cause the hallmark lethal disease known as the acquired immune deficiency syndrome (AIDS), while HTLV-1 infection has been shown to cause sev‐ eral different forms of T-cell leukemia. In addition, both viruses have also been shown to cause a spectrum of neurologic disorders with HIV-1 shown to cause an array of neurologic syndromes referred to as HIV-1-associated neurologic disorders or HAND, while HTLV-1 has been shown to be the etiologic agent of HTLV-1-asso‐ ciated myelopathy/tropical spastic paraparesis or HAM/TSP. The natural history of the coinfection, however, is different from that observed in monoinfections. Several studies have demonstrated utilizing a number of in vitro models of HTLV-1/HIV-1 coinfection that the two viruses interact in a manner that results in enhanced ex‐ pression of both viral genomes. Nevertheless, there remains unresolved controversy regarding the overall impact of each virus on progression of disease caused by both viruses during the course of coinfection. Although combination antiretroviral thera‐ py has been shown to work very effectively with respect to maintaining HIV-1 viral loads in the undetectable range, these therapeutic strategies exhibit no benefit for HTLV-1-infected individuals, unless administered immediately after exposure. Fur‐ thermore, the treatment options for HTLV-1/HIV-1-coinfected patients are very lim‐ ited. In recent years, allogeneic stem cell transplantation (alloSCT) has been used for

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

the treatment of leukemia. In this regard, the case of a leukemic patient positive for HIV-1 who was cured of their HIV-1 infection while treated with alloSCT for acute myeloid leukemia has also been examined with regard to impact on HIV-1 disease.

**Keywords:** HIV-1, HTLV-1, coinfection, ATL/ATLL, HAM/TSP

## **1. Introduction**

HTLV-1/HIV-1 coinfection is very common among drug users, particularly in metropolitan areas [1, 2]. It is estimated that in endemic areas, 10% of HIV-1-infected patients are coinfected with HTLV-1 [3]. The frequency of HTLV-1 and HIV-1 coinfections is on the rise, especially in Africa and South America [4]. Both HTLV-1 and HIV-1 are retroviruses capable of integrating their proviral DNA genome into the host cell chromosome, thereby establishing a latent infection, both of which share quiescent CD4+ T cells as their primary target. However, the life cycle, pathogenesis, and clinical syndromes of these two viruses after infection are very different within this cellular compartment [5, 6]. In the absence of therapeutic intervention, there is a striking difference in the prevalence of disease caused by the two viruses with overt clinical disease far less common after HTLV-1 infection, perhaps because HTLV-1 has existed within the human population for far longer than HIV-1 and therefore may be more highly adapted to its human host. This could explain why HTLV-1 has been shown to cause T-cell transformation and clonal expansion of immortalized cells, whereas HIV-1 induces CD4+ Tcell death [7]. Since the first cases of HIV-1 were reported early in the 1980s, there has been much progress with respect to investigating the viral replication cycle, mechanisms of pathogenesis, as well as the diagnosis and treatment of HIV-1 infection. There are now more than 30 antiretroviral drugs available for the management of HIV-1 infection, with them, their modes of inhibition and novel drug discovery techniques have been reviewed previously [8]. A number of drug combinations have been shown to be quite effective with regard to reducing HIV-1 titers to undetectable levels with subsequent maintenance of this well-controlled state for many years if not decades, yet they do not cure individuals because the drugs are effective against actively replicating virus and not the latent, integrated provirus. In the absence of treatment and subsequent to the development of drug resistance, particularly in the precom‐ bination antiretroviral therapy (cART) era, HIV-1 has been shown to induce severe immuno‐ suppression leading to AIDS with the ultimate development of opportunistic infections and cancers. In the case of HTLV-1 infection, most patients remain asymptomatic for many years before the onset of disease. In contrast to HIV-1, only a small percentage of untreated HTLV-1 carriers develop clinically apparent disease [9, 10]. HTLV-1 may cause neurologic problems, skin and inflammatory disorders, leukemia, and leukemia/lymphoma [9, 10]. The treatments available for HTLV-1-related complications are limited, and the antiretrovirals used to treat HIV-1 infection are not efficacious unless taken early after first contact with the virus [6] when viral replication is responsible for expansion in the number of infected cells as compared to expansion of infected cells by cell division with the associated integrated HTLV-1 provirus expanding within the transformed cell population in the absence of infectious HTLV-1 production [5]. The clinical implications and the molecular interactions between HTLV-1 and HIV-1 remain understudied. The management of patients coinfected with HTLV-1/HIV-1 is clearly a challenge. Although it is well known that both HTLV-1 and HIV-1 may cause progressive diseases within the central nervous system, the focus here will center on the interaction of these two retroviruses within the immune system and more specifically examine the impact of HIV-1 infection on the leukemogenic process induced by HTLV-1 in coinfected individuals as well as the impact of HTLV-1 infection on HIV-1 disease. A summary of the epidemiology of the two viruses within the human population is shown in Table 1.


**Table 1.** Epidemiological comparison of HTLV-1 and HIV-1 infection and disease

the treatment of leukemia. In this regard, the case of a leukemic patient positive for HIV-1 who was cured of their HIV-1 infection while treated with alloSCT for acute myeloid leukemia has also been examined with regard to impact on HIV-1 disease.

HTLV-1/HIV-1 coinfection is very common among drug users, particularly in metropolitan areas [1, 2]. It is estimated that in endemic areas, 10% of HIV-1-infected patients are coinfected with HTLV-1 [3]. The frequency of HTLV-1 and HIV-1 coinfections is on the rise, especially in Africa and South America [4]. Both HTLV-1 and HIV-1 are retroviruses capable of integrating their proviral DNA genome into the host cell chromosome, thereby establishing a latent

cycle, pathogenesis, and clinical syndromes of these two viruses after infection are very different within this cellular compartment [5, 6]. In the absence of therapeutic intervention, there is a striking difference in the prevalence of disease caused by the two viruses with overt clinical disease far less common after HTLV-1 infection, perhaps because HTLV-1 has existed within the human population for far longer than HIV-1 and therefore may be more highly adapted to its human host. This could explain why HTLV-1 has been shown to cause T-cell transformation and clonal expansion of immortalized cells, whereas HIV-1 induces CD4+ Tcell death [7]. Since the first cases of HIV-1 were reported early in the 1980s, there has been much progress with respect to investigating the viral replication cycle, mechanisms of pathogenesis, as well as the diagnosis and treatment of HIV-1 infection. There are now more than 30 antiretroviral drugs available for the management of HIV-1 infection, with them, their modes of inhibition and novel drug discovery techniques have been reviewed previously [8]. A number of drug combinations have been shown to be quite effective with regard to reducing HIV-1 titers to undetectable levels with subsequent maintenance of this well-controlled state for many years if not decades, yet they do not cure individuals because the drugs are effective against actively replicating virus and not the latent, integrated provirus. In the absence of treatment and subsequent to the development of drug resistance, particularly in the precom‐ bination antiretroviral therapy (cART) era, HIV-1 has been shown to induce severe immuno‐ suppression leading to AIDS with the ultimate development of opportunistic infections and cancers. In the case of HTLV-1 infection, most patients remain asymptomatic for many years before the onset of disease. In contrast to HIV-1, only a small percentage of untreated HTLV-1 carriers develop clinically apparent disease [9, 10]. HTLV-1 may cause neurologic problems, skin and inflammatory disorders, leukemia, and leukemia/lymphoma [9, 10]. The treatments available for HTLV-1-related complications are limited, and the antiretrovirals used to treat HIV-1 infection are not efficacious unless taken early after first contact with the virus [6] when viral replication is responsible for expansion in the number of infected cells as compared to expansion of infected cells by cell division with the associated integrated HTLV-1 provirus expanding within the transformed cell population in the absence of infectious HTLV-1

T cells as their primary target. However, the life

**Keywords:** HIV-1, HTLV-1, coinfection, ATL/ATLL, HAM/TSP

**1. Introduction**

42 Leukemias - Updates and New Insights

infection, both of which share quiescent CD4+

## **2. Introduction to Human T-cell Leukemia Virus type 1 (HTLV-1)**

HTLV-1 is a single-stranded positive-sense RNA, a type C retrovirus, and the etiologic agent of adult T-cell leukemia (ATL) or adult T-cell leukemia/lymphoma (ATLL) and a progressive neuroinflammatory disease known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), with some similarities to multiple sclerosis due to the progressive destruction/loss of myelination [11] (also reviewed in [68]). It was the first human retrovirus discovered [10, 12] and isolated in the United States in the late 1970s, with parallel discoveries by investigators in Japan [13–15]. In this regard, in the United States, HTLV-1 particles were detected in fresh peripheral blood lymphocytes and in the T-cell lymphoblastoid cell lines, HUT 102 and CTLC-3, derived from a 28-year-old African American man suffering from cutaneous T-cell lymphoma [13]. At nearly the same time, the Japanese investigators isolated HTLV-1 from a series of cell lines derived from cases of adult T-cell leukemia [13, 15]. Over the years, HTLV-1 has been shown to be associated with the human population for a much longer period of time than the HIV-1 with HTLV-1 being detected in remains of a 1,500 Chilean mummy [16], while HIV-1 sequences have been detected in humans only as far back as the 1920s, many decades before its discovery in the mid-1980s. Based on extensive studies performed over the past three to four decades concerning clinical parameters, epidemiology, molecular biology and virology, immunology, cancer biology, neurobiology, and immuneand neuropathogenesis of HTLV-1 infection, the virus appears to be much more adapted to the human population with greater than 95% of the infected individuals harboring the virus asymptomatically with only small percentages of individuals presenting with symptomatic disease in the form of leukemia or neurologic disease [9, 17]. This epidemiologic pattern is quite different than the widespread highly lethal disease of the immune and nervous systems caused by the HIV-1 in the absence of highly active antiretroviral therapy (HAART) [18] (also reviewed in [19]).

#### **2.1. HTLV-1 genetic architecture and viral replication**

The HTLV-1 genome comprises four main genetic components that include gag, pol, env, and pX sequences (Figure 1). The gag, pol, and env genes encode the structural proteins and enzymatic proteins, with a 5 and 3 long terminal repeat (LTR) at each end of the genome, typical of all viruses in the Retroviridae family. Gag proteins have been designated the group-specific antigens, which encode the inner virion structural components such as matrix, capsid, and nucleocapsid. The pro and pol genes encode the protease, integrase, and viral RNA-dependent DNA polymerase or reverse transcriptase, respectively, and the env gene that encodes the viral surface and transmembrane envelope proteins [63]. What makes HTLV-1 unique compared to other retroviruses is the pX region. This region encodes multiple accessory and regulatory proteins that give HTLV-1 its unique phenotype, one of which is the multifunction oncoprotein Tax. Tax has been shown to alter the course of gene expression at a number of points during the course of viral infection and in this regard has been shown to regulate viral replication, modulate cellular gene expression, induce inflammation, and block apoptosis to pinpoint just a few, with many of its functions being identified by Tax mutagenesis [20]. More importantly, this protein is known for its ability to transform T lymphocytes into cancerous leukemic cells, as described in the following sections. HTLV-1 infection of susceptible cells via transmission involving transfer of virus from an infected cell to an uninfected cell occurs very efficiently as a result of specific cell–cell interactions and less efficiently from cell-free viral particle-driven transmission [21–23]. The replication cycle of HTLV-1 begins with the interaction between the viral Env glycoproteins and the specific cellular receptor proteins. At least three cellular receptors have been found to facilitate viral attachment and entry into the cell, and these include the glucose transporter 1 (GLUT1) [24, 25], neuropilin-1 (NRP-1) [26–28], and heparan sulfate proteoglycans (HSPG) [29–31]. During the membrane fusion process and after entry of the viral capsid into the cytoplasm, the viral genomic RNA is reverse transcribed into DNA, by the viral particle-associated reverse transcriptase. During the process of reverse transcrip‐ tion, the newly synthesized proviral DNA is transported to the nuclear membrane. After the translocation of the proviral DNA into the nucleus, it is subsequently integrated into the host cell chromosomal material, which is catalyzed by the activity of the viral-encoded, particleassociated integrase. Following integration, the transcription of viral RNAs and the translation of viral proteins are carried out by host machinery with the subsequent assembly of viral particles and release of infectious virions into the extracellular environment.

**Figure 1.** HTLV-1 genomic architecture. A schematic representation of the proviral genome organization, open reading frames, and viral products of HTLV-1. The organization of the ~9-kb genome is depicted along with the genes and their transcriptional splicing.

## **2.2. HTLV-1 infectivity, transmission, and pathogenesis**

molecular biology and virology, immunology, cancer biology, neurobiology, and immuneand neuropathogenesis of HTLV-1 infection, the virus appears to be much more adapted to the human population with greater than 95% of the infected individuals harboring the virus asymptomatically with only small percentages of individuals presenting with symptomatic disease in the form of leukemia or neurologic disease [9, 17]. This epidemiologic pattern is quite different than the widespread highly lethal disease of the immune and nervous systems caused by the HIV-1 in the absence of highly active antiretroviral therapy (HAART) [18] (also

The HTLV-1 genome comprises four main genetic components that include gag, pol, env, and pX sequences (Figure 1). The gag, pol, and env genes encode the structural proteins and enzymatic proteins, with a 5 and 3 long terminal repeat (LTR) at each end of the genome, typical of all viruses in the Retroviridae family. Gag proteins have been designated the group-specific antigens, which encode the inner virion structural components such as matrix, capsid, and nucleocapsid. The pro and pol genes encode the protease, integrase, and viral RNA-dependent DNA polymerase or reverse transcriptase, respectively, and the env gene that encodes the viral surface and transmembrane envelope proteins [63]. What makes HTLV-1 unique compared to other retroviruses is the pX region. This region encodes multiple accessory and regulatory proteins that give HTLV-1 its unique phenotype, one of which is the multifunction oncoprotein Tax. Tax has been shown to alter the course of gene expression at a number of points during the course of viral infection and in this regard has been shown to regulate viral replication, modulate cellular gene expression, induce inflammation, and block apoptosis to pinpoint just a few, with many of its functions being identified by Tax mutagenesis [20]. More importantly, this protein is known for its ability to transform T lymphocytes into cancerous leukemic cells, as described in the following sections. HTLV-1 infection of susceptible cells via transmission involving transfer of virus from an infected cell to an uninfected cell occurs very efficiently as a result of specific cell–cell interactions and less efficiently from cell-free viral particle-driven transmission [21–23]. The replication cycle of HTLV-1 begins with the interaction between the viral Env glycoproteins and the specific cellular receptor proteins. At least three cellular receptors have been found to facilitate viral attachment and entry into the cell, and these include the glucose transporter 1 (GLUT1) [24, 25], neuropilin-1 (NRP-1) [26–28], and heparan sulfate proteoglycans (HSPG) [29–31]. During the membrane fusion process and after entry of the viral capsid into the cytoplasm, the viral genomic RNA is reverse transcribed into DNA, by the viral particle-associated reverse transcriptase. During the process of reverse transcrip‐ tion, the newly synthesized proviral DNA is transported to the nuclear membrane. After the translocation of the proviral DNA into the nucleus, it is subsequently integrated into the host cell chromosomal material, which is catalyzed by the activity of the viral-encoded, particleassociated integrase. Following integration, the transcription of viral RNAs and the translation of viral proteins are carried out by host machinery with the subsequent assembly of viral

particles and release of infectious virions into the extracellular environment.

reviewed in [19]).

44 Leukemias - Updates and New Insights

**2.1. HTLV-1 genetic architecture and viral replication**

Although the tropism of HTLV-1 may not represent a direct representation of cells that can be infected *in vivo,* studies performed *in vitro* are often performed to get an approximation regarding the cell types that may be targeted by a virus during the course of *in vivo* infection. If proven susceptible and productive for viral replication, these cell types may then be used for virus propagation or studies concerning viral pathogenesis. In this regard, during *in vitro* propagation of HTLV-1, a wide variety of non-T-cell types have been shown to be susceptible to viral infection, including human primary endothelial cells [32], monocytes [33], microglial cells [33], B cells [34], mammary epithelial cells [35], and dendritic cells (DCs) [36], although the relative level of viral production between the different cell types was shown to differ greatly. In parallel with these observations, HTLV-1 infection *in vivo has* been shown to occur primarily in CD4+ T-cell subsets and to a lesser extent in CD8+ T cells in both asymptomatic and symptomatic HTLV-1-infected patients [37]. Furthermore, Koyanagi and colleagues [37] demonstrated HTLV-1 tropism for CD8+ T cells, monocytes, and B cells in the majority of the asymptomatic HTLV-1-positive individuals studied, as well as in patients with HTLV-1 related ATL or HAM/TSP. Other groups have also confirmed these observations and have reported that HTLV-1 also infects macrophages [37], DCs [38], synovial fluid cells [39], and astrocytes [40] among others when the target cells are examined *in vivo*. Perhaps of great importance in the pathogenesis of HTLV-1-associated neurologic disease is the penetration of the virus into the bone marrow compartment with increasing numbers of HTLV-1 DNA+ / RNAprogenitor cells detected in patients suffering from HAM/TSP as compared to patients with ATL [41], thus further demonstrating another layer of complexity with respect to target cell identification that likely involves latency, immune invasion, and adaptation to the host.

HTLV-1 has been shown to be transmitted primarily via three routes: (i) mother-to-child transmission [42], (ii) sexual intercourse [43], and (iii) parenteral transmission. The vertical transmission of HTLV-1 from mother to child occurs via the transfer of maternally infected lymphocytes to the fetus or newborn through the placenta [44], during delivery [45, 46], or by breastfeeding [47]. Sexual transmission is a common route of HTLV-1 transmission. Sexual transmission of HTLV-1 occurs more efficiently from male to female than from female to male. This might be due in part to the higher numbers of HTLV-1-infected lymphocytes found in semen than in vaginal secretions [48]. The parenteral transmission of HTLV-1 includes blood or cellular blood products transferred during the transfusion process [49], organ transplanta‐ tion [50], and possibly percutaneous exposure of the virus via sharing of contaminated objects such as razor blades and needles, particularly among drug users and healthcare workers [48]. Parenteral transmission represents a large proportion of infected individuals. Regardless of how the virus is acquired, the infected cells produce numerous progeny virion. Particularly in the case of T lymphocytes, they too are activated and clonally proliferate, further driving the expansion and number of cells harboring provirus DNA.

## **2.3. Cancers and other diseases associated with HTLV-1 infection**

Currently, there are approximately 20 million individuals living with HTLV-1 worldwide [7]. In highly endemic regions such as the Caribbean Basin, Central Africa, and southern Japan, more than 1% of the population is infected with HTLV-1 (reviewed in [51] and [52]). Approx‐ imately 95% of the individuals infected with HTLV-1 remain as asymptomatic carriers throughout their lives [9, 17]. As previously indicated, HTLV-1 is the etiological agent for causing two distinct disease phenotypes, ATL and HAM/TSP, the first involving a CD4+ T-cell malignancy and the second involving a progressive neurological disease. Interestingly, the specific response of the immune system to the virus seems to influence which clinical mani‐ festation presents and likely includes other factors such as route of transmission, host genetics, and perhaps aspects of viral genetics.

Less than 5% of the HTLV-1-infected individuals develop ATL after a long period of latency, which in some cases can be greater than 50 years [53]. ATL is more prevalent in men than in women with a median onset age of 55 years. ATL can present as four overlapping clinical manifestations that are broken down into smoldering, chronic, acute, and lymphoma [54, 55]. Approximately 5% of the patients with ATL have been shown to develop the smoldering type of disease presenting with a number of minor symptoms with leukemic cells infiltrating the skin causing surface lesions leading to a breach in the epithelial layer of the skin [56]. It has been estimated that 20% of the HTLV-1-positive patients will develop a chronic form of the disease. These patients experience similar manifestations as individuals with smoldering ATL, but they also develop abnormalities in their viscera, leading to impairment of spleen, liver, and lymphatic functions as well as a slight increase in the levels of leukemic cells [54].

In the acute phase of ATL, the disease progresses quickly, and patients exhibit generalized swelling of the lymph nodes, elevated calcium and lactate dehydrogenase levels, impairment of liver and spleen function, skin lesions, bone wounds, and release of cytokines by malignant cells. All of these abnormalities cause patients to experience fever, cough, malaise, dehydra‐ tion, lethargy, shortness of breath, and inflammation of the lymph nodes. About 55% of patients with ATL experience the acute form of the disease. Furthermore, around 20% of the ATL cases are of the lymphoma type, and they experience inflammation of the lymph nodes, with no evidence of leukemic cells in the periphery, and general suppression of their immune system function as summarized in Figure 2. The survival rate associated with ATL from the time of first disease manifestations is approximately 24.3, 10.2, and 6.2 months for the chronic, lymphoma, and acute types, respectively [55]. The actual process of transforming T lympho‐ cytes and developing ATL is dependent on the HTLV-1 oncoprotein Tax in part as well as a number of other factors indicated above.

transmission of HTLV-1 from mother to child occurs via the transfer of maternally infected lymphocytes to the fetus or newborn through the placenta [44], during delivery [45, 46], or by breastfeeding [47]. Sexual transmission is a common route of HTLV-1 transmission. Sexual transmission of HTLV-1 occurs more efficiently from male to female than from female to male. This might be due in part to the higher numbers of HTLV-1-infected lymphocytes found in semen than in vaginal secretions [48]. The parenteral transmission of HTLV-1 includes blood or cellular blood products transferred during the transfusion process [49], organ transplanta‐ tion [50], and possibly percutaneous exposure of the virus via sharing of contaminated objects such as razor blades and needles, particularly among drug users and healthcare workers [48]. Parenteral transmission represents a large proportion of infected individuals. Regardless of how the virus is acquired, the infected cells produce numerous progeny virion. Particularly in the case of T lymphocytes, they too are activated and clonally proliferate, further driving the

Currently, there are approximately 20 million individuals living with HTLV-1 worldwide [7]. In highly endemic regions such as the Caribbean Basin, Central Africa, and southern Japan, more than 1% of the population is infected with HTLV-1 (reviewed in [51] and [52]). Approx‐ imately 95% of the individuals infected with HTLV-1 remain as asymptomatic carriers throughout their lives [9, 17]. As previously indicated, HTLV-1 is the etiological agent for causing two distinct disease phenotypes, ATL and HAM/TSP, the first involving a CD4+ T-cell malignancy and the second involving a progressive neurological disease. Interestingly, the specific response of the immune system to the virus seems to influence which clinical mani‐ festation presents and likely includes other factors such as route of transmission, host genetics,

Less than 5% of the HTLV-1-infected individuals develop ATL after a long period of latency, which in some cases can be greater than 50 years [53]. ATL is more prevalent in men than in women with a median onset age of 55 years. ATL can present as four overlapping clinical manifestations that are broken down into smoldering, chronic, acute, and lymphoma [54, 55]. Approximately 5% of the patients with ATL have been shown to develop the smoldering type of disease presenting with a number of minor symptoms with leukemic cells infiltrating the skin causing surface lesions leading to a breach in the epithelial layer of the skin [56]. It has been estimated that 20% of the HTLV-1-positive patients will develop a chronic form of the disease. These patients experience similar manifestations as individuals with smoldering ATL, but they also develop abnormalities in their viscera, leading to impairment of spleen, liver, and lymphatic functions as well as a slight increase in the levels of leukemic cells [54].

In the acute phase of ATL, the disease progresses quickly, and patients exhibit generalized swelling of the lymph nodes, elevated calcium and lactate dehydrogenase levels, impairment of liver and spleen function, skin lesions, bone wounds, and release of cytokines by malignant cells. All of these abnormalities cause patients to experience fever, cough, malaise, dehydra‐ tion, lethargy, shortness of breath, and inflammation of the lymph nodes. About 55% of patients with ATL experience the acute form of the disease. Furthermore, around 20% of the

expansion and number of cells harboring provirus DNA.

and perhaps aspects of viral genetics.

46 Leukemias - Updates and New Insights

**2.3. Cancers and other diseases associated with HTLV-1 infection**

**Figure 2.** Summary of pathogenic forms of HTLV-1-induced leukemia. A summary of disease outcomes and sympto‐ matology during the course of HTLV-1-induced leukemia.

Like most other RNA viruses, due to the constraints of genome size, as compared to larger DNA viruses, the proteins they encode usually have multiple functions. The multifunction oncoprotein Tax is a 353 amino acid phosphoprotein that is a transcriptional activator of the LTR, and the protein is primarily responsible for transformation of T lymphocytes. It has been shown that selected domains of Tax are responsible for interacting with the host transcription factors NF-κB, ATF/CREB, Sp1, Ets-1, and many others in conjunction with their cognate binding sites. This leads to enhanced chromatin remodeling, transactivation of the LTR, activation of host genes, and enhanced viral gene expression. More importantly in the process of cellular transformation is its chronic activation of signaling pathways (JAK/STAT), expres‐ sion of cytokines and their receptors (IL-2, IL-2Rα), and interaction with cellular tumor suppressors (p53) and cell cycle kinases and regulators (p15, p16, and p21), to name a few, all of which increase the probability of uncontrolled cell division and transformation exhaustively reviewed [10, 57, 58]. As of now, it is not fully understood how and why Tax, which is a strong inducer of transformation, only induces ATL in 5% of infected individuals. This could be, in part, the immune system efficiently removing infected cells and/or combating Tax with specific host restriction factors. Additionally, Tax has been shown to be secreted from infected cells and have bystander effects, such as pro-inflammatory cytokines and the infiltration of Taxspecific CD8+ T cells into the CNS, thus playing a part in HAM/TSP [59–64].

HTLV-1 infection also causes HAM/TSP, a neuroinflammatory disorder that mostly affects the spinal cord and brain due to chronic proinflammatory cytokines, the destruction of myelin, and the cells that secrete it, oligodendrocytes [64–66]. It was first thought that there might be a hormonal component regulating HAM/TSP, as it occurs more frequently and progresses more rapidly in women than in men, particularly if the first signs of disease occur before menopause [67]. The onset of HAM/TSP usually happens after 20–30 years of latency [4], and the average age in which patients experience the first signs of disease is about 43 years of age [68]. The early phase of HAM/TSP is presented with a profound inflammatory response resulting in lower back pain, weakness in the lower limbs, and impairment of urinary and sexual functions. Eventually, a chronic degenerative disorder develops characterized by the progressive loss of myelin in the thoracic and lumbar regions of the spinal cord [67]. Damage to the central nervous system (CNS) also occurs in patients with HAM/TSP, likely mediated by particular cells of the immune system such as CD4+ T cells, CD8+ T cells, DCs, and cells of the monocyte–macrophage lineage. For instance, it has been proposed (28) that at least three mechanisms participate in the process of myelin degradation that occurs in HAM/TSP: (i) direct injury caused by CD8+ T cells, (ii) damage mediated by an uncontrolled cytokine storm, and (iii) an autoimmune response. Previous studies have shown that infiltrating activated Taxspecific CD8+ cytotoxic T lymphocytes (CTLs) in the peripheral blood and cerebrospinal fluid (CSF) induced lysis of HTLV-1-infected cells triggering a pro-inflammatory cytokine storm. There is evidence that HTLV-1 has been found in the CNS. The release of pro-inflammatory factors such as TNF-α and IFN-γ secreted by activated CD8+ CTLs injures the CNS. Further damage may occur as a result of the molecular mimicry between the Tax protein and the neuronal antigen heterogeneous ribonuclear protein-A1 (hnRNP-A1), which may cause an autoimmune response [69].

Both HTLV-1 and HIV-1 have been shown to penetrate the bone marrow to varying degrees during the course of monoinfection, and it is assumed to be the case during the course of coinfection with both viruses but less information is available in this regard. Clearly, the relative penetration of the two viruses into the bone marrow compartment during the course of coinfection may have dramatic effects on HTLV-1- and HIV-1-induced pathogenesis and disease caused by either viruses, and these pathogenic processes will also be a subject of this review. These interactions may periodically alter the balance between immune control and HTLV-1 infection, and the periodic imbalance has also been associated with the etiology of other inflammatory diseases such as arthropathy, pulmonary alveolitis, uveitis, dermatitis, Sjögren's syndrome, Behçet's disease, thyroid disease, prostatitis, cystitis, hepatitis, polymyo‐ sitis, arthritis, and a sarcoidosis-like disorder [67].

## **3. Introduction to HIV-1 infection, pathogenesis, and disease**

of which increase the probability of uncontrolled cell division and transformation exhaustively reviewed [10, 57, 58]. As of now, it is not fully understood how and why Tax, which is a strong inducer of transformation, only induces ATL in 5% of infected individuals. This could be, in part, the immune system efficiently removing infected cells and/or combating Tax with specific host restriction factors. Additionally, Tax has been shown to be secreted from infected cells and have bystander effects, such as pro-inflammatory cytokines and the infiltration of Tax-

T cells into the CNS, thus playing a part in HAM/TSP [59–64].

by particular cells of the immune system such as CD4+

factors such as TNF-α and IFN-γ secreted by activated CD8+

sitis, arthritis, and a sarcoidosis-like disorder [67].

autoimmune response [69].

HTLV-1 infection also causes HAM/TSP, a neuroinflammatory disorder that mostly affects the spinal cord and brain due to chronic proinflammatory cytokines, the destruction of myelin, and the cells that secrete it, oligodendrocytes [64–66]. It was first thought that there might be a hormonal component regulating HAM/TSP, as it occurs more frequently and progresses more rapidly in women than in men, particularly if the first signs of disease occur before menopause [67]. The onset of HAM/TSP usually happens after 20–30 years of latency [4], and the average age in which patients experience the first signs of disease is about 43 years of age [68]. The early phase of HAM/TSP is presented with a profound inflammatory response resulting in lower back pain, weakness in the lower limbs, and impairment of urinary and sexual functions. Eventually, a chronic degenerative disorder develops characterized by the progressive loss of myelin in the thoracic and lumbar regions of the spinal cord [67]. Damage to the central nervous system (CNS) also occurs in patients with HAM/TSP, likely mediated

the monocyte–macrophage lineage. For instance, it has been proposed (28) that at least three mechanisms participate in the process of myelin degradation that occurs in HAM/TSP: (i) direct injury caused by CD8+ T cells, (ii) damage mediated by an uncontrolled cytokine storm, and (iii) an autoimmune response. Previous studies have shown that infiltrating activated Taxspecific CD8+ cytotoxic T lymphocytes (CTLs) in the peripheral blood and cerebrospinal fluid (CSF) induced lysis of HTLV-1-infected cells triggering a pro-inflammatory cytokine storm. There is evidence that HTLV-1 has been found in the CNS. The release of pro-inflammatory

damage may occur as a result of the molecular mimicry between the Tax protein and the neuronal antigen heterogeneous ribonuclear protein-A1 (hnRNP-A1), which may cause an

Both HTLV-1 and HIV-1 have been shown to penetrate the bone marrow to varying degrees during the course of monoinfection, and it is assumed to be the case during the course of coinfection with both viruses but less information is available in this regard. Clearly, the relative penetration of the two viruses into the bone marrow compartment during the course of coinfection may have dramatic effects on HTLV-1- and HIV-1-induced pathogenesis and disease caused by either viruses, and these pathogenic processes will also be a subject of this review. These interactions may periodically alter the balance between immune control and HTLV-1 infection, and the periodic imbalance has also been associated with the etiology of other inflammatory diseases such as arthropathy, pulmonary alveolitis, uveitis, dermatitis, Sjögren's syndrome, Behçet's disease, thyroid disease, prostatitis, cystitis, hepatitis, polymyo‐

T cells, CD8+ T cells, DCs, and cells of

CTLs injures the CNS. Further

specific CD8+

48 Leukemias - Updates and New Insights

In 1981, the first cases of AIDS were reported in the United States. At the beginning of the epidemic, AIDS was first identified in homosexual men and drug users. However, the epidemic rapidly spread to the general population primarily by heterosexual intercourse [70]. In 1983, a retrovirus was isolated at the Pasteur Institute in France from a lymph node biopsy from a homosexual man with lymphadenopathy presenting with AIDS-like symptoms [71]. Almost in parallel at the National Cancer Institute in Bethesda, United States, the same virus was identified from samples of patients suffering from AIDS [72]. In 1986, the virus was named the human immunodeficiency virus (HIV) and demonstrated to be the causative agent of AIDS [73].

There are two types of HIV, HIV type 1 (HIV-1) and HIV type 2 (HIV-2), which are genetically and morphologically related viruses that share similarities in their mechanisms of replication and transmission. However, HIV-1 and HIV-2 differ in clinical disease progression and geographical distribution. HIV-1 leads to overt disease much faster than HIV-2, with world‐ wide distribution, whereas HIV-2 infections are more prevalent in West Africa [74]. HIV-1 is divided into four genetically different groups: M (main), O (outlier), N (non-M, non-O), and P. The group M, which accounts for 98% of the HIV-1 cases worldwide, is further classified into nine subtypes, with subtype B being the most prevalent within North America [75].

## **3.1. HIV-1 genetic architecture, entry, and viral replication**

HIV-1 is also a type C retrovirus that belongs to the genus lentivirus. On a genomic level, HIV-1 is similar to HTLV-1 in a number of aspects. They both contain 5 and 3 LTR and gag, pol, and env genes. However, HIV-1 lacks the pX region and encodes for other accessory proteins that have some overlapping function with those of HTLV-1. HIV-1 proviral DNA encodes for the accessory proteins Tat, Vpu, Nef, Vif, Rev, and Vpr (Figure 3) [6, 76]. Tat is similar in function to Tax in the sense it can transactivate the LTR but does not function in cell transformation. Similarly, both Rev and Rex function to export unspliced and singly spliced RNAs from the nucleus. While HIV-1 shares sequence similarity at the genome level, it has been shown to utilize a different receptor for entry.

The HIV-1 envelope glycoprotein gp120 is the trimeric spike on the surface of the virion that has been shown to mediate attachment to the host target cells by engaging the CD4 receptor embedded in the plasma membrane. Subsequent structural changes mediate the exposure and interaction of the V3 region of gp120 to the chemokine coreceptors, either CXCR4 or CCR5 (although others have also been identified to facilitate this process as well), depending on V3 sequence and charge, and mediate fusion of the viral plasma membrane with the host cell membrane. Once the particle has been internalized, the capsid and associated viral enzymes and RNA remain associated in the cytoplasm in a capsid-like structure, where the viral reverse transcriptase continues to be transcribed from viral RNA to DNA (a process that is likely initiated as the viral particle interfaces with the cell surface proteins involved in viral entry). As the reverse transcription process continues to completion, the capsid structure transitions to a preintegration complex (PIC) containing the reverse transcriptase, integrase, capsid, and

**Figure 3.** HIV-1 genomic architecture. A schematic representation of the proviral genome organization open reading frame and viral products of HIV-1. The organization of the ~10-kb genome is depicted along with the genes and their transcriptional splicing.

Vpr, and this structure has been shown to be transferred to the nuclear membrane. After entry into the nucleus, the proviral DNA genome is integrated into the host cell chromosome, with recent studies characterizing integration site preference and how it changes with disease progression [77–79]. Once integrated, the proviral DNA becomes part of the cellular chromatin environment and subjected to host machinery involved in the processes of transcription and translation. In conjunction with a number of cellular transcription factors and the viral transactivator proteins Tat and Vpr, transactivation directed by the LTR-driven contained within the integrated provirus is initiated and used as a template for the transcription of viral genomic RNAs and viral mRNAs with the subsequent translation of these viral mRNA into structural, enzymatic, and accessory proteins. From there, the viral polyproteins an proteins are recruited and aggregate primarily in the vicinity of the plasma membrane, resulting in the formation and release of mature infectious virions as previously reviewed [80, 81].

#### **3.2. HIV-1 infectivity, transmission, and pathogenesis**

As a result of studies performed in cell lines and primary human cells cultured *in vitro*, transplanted human cells maintained *in vivo* in engineered animals, and primary cells examined in an *ex vivo* experimental environment, HIV-1 has been shown to most efficiently infect activated CD4+ T cells [82], although these cells do not need to be activated in order for infection to occur [83]. Both HIV and HTLV-1 share CD4+ T cells as their cell target, which was further demonstrated by complementation viral envelopes for each other [84]. In addition to the CD4+ T-cell compartment, a number of other human cellular compartments are infected by HIV-1, including cells of the monocyte–macrophage lineage, some subsets of dendritic cells, microglial cells and astrocytes within the brain, hematopoietic progenitor cells, and endothelial cells lining the blood-brain barrier [85, 86].

Studies performed *in vitro* have shown that all HIV-1 isolates infect activated peripheral blood mononuclear cells (PBMCs). Some isolates are able to infect CD4+ T-cell lines, T-cell leukemia cell lines, and monocyte-derived macrophages (MDMs). The target cell population and coreceptor utilization seems to change during disease progression, and the once clear distinc‐ tion between CXCR4-utilizing or X4 versus the CCR5-utilizing or R5 viruses as compared to the designations referred to as T-tropic and M-tropic now seems to be also guided by the relative utilization of different levels of the CD4 receptor present on T cells and cells of the monocyte–macrophage lineage [87]. During the acute phase of infection and to a lesser extent at later times during the course of disease, primarily due to cART combating replication competent virus, HIV-1 infects CD4+ T-cell populations, with the resting memory CD4+ T cells establishing a latent HIV-1 reservoir. The majority of activated and infected CD4+ T cells will be eliminated, and during this T-cell depletion phase, a portion of the activated CD4+ T cells will undergo a reprogramming of transcription and translation to allow them to survive and differentiate into a resting memory cell phenotype. This resting memory CD4+ T-cell can contribute to the latent reservoir because it was infected and then differentiated into a resting memory cell or as the cell was differentiated into a memory cell it became infected; regardless of the mode of infection, the HIV-1 provirus survives, along with a substantial number of defective proviral genotypes [78, 79]. Because memory CD4+ T cells live for many years, with a predicted half-life of 44 months [79, 88], these cells maintain one of the critical latent reservoirs of HIV-1 and a complicating factor in the pursuit to identify an effective means to cure the HIV-1-infected individual by a new generation of treatment strategies. However, there are very likely a number of additional cellular reservoirs that facilitate the persistence of HIV-1 during prolonged cART. For example, cells of the monocyte–macrophage lineage in the peripheral blood and other lymphoid tissues, resident macrophages of the central nervous system, astrocytes, DCs, follicular dendritic cells (FDCs), hematopoietic progenitor cells (HPCs) in the bone marrow, and specialized epithelial cells within the kidney likely play a role in maintaining HIV-1 during clinical latency prior to the start of any form of therapy despite ongoing immune surveillance and after the start of cART [85]. Each of these cell types present a unique challenge with respect to a cure based on the IR rate of cell turnover, the relative proviral genome transcriptional competency, the innate capacity of the virus to move out of the reservoir, the continued production of infected cells from infected precursors, and the poor drug penetrating ability into these physiological niches, with resting memory CD4+ T cells and cells within the brain and bone marrow being prime examples.

Vpr, and this structure has been shown to be transferred to the nuclear membrane. After entry into the nucleus, the proviral DNA genome is integrated into the host cell chromosome, with recent studies characterizing integration site preference and how it changes with disease progression [77–79]. Once integrated, the proviral DNA becomes part of the cellular chromatin environment and subjected to host machinery involved in the processes of transcription and translation. In conjunction with a number of cellular transcription factors and the viral transactivator proteins Tat and Vpr, transactivation directed by the LTR-driven contained within the integrated provirus is initiated and used as a template for the transcription of viral genomic RNAs and viral mRNAs with the subsequent translation of these viral mRNA into structural, enzymatic, and accessory proteins. From there, the viral polyproteins an proteins are recruited and aggregate primarily in the vicinity of the plasma membrane, resulting in the

**Figure 3.** HIV-1 genomic architecture. A schematic representation of the proviral genome organization open reading frame and viral products of HIV-1. The organization of the ~10-kb genome is depicted along with the genes and their

formation and release of mature infectious virions as previously reviewed [80, 81].

As a result of studies performed in cell lines and primary human cells cultured *in vitro*, transplanted human cells maintained *in vivo* in engineered animals, and primary cells examined in an *ex vivo* experimental environment, HIV-1 has been shown to most efficiently

further demonstrated by complementation viral envelopes for each other [84]. In addition to

by HIV-1, including cells of the monocyte–macrophage lineage, some subsets of dendritic cells, microglial cells and astrocytes within the brain, hematopoietic progenitor cells, and endothelial

Studies performed *in vitro* have shown that all HIV-1 isolates infect activated peripheral blood

T-cell compartment, a number of other human cellular compartments are infected

T cells [82], although these cells do not need to be activated in order for

T cells as their cell target, which was

T-cell lines, T-cell leukemia

**3.2. HIV-1 infectivity, transmission, and pathogenesis**

infection to occur [83]. Both HIV and HTLV-1 share CD4+

mononuclear cells (PBMCs). Some isolates are able to infect CD4+

cells lining the blood-brain barrier [85, 86].

infect activated CD4+

transcriptional splicing.

50 Leukemias - Updates and New Insights

the CD4+

It has been suggested that HIV-1 can be transmitted via free virus or HIV-1-infected cells present in infected blood and body fluids that enter the blood stream of an uninfected individual. The three routes of HIV-1 transmission are (i) via sexual intercourse, (ii) from mother to child, and (iii) parenteral transmission [76]. Sexual transmission is the most common mode of HIV-1 transmission and accounts for the 80% of infections in adults [89] and includes vaginal, anal, and oral unprotected sex between an infected individual and his or her unin‐ fected partner. The risk of HIV-1 transmission is higher between homosexual men as compared to the risk during heterosexual intercourse and individuals whom engage in high risk behaviors [90]; however, this trend can be steadily reduced by prophylactic antiviral use and educating the public. Parenteral transmission of HIV-1 is usually associated with the transfu‐ sion of contaminated blood, transplant of infected organs, and sharing of infected sharps, needles, or syringes [76]. Mother-to-child HIV-1 transmission occurs during pregnancy, delivery, or breastfeeding. The presence of higher levels of HIV-1 RNA in blood/body fluids of the infected host has been associated with greater probabilities of transmission [91].

#### **3.3. Diseases caused by HIV-1**

According to the World Health Organization (WHO), by the end of 2013, there were 35 million people living with HIV worldwide, and 1.5 million people died as a consequence of AIDSassociated diseases [92]. The course of HIV-1 infection consists of three phases of disease: primary or acute, asymptomatic/chronic, and AIDS. After 2 weeks of initial exposure to HIV-1, approximately 50–70% of the infected patients experience nonspecific symptoms that do not last for more than 4 weeks. These symptoms include increase in body temperature, sore throat, cephalea, joint and muscle pain, general discomfort, and weight loss. Around 70% of patients will develop a rash on trunk and face [6]. This is followed by a long-term period (in many individuals, this highly variable period has been thought to be longer than 10 years but may be altered by many host and comorbidity factors) of asymptomatic chronic infection [93]. This phase is marked by a loss of CD4+ T cells at an annual rate of 30 to 60 cells/mm3 [6]. HIV-1 titers in the peripheral blood, and antibodies to HIV-1 become readily detectable [76]. Most patients do not present major symptoms; however, some experience tiredness and swollen lymph nodes. Less than 1% of patients in this phase develop AIDS within a period of 1–2 years. The more advanced disease symptoms begin when the CD4+ T-cell levels drop below 500 cell/ mm3 . During this stage, HIV-1-infected patients become immunocompromised, developing opportunistic infections such as oral candidiasis, pneumococcal infections, tuberculosis, and infections caused by the herpes simplex and varicella zoster viruses. When the CD4 counts decrease below 200 cells/mm3 , they are clinically diagnosed as having progressed to AIDS. Here HIV-1-infected patients are at high risk of serious diseases like systemic fungal infections, toxoplasma encephalitis, and cryptococcal meningitis, reactivation of other latent viruses such as cytomegalovirus (CMV), and other opportunistic infections, [94], which is primarily due to the decrease in T-cell count. AIDS patients are also susceptible to developing AIDS- and non-AIDS-defining cancers. Kaposi sarcoma (KS) induced by human herpes virus 8 and non-Hodgkin's lymphoma are two examples of AIDS-defining cancers. Importantly, the incidence of AIDS-defining cancers and opportunistic infections in HIV-1-infected patients has dropped since the introduction in 1996 of the highly active antiretroviral therapy (HAART) for the treatment of HIV-1 in North America, Europe, and Australia [95]. In contrast, the frequency of non-AIDS-defining cancers such as cervical and anal cancer caused by human papilloma virus, liver cancer, Hodgkin's lymphoma, lung cancer, and prostate cancer has increased among the HIV-1-infected population [95].

## **4. Impact of HIV-1 on HTLV-1 disease progression**

The effect that HIV-1 has on the progression of HTLV-1 infection remains controversial, as there are very few studies that have directly examined the process interaction. However, it is likely that the periods of immunosuppression observed during the course of HIV-1 disease include the first 3–6 months of the primary infection, a time when the CD4+ T-cell compartment is acutely targeted by HIV-1, the period involving the transition from asymptomatic clinical latency to symptomatic disease prior to therapeutic intervention, and last during the final progressive decrease in the CD4+ T-cell count prior to the availability of therapy or after the development of drug resistance without the availability of alternative therapies, which may very likely alter the course of primary HTLV-1 infection, the development and control of ATL, or the etiology and progression of HAM/TSP. Although HIV-1 has not been shown to infect bone marrow stem cells, it has been shown to infect more differentiated progenitor cells. In addition, HTLV-1 has also been shown by Jacobson and coworkers [96] to penetrate the bone marrow compartment with the detection of HTLV-1 DNA in the absence or presence of detectable transcription. Given these observations, it is possible that HIV-1 infection of similar cell populations in the bone marrow may impact HTLV-1 gene expression programming and alter the functional course of these cell populations with respect to the development and control of ATL and HAM/TSP.

#### **4.1. Incidence of HAM/TSP among HTLV-1/HIV-1-coinfected patients**

delivery, or breastfeeding. The presence of higher levels of HIV-1 RNA in blood/body fluids of the infected host has been associated with greater probabilities of transmission [91].

According to the World Health Organization (WHO), by the end of 2013, there were 35 million people living with HIV worldwide, and 1.5 million people died as a consequence of AIDSassociated diseases [92]. The course of HIV-1 infection consists of three phases of disease: primary or acute, asymptomatic/chronic, and AIDS. After 2 weeks of initial exposure to HIV-1, approximately 50–70% of the infected patients experience nonspecific symptoms that do not last for more than 4 weeks. These symptoms include increase in body temperature, sore throat, cephalea, joint and muscle pain, general discomfort, and weight loss. Around 70% of patients will develop a rash on trunk and face [6]. This is followed by a long-term period (in many individuals, this highly variable period has been thought to be longer than 10 years but may be altered by many host and comorbidity factors) of asymptomatic chronic infection [93]. This

in the peripheral blood, and antibodies to HIV-1 become readily detectable [76]. Most patients do not present major symptoms; however, some experience tiredness and swollen lymph nodes. Less than 1% of patients in this phase develop AIDS within a period of 1–2 years. The

. During this stage, HIV-1-infected patients become immunocompromised, developing opportunistic infections such as oral candidiasis, pneumococcal infections, tuberculosis, and infections caused by the herpes simplex and varicella zoster viruses. When the CD4 counts

Here HIV-1-infected patients are at high risk of serious diseases like systemic fungal infections, toxoplasma encephalitis, and cryptococcal meningitis, reactivation of other latent viruses such as cytomegalovirus (CMV), and other opportunistic infections, [94], which is primarily due to the decrease in T-cell count. AIDS patients are also susceptible to developing AIDS- and non-AIDS-defining cancers. Kaposi sarcoma (KS) induced by human herpes virus 8 and non-Hodgkin's lymphoma are two examples of AIDS-defining cancers. Importantly, the incidence of AIDS-defining cancers and opportunistic infections in HIV-1-infected patients has dropped since the introduction in 1996 of the highly active antiretroviral therapy (HAART) for the treatment of HIV-1 in North America, Europe, and Australia [95]. In contrast, the frequency of non-AIDS-defining cancers such as cervical and anal cancer caused by human papilloma virus, liver cancer, Hodgkin's lymphoma, lung cancer, and prostate cancer has increased

The effect that HIV-1 has on the progression of HTLV-1 infection remains controversial, as there are very few studies that have directly examined the process interaction. However, it is likely that the periods of immunosuppression observed during the course of HIV-1 disease

T cells at an annual rate of 30 to 60 cells/mm3 [6]. HIV-1 titers

, they are clinically diagnosed as having progressed to AIDS.

T-cell levels drop below 500 cell/

T-cell compartment

**3.3. Diseases caused by HIV-1**

52 Leukemias - Updates and New Insights

phase is marked by a loss of CD4+

decrease below 200 cells/mm3

among the HIV-1-infected population [95].

**4. Impact of HIV-1 on HTLV-1 disease progression**

include the first 3–6 months of the primary infection, a time when the CD4+

mm3

more advanced disease symptoms begin when the CD4+

Much knowledge of viral coinfection and HAM/TSP has come from longitudinal and crosssectional studies of patient cohorts. While much still needs to be understood on a molecular biologic and immunologic level, these human studies are invaluable with respect to identifying correlations that allow one to develop experimental designs to explore mechanistic avenues to determine the role of virus–virus interactions. Based on these studies, it was determined that less than 2% of the individuals were infected with HTLV-1 develop HAM/TSP [97, 98]. Previous studies have suggested that HIV-1 increases the risk of HAM/TSP in HTLV-1/HIV-1 coinfected individuals. For example, the incidence of HAM/TSP is 9.7% among HTLV-1/HIV-1 coinfected individuals in a cohort of patients from New Orleans, Louisiana. These patients did not present with AIDS, and their CD4+ T-cell levels were normal or slightly elevated [99]. The occurrence of myelopathy in HTLV-1/HIV-1-coinfected individuals was estimated in a casecontrol study in Rio de Janeiro, Brazil. The results indicated that 73% of the coinfected patients and 16% of the patients infected with only HIV-1 developed myelopathy [100]. Another group reported that the prevalence of HAM/TSP among HTLV-1/HIV-1-coinfected patients in Brazil was 8% [101]. Schutte and coworkers [102] observed in a cohort of patients in Pretoria, South Africa, that HTLV-1/HIV-1-coinfected individuals were prone to developing HAM/TSP at an earlier age than when infected with HTLV-1. Furthermore, the period of time in which the coinfected patients remained asymptomatic was shorter than the monoinfected patients (less than 3 years) [102]. Furthermore, Casseb and colleagues [101] demonstrated that the levels of HTLV-1 proviral DNA load in coinfected patients with HAM/TSP were five times higher than in asymptomatic coinfected individuals. HTLV-1 proviral DNA levels in PBMCs varied during the course of HTLV-1 infection [103]. High proviral DNA levels [104] along with the replication or migration of HTLV-1-infected lymphocytes to the CNS have been associated with the development of HAM/TSP [103]. Indeed, Bassi et al. [105] proposed the use of HTLV-1 proviral DNA loads as a diagnostic tool for the early detection of HAM/TSP. In this regard, other studies have established the lower limits of detection of HTLV-1 proviral DNA, and these efforts facilitated studies to distinguish between asymptomatic HTLV-1-infected patients and HAM/ TSP patients. With regard to coinfection, studies have reported that HIV-1 infection increased the HTLV-1 proviral DNA levels in HTLV-1-infected patients. Yet, Césaire and colleagues [106] found no difference between the levels of HTLV-1 proviral DNA in the coinfected patients compared to those infected with only HTLV-1. Even without understanding the molecular mechanism of how one retrovirus influences pathology and disease, what is obvious is the strong association of HAM/TSP and HTLV-1/HIV-1 coinfection (Table 2).


**Table 2.** Points of intersection between HTLV-1 and HIV-1

The levels of CD4+ T-cell counts and HTLV-1 disease progression in HTLV-1/HIV-1 coinfection were evaluated in a study conducted in Brazil by Casseb and coworkers. One hundred and fifty HTLV-1-infected patients were enrolled in the study; 27 of them were coinfected with HIV-1, and 15 of the coinfected patients had already reported an AIDS-defining event. CD4+ T-cell counts were higher in coinfected individuals with AIDS than in HIV-1-monoinfected patients (median = 189 cells/mm3 and 89 cells/mm3 , respectively; *p* = 0.036). Moreover, five of the coinfected subjects who had AIDS and three of the coinfected patients without AIDS showed signs of HAM/TSP. Three of the eight patients with signs of HAM/TSP also developed an opportunistic infection. Importantly, the incidence of HAM/TSP in coinfected patients with AIDS was 20 times higher than those infected with only HTLV-1 infection. These results supported previous observations that HTLV-1/HIV-1 coinfection was associated with a higher probability of a more severe HTLV-1 infection along with an increase in the levels of CD4+ T cells [3]. These results have suggested the possibility that HTLV-1 may inhibit HIV-1 replica‐ tion with a subsequent increase in CD4+ T-cell counts, thereby enhancing HTLV-1 disease progression. Based on these observations, current research has centered on a more in-depth molecular analysis with respect to how HTLV-1 and HIV-1 impact each other during the course of dual infection.

## **5. Impact of HTLV-1 on HIV-1 disease progression**

the HTLV-1 proviral DNA levels in HTLV-1-infected patients. Yet, Césaire and colleagues [106] found no difference between the levels of HTLV-1 proviral DNA in the coinfected patients compared to those infected with only HTLV-1. Even without understanding the molecular mechanism of how one retrovirus influences pathology and disease, what is obvious is the

HAM/TSP and ATL

influence HAM/TSP

T-cell counts and HTLV-1 disease progression in HTLV-1/HIV-1 coinfection

were evaluated in a study conducted in Brazil by Casseb and coworkers. One hundred and fifty HTLV-1-infected patients were enrolled in the study; 27 of them were coinfected with HIV-1, and 15 of the coinfected patients had already reported an AIDS-defining event. CD4+ T-cell counts were higher in coinfected individuals with AIDS than in HIV-1-monoinfected

the coinfected subjects who had AIDS and three of the coinfected patients without AIDS showed signs of HAM/TSP. Three of the eight patients with signs of HAM/TSP also developed an opportunistic infection. Importantly, the incidence of HAM/TSP in coinfected patients with AIDS was 20 times higher than those infected with only HTLV-1 infection. These results supported previous observations that HTLV-1/HIV-1 coinfection was associated with a higher probability of a more severe HTLV-1 infection along with an increase in the levels of CD4+

cells [3]. These results have suggested the possibility that HTLV-1 may inhibit HIV-1 replica‐

progression. Based on these observations, current research has centered on a more in-depth molecular analysis with respect to how HTLV-1 and HIV-1 impact each other during the course

and 89 cells/mm3

and infiltration of Tax specific CD8+

Some studies have suggested that there is minimal effect by Tat on HTLV-1 infection; other studies have suggested that HIV-1 Rev is the protein that potentially enhances gene expression; some studies have shown that HIV-1 does not affect proviral load in PBMCs; there has been a link between coinfection and increased risk to develop

Transactivator protein that enhances viral transcription; largely implicated in the oncogenic potential of HTLV-1; can be secreted from infected cells resulting in bystander effects such as upregulation of cytokines and chemokines,

, respectively; *p* = 0.036). Moreover, five of

T-cell counts, thereby enhancing HTLV-1 disease

T cells, which can

T

strong association of HAM/TSP and HTLV-1/HIV-1 coinfection (Table 2).

**Viral protein Effect on HIV-1 infection Effect on HTLV-1 infection**

Transactivator protein that enhances viral transcription; can be secreted and cause apoptosis in uninfected bystander cells

Has been shown to be overexpressed in HTLV-1/HIV-1 coinfection; promotes nuclear transport of the reverse transcribed HIV-1 DNA; stimulates HIV-1 via activation of NFκB (both alone and synergistically with Tat); has been shown to interact with CCR5, a major coreceptor of HIV-1, although a role in

disease progression is controversial

**Table 2.** Points of intersection between HTLV-1 and HIV-1

**Tat**

54 Leukemias - Updates and New Insights

**Tax**

The levels of CD4+

of dual infection.

patients (median = 189 cells/mm3

tion with a subsequent increase in CD4+

The influence of HTLV-1 infection on the development of AIDS in HIV-1-coinfected patients is not well understood. Several studies have indicated that HTLV-1 infection promotes HIV-1 replication, accelerating the development of AIDS, while other reports have shown that HTLV-1 actually inhibits HIV-1 infection [4]. The conflicting results reported are likely due in part to the diverse antiretroviral regimens used to treat HTLV-1/HIV-1-coinfected patients [104]. In addition, the timing with respect to the introduction of the second virus (HTLV-1 or HIV-1) may have great impact on HIV-1 replication and disease.

Prior to the HAART era, Bartholomew and colleagues [107] reported the results of a study conducted in Trinidad with 40 HIV-1-positive homosexual men, 6 of them coinfected with HTLV-1. The coinfected individuals were severely immunocompromised compared to the HIV-1-monoinfected patients. Irrespective of sex and CD4+ T-cell counts, a retrospective casecontrol study performed in Bahia, Brazil, showed that people living with HTLV-1/HIV-1 coinfection exhibited a shorter lifespan than HIV-1-monoinfected patients. The mean survival time for controls was 2,430 days, whereas for HTLV-1/HIV-1-coinfected patients, it was 1,849 days, with a *p* = 0.02 when comparing the two groups [108]. The reduced survival was also observed in children [109].

Scapellato and colleagues [110] reported that in HTLV-1/HIV-1-coinfected patients naive to treatment, CD4+ T-cell counts were higher in the coinfected patients than in HIV-1 monoinfected patients at the time of an AIDS-defining illness. A case-control study to characterize the phenotype of CD4+ T cells during HTLV-1/HIV-1 coinfection was conduct‐ ed with 701 HAART-naïve, HIV-1-positive African adults. Within this patient cohort, 29 patients were found to be coinfected with HTLV-1. Each coinfected patient was matched by age and sex with two HIV-1-monoinfected individuals. The study also included unmatched healthy controls. CD4+ T-cell levels, markers of CD4+ T-cell activation, and HIV viral load were the parameters used to assess HIV-1 disease progression. The results showed that coinfected patients exhibited higher levels of CD4+ T cells (median = 525 cells/mm3 and 274 cell/mm3 , respectively; *p* < 0.05) with higher levels of expression of the activation markers CD25 and CD45RO and lower expression levels of CD45RA and CD62L (markers of naïve T cells) in coinfected individuals as compared to monoinfected individuals. Furthermore, coinfected patients exhibited an increase in HIV-1 proviral DNA load as compared to monoinfected subjects. Despite the normal or higher levels of CD4+ T cells, coinfected patients still progressed to AIDS [111]. These observations imply that HTLV-1 infection enhances HIV-1 progression via loss of naïve CD4+ T cells, with an overall increase in total CD4+ T cells and an increase in HIV-1 viral load, key features with respect to the develop‐ ment of AIDS. The lymphocytosis observed in coinfected patients might have been caused by the Tax oncoprotein encoded by HTLV-1. Tax inhibits the cellular mechanisms in‐ volved in DNA repair and induces cell transformation and immortalization [111, 112].

The impact of HTLV-1 on the immune response during coinfection with HIV-1 was evaluated using quiescent PBMCs from HTLV-1/HIV-1-coinfected patients as well as from HIV-1 and HTLV-1-monoinfected individuals. The Th1 cytokine pathway appeared to be overstimulated during HTLV-1/HIV-1 coinfection, as PBMCs from coinfected patients produced increased levels of IL-2 and IFN-γ compared to PBMCs from HIV-1 and HTLV-1-monoinfected individ‐ uals. These results implied that overproduction of Th1 cytokines during the course of HTLV-1/ HIV-1 coinfection could be augmenting the overall negative impairment of the immune system induced by HIV-1 [113], which normally influences a Th2 response during chronic infection. Curiously, the correction to a Th1 response does not seem to correct for the shortened lifespan and a possible increase in progression to AIDS. It should be noted that there was obviously patient-to-patient variability, differences in phenotype depending on viral genotypes, and the length of time involving mono or dual infection.

## **6. Molecular interactions between HTLV-1 and HIV-1**

To this point, we have discussed the impact that HIV-1 has on HTLV-1 disease progression, particularly on the occurrence of HAM/TSP, as well as the effect of HTLV-1 on HIV-1 infection. Clearly, HTLV-1/HIV-1 coinfection alters the course of disease caused by either virus along as assessed by proviral DNA loads, CD4+ T-cell death and proliferation assessments, and overall immunologic assessment, pathogenesis, and disease indicates that the presence of both viruses negatively impacts human health as compared to the presence of either virus alone. Based on these observations, investigators have also explored the molecular interactions between HTLV-1 and HIV-1 that could be influencing the development of HTLV-1 disease or HIV/AIDS during HTLV-1/HIV-1 coinfection. *In vitro* experiments involving superinfection with the HIV-1 molecular clone HIV-1IIIB on two HTLV-1-transformed cell lines, MT2 (an HTLV-1 producer cell line) and 81-66/45 cell line (an HTLV-1 nonproducer cell line), have demonstrated that HIV-1 infection activates HTLV-1 and increases the levels of HTLV-1 proviral DNA in both HTLV-1-transformed cell lines [114]. Additional studies performed by Zsabó and colleagues [115] have shown that *in vitro* HTLV-1/HIV-1 coinfection of macrophages by both viruses results in increased replication of both viruses. The presence of the HTLV-1 Tax protein promoted nuclear transport of the newly reverse transcribed HIV-1 DNA, whereas the mechanism by which HIV-1 infection enhanced HTLV-1 gene expression did not appear to involve the HIV-1 Tat protein.

In a tripartite coculture assay using Jurkat T cells transfected with an HTLV-1LTR-driven reporter construct designated Jurkat/HTLV-1-Luc with a chronically infected HTLV-1 cell line, HTLV-1-MT2, that has also been infected with HIV-1IIIB via cell-to-cell transfer of virus from HIV-1IIIB-infected H9 cells, Sun and coworkers demonstrated that HIV-1 infection induced an 80-fold increase in LTR-dependent HTLV-1 gene expression. It was also demonstrated that the increase in transcriptional activation of HTLV-1 genes occurred in a mechanism that was dependent on the HTLV-1 Tax protein, the HIV-1 gp120/gp41 complex, and CD40. These results suggested that HIV-1 infection promoted the development of syncytium among the cell lines examined in these studies, thereby acting as a channel for HTLV-1 Tax to translocate from the HTLV-1-infected MT2 cells to the HTLV-1-LTR-Jurkat cells, thereby providing an explanation as to how coinfection with HIV-1 and HTLV-1 may transactivate the LTR of latent provirus in neighboring cells [116]. Using an *in vitro* model of HTLV-1/HIV-1 coinfection, Roy and colleagues confirmed that HIV-1 virus alone or the accessory protein Tat can enhance HTLV-1 gene expression. Culturing the NO-HTLV-1 cell line, an HTLV-1-infected cell line established by exposure of the cells to an HTLV-1 clinical isolate, in the presence of cell-free HIV-1 virion alone (HIV-1IIIB), doubled the amount of HTLV-1 gene expression [117–119]. Additionally, the NO-HTLV-1 cells were exposed to recombinant HIV-1 Tat protein alone, subsequently resulting in an increase in the expression of HTLV-1 matrix protein expression (p. 19). Furthermore, the majority of HTLV-1-infected cells colocalized with HIV-1 virions, indicating HLTV-1 gene expression and transactivation, was dependent and correlated with the presence of HIV-1 virion or Tat [2].

With respect to the interactions between HTLV-1 and HIV-1 that affect HIV-1 expression, Leung and coworkers [120] first reported that the HTLV-1 Tat protein stimulates HIV-1 via activation of NF-κB. Later, studies by Böhnlein and colleagues [121] confirmed that in vitro HTLV-1/HIV-1 coinfection assays indicated that HTLV-1 enhanced HIV-1 expression utilizing a mechanism dependent on HTLV-1 Tax. These experiments revealed that HTLV-1 Tax protein stimulates T cells and promotes transcriptional activation of the HIV-1LTR via interaction with the cellular protein HIVEN86A. Additional studies indicated that HTLV-1 Tax also works synergistically with HIV-1 Tat to increase HIV-1 via stimulation of NF-κB [122]. Culturing HTLV-1-producing MT2 cells with HIV-1 isolates from quiescent CD4+ T cells from HIV-1 infected patients treated with HAART upregulated HIV-1 expression. HTLV-1 Tax or the Env glycoprotein alone was sufficient to induce HIV-1 replication [123].

## **7. Treatments for HIV-1 and HTLV-1 infections**

during HTLV-1/HIV-1 coinfection, as PBMCs from coinfected patients produced increased levels of IL-2 and IFN-γ compared to PBMCs from HIV-1 and HTLV-1-monoinfected individ‐ uals. These results implied that overproduction of Th1 cytokines during the course of HTLV-1/ HIV-1 coinfection could be augmenting the overall negative impairment of the immune system induced by HIV-1 [113], which normally influences a Th2 response during chronic infection. Curiously, the correction to a Th1 response does not seem to correct for the shortened lifespan and a possible increase in progression to AIDS. It should be noted that there was obviously patient-to-patient variability, differences in phenotype depending on viral genotypes, and the

To this point, we have discussed the impact that HIV-1 has on HTLV-1 disease progression, particularly on the occurrence of HAM/TSP, as well as the effect of HTLV-1 on HIV-1 infection. Clearly, HTLV-1/HIV-1 coinfection alters the course of disease caused by either virus along as assessed by proviral DNA loads, CD4+ T-cell death and proliferation assessments, and overall immunologic assessment, pathogenesis, and disease indicates that the presence of both viruses negatively impacts human health as compared to the presence of either virus alone. Based on these observations, investigators have also explored the molecular interactions between HTLV-1 and HIV-1 that could be influencing the development of HTLV-1 disease or HIV/AIDS during HTLV-1/HIV-1 coinfection. *In vitro* experiments involving superinfection with the HIV-1 molecular clone HIV-1IIIB on two HTLV-1-transformed cell lines, MT2 (an HTLV-1 producer cell line) and 81-66/45 cell line (an HTLV-1 nonproducer cell line), have demonstrated that HIV-1 infection activates HTLV-1 and increases the levels of HTLV-1 proviral DNA in both HTLV-1-transformed cell lines [114]. Additional studies performed by Zsabó and colleagues [115] have shown that *in vitro* HTLV-1/HIV-1 coinfection of macrophages by both viruses results in increased replication of both viruses. The presence of the HTLV-1 Tax protein promoted nuclear transport of the newly reverse transcribed HIV-1 DNA, whereas the mechanism by which HIV-1 infection enhanced HTLV-1 gene expression did not appear to

In a tripartite coculture assay using Jurkat T cells transfected with an HTLV-1LTR-driven reporter construct designated Jurkat/HTLV-1-Luc with a chronically infected HTLV-1 cell line, HTLV-1-MT2, that has also been infected with HIV-1IIIB via cell-to-cell transfer of virus from HIV-1IIIB-infected H9 cells, Sun and coworkers demonstrated that HIV-1 infection induced an 80-fold increase in LTR-dependent HTLV-1 gene expression. It was also demonstrated that the increase in transcriptional activation of HTLV-1 genes occurred in a mechanism that was dependent on the HTLV-1 Tax protein, the HIV-1 gp120/gp41 complex, and CD40. These results suggested that HIV-1 infection promoted the development of syncytium among the cell lines examined in these studies, thereby acting as a channel for HTLV-1 Tax to translocate from the HTLV-1-infected MT2 cells to the HTLV-1-LTR-Jurkat cells, thereby providing an explanation as to how coinfection with HIV-1 and HTLV-1 may transactivate the LTR of latent provirus in neighboring cells [116]. Using an *in vitro* model of HTLV-1/HIV-1 coinfection, Roy

length of time involving mono or dual infection.

56 Leukemias - Updates and New Insights

involve the HIV-1 Tat protein.

**6. Molecular interactions between HTLV-1 and HIV-1**

The current approach to effectively treat HIV-1-infected patients involves the use of a combi‐ nation of antiretroviral drugs that inhibit a number of steps within the HIV-1 replication cycle, including the reverse transcription process, integration, and proteolytic processing of struc‐ tural polyproteins, thereby reducing the production of mature infectious HIV-1 virions that involves the HIV-1-encoded protease [124]. By 2011, the FDA had approved 26 antiretrovirals for the treatment of HIV-1 [6] with now more than 30 agents approved for use in humans to treat HIV-1 infection [8]. The rationale of using highly active antiretroviral therapy (HAART) or also referred as combination antiretroviral therapy (cART) in HIV-1-infected individuals has been to minimize the development of drug-resistant virus while effectively reducing the viral load to undetectable levels for prolonged periods of time, in essence, the establishment of a manageable chronic disease. As mentioned previously, although cART reduced actively replicating virus, it does not remove active or defective integrated and latent provirus. To affectively "cure" and HIV-infected individual, every copy of proviral DNA must be removed. Recently, much attention has been focused on purging and excising the latent reservoir by the shock and kill method (HDAC inhibitors) and gene-editing enzymes (zinc-finger nucleases, TALENS and CRISPR/Cas9) [125–128], as previously reviewed [129, 130]. The shock and kill strategy has involved reactivating latent virus reservoirs from resting memory CD4+ T cells with this process leading to a contraction in the size of the reservoir post activation in combi‐ nation with cART therapy that leads to the destruction of the activated cell and the prevention of infection of uninfected T cells. Additional therapeutic strategies have recently focused on the use of gene-editing systems involving protein-based enzymes that have been designed to seek out specific HIV-1 proviral DNA sequences and induce double-stranded DNA breaks that can induce nucleotide deletions and removal of whole gene segments [126, 130]. Many of these techniques are in basic discovery phases for the treatment of infected cells with the goal of eliminating integrated provirus. These technologies hold great promise to eliminate integrated retroviral genetic information thereby curing the infected cell.

In the case of HTLV-1 infection, reverse transcriptase inhibitors stop HTLV-1 replication *in vivo* but can only prevent infection if they are taken immediately after first contact with the virus [131, 132]. The current therapeutic options for HTLV-1 infection consist of a number of chemotherapeutic agents for lymphoma and the combination of zidovudine and interferon-α (IFN-α) for the management of acute, smoldering, and chronic ATL. The clinical management of ATL continues to be challenging because it has been shown to be a highly lethal type of cancer resistant to many of the currently available anticancer drugs (Figure 4) [124]. In 1996, Borg and colleagues [133] successfully treated and caused cancer rejection in an Afro-Carib‐ bean female with ATL using a combination of chemotherapeutic agents (cyclophosphamide, doxorubicin, and etoposide) and allogeneic bone marrow stem cell transplantation (alloSCT). In the case of people older than 50 years, alloSCT treatment is given with low doses of chemotherapeutic drugs. Retrospective studies indicate that 30–40% of ATL patients who have undergone alloSCT treatment become long-term survivors [134]. The use of the humanized defucosylated antiCCR4 antibody in patients with ATL has also had promising results [135, 136] with HAM/TSP [137]. Concurrently, the treatment for HAM/TSP and its secondary effects involves the use of spasmolytic drugs, prednisone, danazol [138], valproic acid [139], prosul‐ tiamine [140], IFN-α [141–143], IFN-β-1a [144–146], or vitamin B1 [147].

The management of HTLV-1/HIV-1 coinfection can be challenging due to the lack of efficacy of antiretroviral therapy with respect to the inhibition of HTLV-1 replication as well as the disparate effects one therapy or compound may have on the other virus. One of the drugs commonly used for HTLV-1 infection is IFN-α. However, the clinical benefits of IFN-α for HIV-1-infected patients are controversial. *In vitro*, IFN-α downregulates HIV-1 replication in macrophages and T cells and halts the formation of mature HTLV-1 virions. Importantly, IFNα treatment induces caspase-3-mediated apoptosis of HTLV-1/HIV-1IIIB-coinfected MT-4 cells, but not HTLV-1-monoinfected MT-4 cells. Interestingly, IFN-α treatment did not affect HTLV-1 infectivity but markedly reduced HIV-1 replication, with an approximately 1000-fold decrease in HIV-1 p24 antigen expression [124]. These immediate differential affects on therapy of these two viruses also project a possible role of coinfection in influencing ATL and HAM/TSP prominence and therapeutic strategy for treatment.

The incidence of ATL in HTLV-1/HIV-1-coinfected individuals has not been widely reported yet. Shibata and colleagues reported a case involving a 43-year-old African American male with ATL that was positive for both HTLV-1 and HIV-1. The patient underwent three phases of treatment achieving at least 12 months of remission. The first stage consisted of daunoru‐ bicin, prednisone, and vincristine. Then the patient was placed on *cis*-platinum, etoposide, cytosine arabidoside, and dexamethasone. During the third phase, the patient was treated with

of infection of uninfected T cells. Additional therapeutic strategies have recently focused on the use of gene-editing systems involving protein-based enzymes that have been designed to seek out specific HIV-1 proviral DNA sequences and induce double-stranded DNA breaks that can induce nucleotide deletions and removal of whole gene segments [126, 130]. Many of these techniques are in basic discovery phases for the treatment of infected cells with the goal of eliminating integrated provirus. These technologies hold great promise to eliminate

In the case of HTLV-1 infection, reverse transcriptase inhibitors stop HTLV-1 replication *in vivo* but can only prevent infection if they are taken immediately after first contact with the virus [131, 132]. The current therapeutic options for HTLV-1 infection consist of a number of chemotherapeutic agents for lymphoma and the combination of zidovudine and interferon-α (IFN-α) for the management of acute, smoldering, and chronic ATL. The clinical management of ATL continues to be challenging because it has been shown to be a highly lethal type of cancer resistant to many of the currently available anticancer drugs (Figure 4) [124]. In 1996, Borg and colleagues [133] successfully treated and caused cancer rejection in an Afro-Carib‐ bean female with ATL using a combination of chemotherapeutic agents (cyclophosphamide, doxorubicin, and etoposide) and allogeneic bone marrow stem cell transplantation (alloSCT). In the case of people older than 50 years, alloSCT treatment is given with low doses of chemotherapeutic drugs. Retrospective studies indicate that 30–40% of ATL patients who have undergone alloSCT treatment become long-term survivors [134]. The use of the humanized defucosylated antiCCR4 antibody in patients with ATL has also had promising results [135, 136] with HAM/TSP [137]. Concurrently, the treatment for HAM/TSP and its secondary effects involves the use of spasmolytic drugs, prednisone, danazol [138], valproic acid [139], prosul‐

The management of HTLV-1/HIV-1 coinfection can be challenging due to the lack of efficacy of antiretroviral therapy with respect to the inhibition of HTLV-1 replication as well as the disparate effects one therapy or compound may have on the other virus. One of the drugs commonly used for HTLV-1 infection is IFN-α. However, the clinical benefits of IFN-α for HIV-1-infected patients are controversial. *In vitro*, IFN-α downregulates HIV-1 replication in macrophages and T cells and halts the formation of mature HTLV-1 virions. Importantly, IFNα treatment induces caspase-3-mediated apoptosis of HTLV-1/HIV-1IIIB-coinfected MT-4 cells, but not HTLV-1-monoinfected MT-4 cells. Interestingly, IFN-α treatment did not affect HTLV-1 infectivity but markedly reduced HIV-1 replication, with an approximately 1000-fold decrease in HIV-1 p24 antigen expression [124]. These immediate differential affects on therapy of these two viruses also project a possible role of coinfection in influencing ATL and

The incidence of ATL in HTLV-1/HIV-1-coinfected individuals has not been widely reported yet. Shibata and colleagues reported a case involving a 43-year-old African American male with ATL that was positive for both HTLV-1 and HIV-1. The patient underwent three phases of treatment achieving at least 12 months of remission. The first stage consisted of daunoru‐ bicin, prednisone, and vincristine. Then the patient was placed on *cis*-platinum, etoposide, cytosine arabidoside, and dexamethasone. During the third phase, the patient was treated with

integrated retroviral genetic information thereby curing the infected cell.

58 Leukemias - Updates and New Insights

tiamine [140], IFN-α [141–143], IFN-β-1a [144–146], or vitamin B1 [147].

HAM/TSP prominence and therapeutic strategy for treatment.

**Figure 4.** Current diagnosis and treatment of HTLV-1-induced ATL. Summary of criteria used to diagnose previously identifiable forms of ATL and a brief overview of currently available treatments are shown.

zidovudine. PCR analysis of PBMCs detected HTLV-1 in 1/1,000 cells and HIV-1 in a similar fraction [148].

Furthermore, Hütter and colleagues used alloSCT to treat a 40-year-old white male who suffered from acute myeloid leukemia and was also HIV-1-positive. The patient received the transplant from an HLA-matched donor who was homozygous for the CCR5 delta32 deletion. Importantly, a homozygous 32-bp deletion in the CCR5 allele has been shown to confer longterm resistance to HIV-1 infection with CCR5-utilizing viruses but not CXCR4-utilizing viruses. The patient was infected with HIV-1 more than 10 years earlier and had received cART for the previous 4 years. At the time of his leukemia, he was asymptomatic with respect to HIV disease. Initially, the patient was treated with chemotherapy, after which time he suffered from a rebound in his HIV-1 viral load and a relapse in his leukemia. At this time, he received the alloSCT, which successfully treated the leukemia. In addition, the patient stopped taking the cART and his HIV-1 viral loads were undetectable. After 20 months of follow-up, this patient was free of both the HIV-1 infection and the leukemia [149]. While these *in vitro* and *in vivo* studies and clinical trials have revealed a great deal of information about co- and monoinfec‐ tion of HTLV-1/HIV-1-infected individuals, much more research is needed to help manage these lifelong chronic infections.

#### **8. Summary and concluding remarks**

We have discussed a number of diseases caused by infection with two common human retroviruses, HTLV-1 and HIV-1, alone or within the context of coinfection. As previously shown by epidemiological studies, coinfection with these two viral pathogens occurs fre‐ quently among illicit drug users, and its incidence is on the rise particularly in regions around the world where both viruses are endemic. Although HTLV-1 and HIV-1 are both retroviruses and as such share a number of genomic structural features, similar events within the replication cycle, and common modes of transmission, the overall pathogenic outcomes and the associated diseases they cause are very different except, for the most part, they all occur within the context of the immune and nervous systems with other end organs also involved. Additional infor‐ mation relevant to epidemiology, virology, immunology, immune- and neuropathogenesis, diagnosis, molecular mechanisms of disease, treatment, and clinical management are also discussed within the context of mono- and coinfection with these two important human retroviruses. Despite large bodies of information available concerning the molecular patho‐ genesis and disease resulting from monoinfections, there is much less information concerning the molecular interactions between HTLV-1 and HIV-1 replication machinery as well as the implications each virus has on disease progression resulting from the pathogenic outcomes when both viruses are replication in the same cells or neighboring cells within the same tissue compartment. Clearly, studies of HTLV-1/HIV-1 coinfection have been complicated by the fact that most of these studies have been conducted after the initiation of cART for suppression of HIV-1 infection (although many of the coinfection studies have been performed in countries where delivery of optimal cART has been difficult). With regard to the molecular interactions between the two viruses, even though both viruses target CD4+ T cells, HIV-1 infection usually results in lytic replication in activated T-cell populations, whereas HTLV-1 infection usually results in more limited replication and gene expression and ultimately induces clonal expan‐ sion of selected CD4+ T-cell populations. Interestingly, the bone marrow compartment is penetrated by both HTLV-1 and HIV-1 during the course of disease; the penetration of this compartment appears to seed each virus into different cellular compartments [41, 85, 150]. With respect to HIV-1, the stem cell population within this compartment appears to be spared of viral infection with virus only seeded into more committed or differentiated progenitor cell compartments. The binding of HIV-1 particles to stem cells has been shown to alter their functional properties despite the absence of detectable levels of viral entry into stem cells. However, following HTLV-1 infection, there appears to be differential gene expression within cellular compartments within the bone marrow with much greater numbers of DNA+ RNA+ progenitor cells in individuals suffering from HAM/TSP as compared to individuals with ATL where there are far fewer DNA+ RNA+ progenitor cells [61, 64]. The significance of these molecular interactions within the context of bone marrow cell populations remains unresolved during the course of monoinfections and remains to be examined in HTLV-1/HIV-1 coinfec‐ tions. The interaction of both viruses with the bone marrow compartment during the course of mono- and coinfection within the same or different bone marrow-infected cell populations will likely play an important role in the pathogenesis of diseases caused by both viruses. Clearly, studies with a greater number of larger coinfection cohorts will be required to approach defining molecular mechanisms, diagnosis, treatment, prevention, and overall clinical management of diseases caused by HTLV-1/HIV-1 coinfection. It would also seem important to obtain a better understanding concerning the relationship between the timing of HIV-1 infection relative to the course of HTLV-1-induced disease. Interestingly and perhaps relevant to thinking about this problem is the epidemiologic data suggesting that individuals that suffer from ATL as compared to HAM/TSP are more likely to have been infected by mucosal membrane exposure early in life often as a result of vertical transmission of HTLV-1 as compared to a blood stream exposure of the virus as a result of IV transmission associated with illicit drug abuse [151]. Clearly, the immunosuppression that occurs as a result of primary HIV-1 infection and later during the course of disease with individuals that first seek medical attention for symptoms consistent with HIV-1 disease prior to the start of cART may have great impact on the initial phases of HTLV-1 disease depending on the size and functionality of the T-cell compartment during the primary HTLV-1 infection. If these predictable periods of immunosuppression occur at critical phases of already ongoing HTLV-1-induced disease, the impact of HIV-1 on the course of HTLV-1 disease could be significant whether the individual is headed toward neuroinflammatory or leukemic disease. Clearly, these interactions will pose significant challenges with respect to the clinical management of HTLV-1-induced disease, while HTLV-1 infection could alter the course of HIV-1 disease depending on what impact the neuroinflammatory state associated with the development of HAM/TSP may have on HIV-1 infection of the CD4+ T-cell compartment and what impact of polyclonal or monoclonal expansion of the CD4+ T-cell compartment associated with HTLV-1-induced leukemogenesis may have on productive HIV-1 infection and replication in these T-cell compartments. Finally, the discussion of an HIV-1-positive patient suffering from ATL and another case with an HIV-1-positive patient with acute myeloid leukemia with respect to the impact of bone marrow stem cell replacement therapy on controlling HTLV-1-induced cancer and impact on ongoing HIV-1 disease was examined. In both cases, HIV-1 disease was well controlled at the time of the bone marrow transplant. Interestingly, they both achieved remission of the leukemia, and the HIV-1 and HTLV-1 titers of the first case were very low after treatment, and in the second case, the HIV-1 infection has been apparently cured. This is clearly a better understanding of the molecular interactions between HTLV-1 and HIV-1 and their respective host cell targets with regard to cellular coinfection or cellular interactions altered by viral coinfection of different cellular compartments.

**8. Summary and concluding remarks**

60 Leukemias - Updates and New Insights

between the two viruses, even though both viruses target CD4+

sion of selected CD4+

where there are far fewer DNA+

We have discussed a number of diseases caused by infection with two common human retroviruses, HTLV-1 and HIV-1, alone or within the context of coinfection. As previously shown by epidemiological studies, coinfection with these two viral pathogens occurs fre‐ quently among illicit drug users, and its incidence is on the rise particularly in regions around the world where both viruses are endemic. Although HTLV-1 and HIV-1 are both retroviruses and as such share a number of genomic structural features, similar events within the replication cycle, and common modes of transmission, the overall pathogenic outcomes and the associated diseases they cause are very different except, for the most part, they all occur within the context of the immune and nervous systems with other end organs also involved. Additional infor‐ mation relevant to epidemiology, virology, immunology, immune- and neuropathogenesis, diagnosis, molecular mechanisms of disease, treatment, and clinical management are also discussed within the context of mono- and coinfection with these two important human retroviruses. Despite large bodies of information available concerning the molecular patho‐ genesis and disease resulting from monoinfections, there is much less information concerning the molecular interactions between HTLV-1 and HIV-1 replication machinery as well as the implications each virus has on disease progression resulting from the pathogenic outcomes when both viruses are replication in the same cells or neighboring cells within the same tissue compartment. Clearly, studies of HTLV-1/HIV-1 coinfection have been complicated by the fact that most of these studies have been conducted after the initiation of cART for suppression of HIV-1 infection (although many of the coinfection studies have been performed in countries where delivery of optimal cART has been difficult). With regard to the molecular interactions

results in lytic replication in activated T-cell populations, whereas HTLV-1 infection usually results in more limited replication and gene expression and ultimately induces clonal expan‐

penetrated by both HTLV-1 and HIV-1 during the course of disease; the penetration of this compartment appears to seed each virus into different cellular compartments [41, 85, 150]. With respect to HIV-1, the stem cell population within this compartment appears to be spared of viral infection with virus only seeded into more committed or differentiated progenitor cell compartments. The binding of HIV-1 particles to stem cells has been shown to alter their functional properties despite the absence of detectable levels of viral entry into stem cells. However, following HTLV-1 infection, there appears to be differential gene expression within cellular compartments within the bone marrow with much greater numbers of DNA+

progenitor cells in individuals suffering from HAM/TSP as compared to individuals with ATL

molecular interactions within the context of bone marrow cell populations remains unresolved during the course of monoinfections and remains to be examined in HTLV-1/HIV-1 coinfec‐ tions. The interaction of both viruses with the bone marrow compartment during the course of mono- and coinfection within the same or different bone marrow-infected cell populations will likely play an important role in the pathogenesis of diseases caused by both viruses. Clearly, studies with a greater number of larger coinfection cohorts will be required to

RNA+

T-cell populations. Interestingly, the bone marrow compartment is

progenitor cells [61, 64]. The significance of these

T cells, HIV-1 infection usually

RNA+

The clinical management of HTLV-1 and HIV-1 mono- and HTLV-1/HIV-1 coinfection will be greatly enhanced by the identification of additional druggable viral or cellular targets to enhance the effective long-term clinical management of HTLV-1- and HIV-1-induced disease outcomes stemming from monoinfections (long-term suppression of viral gene expression in either case with minimal impact of viral infection on host cell function) prior to the encounter of the second virus. A second therapeutic approach will involve curing HTLV-1- and HIV-1 infected patients by elimination of susceptible target cell populations by targeted elimination of cellular receptor epitopes rendering normally susceptible cells refractile to viral infection while maintaining the normal cellular function of these host cell proteins. In parallel with these types of experimental studies, additional types of experimentation will involve the eradication of HTLV-1 and HIV-1 infections by site-specific excision of integrated HTLV-1 and HIV-1 proviral DNA with minimal off target impact on host cell function. Clearly, this is exciting technology with great promise to completely eliminate latent or persistent viral infections without having to activate latent viral gene expression to kill latently infected cells [125–130, 152]. The goal of eliminating both defective and completely functional HIV-1 and HTLV-1 will likely be very critical since it is entirely possible that nonactivatable defective proviruses may still be able to drive the expression of viral proteins (gp120, Tat, Vpr, and Nef) that may cause detrimental effects to neighboring or distant cells in the absence of lytic infectious virus production. Many challenges await this experimental approach, including the exact nature of existing viral reservoirs, the genetic variability of the latent virus, and the delivery of excision technologies to tissue-specific reservoirs, including memory CD4+ T-cell subpopulations, specific cell populations within the monocyte–macrophage lineage, as well as cell populations within the brain and other tissues. Perhaps central to basic and translational science is the development of tomorrow's translational solutions to today's challenges leading to effective solutions to clinical problems.

## **Author details**

Lorena Loarca1 , Neil Sullivan2,3, Vanessa Pirrone2,3 and Brian Wigdahl2,3\*

\*Address all correspondence to: brian.wigdahl@drexelmed.edu

1 Division of Gastroenterology and Hepatology, Mayo Clinic and Medical School, Rochester, MN, USA

2 Center for Molecular Virology and Translational Neuroscience, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, USA

3 Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, USA

## **References**


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specific cell populations within the monocyte–macrophage lineage, as well as cell populations within the brain and other tissues. Perhaps central to basic and translational science is the development of tomorrow's translational solutions to today's challenges leading to effective

, Neil Sullivan2,3, Vanessa Pirrone2,3 and Brian Wigdahl2,3\*

1 Division of Gastroenterology and Hepatology, Mayo Clinic and Medical School, Rochester,

2 Center for Molecular Virology and Translational Neuroscience, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, USA

3 Department of Microbiology and Immunology, Drexel University College of Medicine,

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**Author details**

Lorena Loarca1

MN, USA

Philadelphia, USA

2008;80:494–500.

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Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60928

#### **Abstract**

Acute myeloid leukemia (AML) is a clonal disorder affecting pluripotent stem cells and is characterized by ineffective hematopoiesis. Most AML patients harbor cytogenetic and molecular defects that identify entities with peculiar biologic and clinical data and distinct therapeutic responses. Approximately 50%–60% of de novo AML and 80%–95% of secondary AML patients display chromosomal aberrations. Structural chromosomal rearrangements are the most common cytogenetic abnor‐ malities in de novo AML, with an incidence of 40%. Last years, large collaborative studies have demonstrated the importance of cytogenetic aberrations for the prognosis of AML patients.

The large group of patients with cytogenetically normal (CN) AML refers to the intermediate risk category. It is known that this group of patients is very heterogene‐ ous with respect to prognosis. The recent large-scale sequencing of AML genomes is now providing opportunities for patient stratification and personalized approaches to treatments that are based on individual mutation profiles. Genes recurrently mutated in AML belong to distinct functional groups or pathways. A few recurring gene mutations with prognostic relevance in AML have been identified and have become incorporated into current prognostication models. For patients with CN AML, prognosis can be specified by mutational status of the genes *NPM1*, *FLT3*, and *CEBPA*. CN AML patients with *NPM1* mutation, but no *FLT3*-ITD, or with *CEBPA* mutation, have a favorable prognosis. In contrast, CN AML patients with *FLT3*-ITD mutation have a poor prognosis.

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Recently a new class of mutations affecting genes for DNA methylation and posttranslational histone modification was identified in AML. These mutations frequently occur in the DNA nucleotide methyltransferase 3A gene (*DNMT3A*) and isocitrate dehydrogenase 1/2 gene (*IDH1/2*). Different studies have shown a negative impact of *DNMT3A* mutations on outcomes in patients with AML. The prognostic effect is known to depend on certain biological factors as well as a combination of cytogenetics and other mutations such as those in *FLT3* and *NPM1.* In contrast, the impact of *IDH1/2* mutations on prognosis is not completely understood. It appears that prognosis may depend on specific patient populations and a combination with *NPM1* mutations.

Moreover, a growing number of recurrent mutations in additional genes have recently been identified. Increasing evidence suggests that AML develops throughout the process of branching evolution.

**Keywords:** AML, mutations, prognosis, *FLT3*, *NPM1*, *DNMT3A*, *IDH1/2*

## **1. Introduction**

Acute myeloid leukemia (AML) is the most common type of leukemia among hematologic malignancies in adults. In the last years, progresses in molecular technologies have led to identify AML as a highly heterogeneous disorder. AML is a clonal hematopoietic disease that arises from multiple acquired genetic lesions accumulating in hematopoietic progenitors. The mutations give rise to a malignant clone [1]. In the initial development, Knudson's two-hit hypothesis has provided important insights into the pathogenesis of leukemia. Later studies using mouse models have confirmed that genetic abnormalities in leukemia could be divided into two classes. Class I mutations confer a proliferative or survival advantage to blast cells, while class II mutations block myeloid differentiation and give self-renewability [2-4]. Recently, next-generation sequencing methods provided more complete insight in oncogenic events. Early mutations may be present many years before disease develops [5]. Evidence from many murine models has confirmed that early mutations lead to clonal expansions by progenitor cells. Later, cooperating mutations would arise in cells that already contain initiating early mutations [6].

Karyotype analysis allows detecting genetic changes on a chromosomal level by visual assessment of chromosomal banding. Recurrent chromosomal abnormalities are found in about 55% of adults with AML. Some, not all, of the chromosomal aberrations are strong independent predictors of outcome and are the mainstay of the World Health Organization (WHO) classification of AML risk groups [7]. In AML patients with cytogenetically normal karyotype (CN AML) who have an intermediate-risk cytogenetics, clinical outcomes vary greatly. Identification of recurrent mutations in AML improved the understanding of the molecular pathogenesis. Later studies revealed recurrent genetic markers in more than 85% of CN AML patients [8]. Some of the mutations add important prognostic information and also indicate potential therapeutic targets. The more detailed insight into the genetic architec‐ ture of AML is challenging the established classification and prognostication systems [8]. Particular mutations have already been included in the latest WHO classification that was established in 2008 and in subsequent recommendations for diagnosis of AML by an interna‐ tional expert panel [9].

## **2. Nucleophosmin 1 (***NPM1***) mutations**

Recently a new class of mutations affecting genes for DNA methylation and posttranslational histone modification was identified in AML. These mutations frequently occur in the DNA nucleotide methyltransferase 3A gene (*DNMT3A*) and isocitrate dehydrogenase 1/2 gene (*IDH1/2*). Different studies have shown a negative impact of *DNMT3A* mutations on outcomes in patients with AML. The prognostic effect is known to depend on certain biological factors as well as a combination of cytogenetics and other mutations such as those in *FLT3* and *NPM1.* In contrast, the impact of *IDH1/2* mutations on prognosis is not completely understood. It appears that prognosis may depend on specific patient populations and a combination with *NPM1* mutations.

Moreover, a growing number of recurrent mutations in additional genes have recently been identified. Increasing evidence suggests that AML develops throughout the

Acute myeloid leukemia (AML) is the most common type of leukemia among hematologic malignancies in adults. In the last years, progresses in molecular technologies have led to identify AML as a highly heterogeneous disorder. AML is a clonal hematopoietic disease that arises from multiple acquired genetic lesions accumulating in hematopoietic progenitors. The mutations give rise to a malignant clone [1]. In the initial development, Knudson's two-hit hypothesis has provided important insights into the pathogenesis of leukemia. Later studies using mouse models have confirmed that genetic abnormalities in leukemia could be divided into two classes. Class I mutations confer a proliferative or survival advantage to blast cells, while class II mutations block myeloid differentiation and give self-renewability [2-4]. Recently, next-generation sequencing methods provided more complete insight in oncogenic events. Early mutations may be present many years before disease develops [5]. Evidence from many murine models has confirmed that early mutations lead to clonal expansions by progenitor cells. Later, cooperating mutations would arise in cells that already contain

Karyotype analysis allows detecting genetic changes on a chromosomal level by visual assessment of chromosomal banding. Recurrent chromosomal abnormalities are found in about 55% of adults with AML. Some, not all, of the chromosomal aberrations are strong independent predictors of outcome and are the mainstay of the World Health Organization (WHO) classification of AML risk groups [7]. In AML patients with cytogenetically normal karyotype (CN AML) who have an intermediate-risk cytogenetics, clinical outcomes vary greatly. Identification of recurrent mutations in AML improved the understanding of the molecular pathogenesis. Later studies revealed recurrent genetic markers in more than 85% of CN AML patients [8]. Some of the mutations add important prognostic information and

**Keywords:** AML, mutations, prognosis, *FLT3*, *NPM1*, *DNMT3A*, *IDH1/2*

process of branching evolution.

**1. Introduction**

76 Leukemias - Updates and New Insights

initiating early mutations [6].

The nucleophosmin/nucleoplasmin (NPM) family of chaperones has diverse functions in the cell. The *NPM1* gene maps to chromosome 5q35 and encodes a phosphoprotein that moves between the nucleus and the cytoplasm. The gene product involves a number of cellular processes such as chromatin remodeling, genome stability, ribosome biogenesis, DNA duplication, and transcriptional regulation. *NPM1* also interacts with a number of proteins at the mitotic spindle and in the nucleolus and includes in regulation of the ARF/ p53 pathway [10, 11]. *NPM1* is clearly having both growth promoting and tumor suppres‐ sive functions [11, 12].

In AML, there are some chromosomal translocations involving *NPM1* gene. These genetic alterations usually disturb the cellular transport of *NPM1* [13]. In AML carrying the t(3;5) (q25;q35) translocation, leukemic cells display fusion protein *NPM1*–*MLF1* (myelodysplasia/ myeloid leukemia factor 1) and show aberrant *NPM1* expression in cytoplasm [14]. In rare cases of acute promyelocytic leukemia carrying the translocation t(5;17), the *NPM1–RARα* fusion protein was detected [15]. The transforming role of partner genes in these cases is well established [13]. Nevertheless, *NPM1* moiety seems to be not only provides a dimerization substrate for the C-terminal onco-protein. *In vivo* studies using mouse model have shown that *NPM1* is a haploinsufficient tumor suppressor gene [16]. Therefore, loss of *NPM1* could also contribute to the pathogenesis of AML [17, 18].

Mutations in the *NPM1* gene represent one of the most common gene mutations in AML [19]. Approximately 25%–30% of AML patients and about 60%–85% of CN AML patients display *NPM1* mutation [13, 20]. *NPM1* mutations are heterogeneous; more than 50 different variants of mutations are identified. A more common variant of mutations is the insertion of four nucleotides at position 288-290 at exon 12. Mutations type A with insertion of "TCTG" at position 288 is the most frequent aberration (75%–80% of cases) [14, 19, 21]. In most cases (about 95%), three mutations types (A, B, and D) are found [13]. *NPM1* mutations result in common changes at the C-terminus end of the *NPM1* protein, that is, changes of tryptophans and insertion of a new nuclear export signal motif. These changes cause aberrant cytoplasmic accumulation of *NPM1* mutants, thus preventing or decreasing *NPM1* binding to the nucleo‐ lus. Aberrant *NPM1* expression in cytoplasm is easily detectable by immunohistochemistry [21, 22]. *NPM1* mutations often combined with other AML-associated mutations, especially with *FLT3* (fms-related tyrosine kinase 3), *DNMT3A* (DNA (cytosine-5-methyltransferase 3 alpha), *IDH1* and *IDH2* (isocitrate dehydrogenase 1 and 2 (NADP+)), *NRAS* (neuroblastoma RAS viral (v-ras) oncogene homolog), and others. Most likely, these mutations do not accu‐ mulate in a random order but instead could be allocated to early and late events in the transformation process [5, 23].

The prognostic status of *NPM1* depends on the presence of other concurrent genetic alterations. In the absence of *FLT3-*internal tandem duplication (ITD) mutations, *NPM1* mutations are associated with improved outcome for CN AML patients, even in those older than 60 years [9]. Current European Leukaemia Net (ELN) recommendations for diagnosis and treatment of AML determines CN AML with *NPM1* mutation without *FLT3* mutation as a favorable risk and does not recommend allogeneic stem cell transplantation (alloSCT) in first complete remission (CR) [9]. Recently, a beneficial prognostic effect of *NPM1* mutations was reported in AML patients with simultaneous mutations in *IDH1* or *IDH2* [8], whereas a worse prognosis of CN AML without *FLT3*-ITD but with mutations in *IDH1* or *IDH2* has also been described [24]. *NPM1* mutations inform treatment decisions also in elderly patients because it identifies those who might benefit from intensive chemotherapy.

*NPM1* mutation provides a sensitive marker for minimal residual disease (MRD) detection by qPCR. Because of their stability in the course of disease and relative homogeneity of mutation pattern, *NPM1* represent a useful target for MRD monitoring, in particular in CN AML. The applicability of an RNA- or DNA-based q-PCR assay for *NPM1*mutation monitoring has been shown by several groups [25-28]. Many authors have shown that *NPM1* mutation as MRD marker is a relevant factor for the identification of patients at high and low risk of relapse [28].

## **3.** *FLT3* **mutations in AML**

The *FLT3* gene in chromosome band 13q12 encodes a protein known as fms-like tyrosine kinase 3, which belongs to the family of receptor tyrosine kinases (RTK). RTKs transmit signals from the cell surface into the cell through a signal transduction. RTK3 family members are charac‐ terized by an extracellular domain comprised of 5 immunoglobulin-like domains and by a cytoplasmic domain with a split tyrosine kinase motif [29, 30]. The FLT3 protein is located in the membrane of certain cell types where FLT3 ligand binds it. *FLT3* is highly expressed in CD34+ hematopoietic progenitor cells and variable express in the more mature monocytic lineage. *FLT3* expression has been described in lymphohematopoietic organs such as the liver, spleen, thymus, and placenta [30, 31].

The binding with ligand activates the FLT3 protein, which subsequently activates a series of proteins inside the cell that are part of multiple signaling pathways and leads to receptor oligomerization and transphosphorylation of specific tyrosine residues, which activates the downstream signaling pathways including STAT5, RAS/mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/AKT. The signaling pathways stimulated by the FLT3 protein control many important cellular processes such as the growth, proliferation, and survival of cells, particularly of hematopoietic progenitor cells [32, 33].

The *FLT3* receptor consists of an extracellular domain composed of a transmembrane region, a cytoplasmic juxtamembrane domain (JMD), and 2 cytoplasmic tyrosine kinase domains (TKD; TKD1 and TKD2) interrupted by a short kinase insert. The JMD can be subdivided into 3 distinct parts: the binding motif, which is implicated in activation and in stabilizing the inactive kinase conformation; the switch motif, which consists of 2 phosphorylation sites and contains the STAT5-binding motif; and the linker/zipper peptide segment, which can undergo large amplitude rotations by pivoting about its attachment point [34].

mulate in a random order but instead could be allocated to early and late events in the

The prognostic status of *NPM1* depends on the presence of other concurrent genetic alterations. In the absence of *FLT3-*internal tandem duplication (ITD) mutations, *NPM1* mutations are associated with improved outcome for CN AML patients, even in those older than 60 years [9]. Current European Leukaemia Net (ELN) recommendations for diagnosis and treatment of AML determines CN AML with *NPM1* mutation without *FLT3* mutation as a favorable risk and does not recommend allogeneic stem cell transplantation (alloSCT) in first complete remission (CR) [9]. Recently, a beneficial prognostic effect of *NPM1* mutations was reported in AML patients with simultaneous mutations in *IDH1* or *IDH2* [8], whereas a worse prognosis of CN AML without *FLT3*-ITD but with mutations in *IDH1* or *IDH2* has also been described [24]. *NPM1* mutations inform treatment decisions also in elderly patients because it identifies

*NPM1* mutation provides a sensitive marker for minimal residual disease (MRD) detection by qPCR. Because of their stability in the course of disease and relative homogeneity of mutation pattern, *NPM1* represent a useful target for MRD monitoring, in particular in CN AML. The applicability of an RNA- or DNA-based q-PCR assay for *NPM1*mutation monitoring has been shown by several groups [25-28]. Many authors have shown that *NPM1* mutation as MRD marker is a relevant factor for the identification of patients at high and low risk of relapse [28].

The *FLT3* gene in chromosome band 13q12 encodes a protein known as fms-like tyrosine kinase 3, which belongs to the family of receptor tyrosine kinases (RTK). RTKs transmit signals from the cell surface into the cell through a signal transduction. RTK3 family members are charac‐ terized by an extracellular domain comprised of 5 immunoglobulin-like domains and by a cytoplasmic domain with a split tyrosine kinase motif [29, 30]. The FLT3 protein is located in the membrane of certain cell types where FLT3 ligand binds it. *FLT3* is highly expressed in CD34+ hematopoietic progenitor cells and variable express in the more mature monocytic lineage. *FLT3* expression has been described in lymphohematopoietic organs such as the liver,

The binding with ligand activates the FLT3 protein, which subsequently activates a series of proteins inside the cell that are part of multiple signaling pathways and leads to receptor oligomerization and transphosphorylation of specific tyrosine residues, which activates the downstream signaling pathways including STAT5, RAS/mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/AKT. The signaling pathways stimulated by the FLT3 protein control many important cellular processes such as the growth, proliferation, and

The *FLT3* receptor consists of an extracellular domain composed of a transmembrane region, a cytoplasmic juxtamembrane domain (JMD), and 2 cytoplasmic tyrosine kinase domains

survival of cells, particularly of hematopoietic progenitor cells [32, 33].

transformation process [5, 23].

78 Leukemias - Updates and New Insights

**3.** *FLT3* **mutations in AML**

spleen, thymus, and placenta [30, 31].

those who might benefit from intensive chemotherapy.

Two predominant types of *FLT3*-activating mutations have been described in association with AML. The first involves ITD mutations that are found in about 25% of adults and 15% of pediatric AML cases [9, 35-37]. The *FLT3* mutations lead to constitutive activation by auto‐ phosphorylation of the *FLT3* RTK that could activate multiple signaling pathways and lead to cell proliferation [38, 39]. ITDs are located in exons 14 and 15 of the *FLT3* gene and show a broad variation in the position of insertion site, as well as in the number and sizes of the duplicated fragments. The length of the duplicated JMD region varies from 3 to 400 nucleotides but, despite this heterogeneity, the resultant transcripts are always in-frame [40]. These mutations are mostly located in the JMD. Localization outside the JMD is present in about 25% of the cases [41-43].

*FLT3*-ITD mutations have a significantly adverse impact on prognosis due to a high relapse rate, which translates into an inferior overall survival (OS) [23, 36, 44-46]. The effect on prognosis is modulated by the mutated to wild-type allele ratio, with inferior outcome in the presence of a higher load of ITDs in *FLT3.* The high *FLT3*-ITD/*FLT3*-WT ratio predict for low CR rate and OS [42-44]. Localization outside the JMD was associated with inferior outcome [42]. Many groups studied the role of alloSCT to overcome the negative impact of *FLT3*-ITD in AML patients. Some data suggest that *FLT3*-ITD positivity also outweighs other conven‐ tional prognostic markers in predicting relapse [47]. Recently published data from the German-Austrian AML Study Group showed that the high allelic ratio is a predictive factor for the beneficial effect of alloSCT [43].

The second type of *FLT3* mutation is point mutations, which most frequently occur in the activation loop of the TKD. *FLT3*-TKD mutations occur in 10% of both adult and pediatric AML patents [48]. These mutations also lead to constitutive tyrosine kinase activation. The most common TKD mutation occurs at codon 835, converting aspartic acid to tyrosine (D835Y). Also seen are mutations D835V, D835E, and D835H, converting aspartic acid to valine, glutamic acid, and histidine at residue 835, respectively. The rare mutations convert glycine to glutamic acid at residue 831 (G831E) and arginine to glutamine at residue 834 (R834Q), as well as the deletion of isoleucine at residue 836 [36, 45, 48]. TKD mutations differ from ITD in *FLT3* in their biologically transforming potency. Prognostic impact of FLT3-TKD mutations remains controversial [49, 50].

Specific gene expression signatures have been reported for CN AML with both *FLT3*-ITD and *FLT3*-TKD1 mutations. The *FLT3*-ITD signature predicts a less favorable outcome analogous to the *FLT3*-ITD mutation. High expression of wild-type *FLT3* also seems to adversely affect prognosis [51].

Sequencing studies show that *FLT3* mutations frequently occur together with mutations and alterations of other genes, especially *DNMT3A* (13.3%), *NPM1* (6.8%), Wilms tumor 1 (*WT1*, 5%), runt-related transcription factor 1 (*RUNX1*, 3.5%), mixed-lineage leukemia (*MLL*, 2.5%), CCAAT/enhancer binding protein alpha (*C/EBPα*, 1.5%), and core-binding factor (1.5%) [8, 52].

In addition, *FLT3*-ITD mutation status is different approximately in 30% of AML patients at the time of diagnosis and at relapse. *FLT3*-ITD mutations may be present in only a subset of leukemic blasts, consistent with a role in disease progression. This data suggest that *FLT3*-ITD may contribute as the initial transforming event in relapse of AML and it can reflect the selection and outgrowth of a mutant clone or evolution of a new clone harbor‐ ing this mutation [53, 54].

Recent studies also show that both the *FLT3* mutations, as well as the collaborating mutations, can have prognostic significance. Recently published data submit that *FLT3*-ITD retains its negative prognostic impact in intermediate-risk AML, even in the context of other genetic abnormalities, such as *NPM1*, *DNMT3A*, and *TET2* [8, 55].

The prevalence and prognostic implications of *FLT3* mutations make them a promising therapeutic target in AML. A number of tyrosine kinase inhibitors (TKI) against *FLT3* are currently in clinical trials, with varying degrees of clinical responses, but even those patients who respond develop resistance to monotherapy [56]. One of the mechanisms of acquired resistance to several *FLT3* TKIs is the selection for *FLT3*-TKD mutations documented in relapsed patients [57]. Moreover, some data reported that *FLT3*-TKD AML blasts do not confer increased sensitivity to tyrosine kinase inhibition [58].

## **4. CCAAT/Enhancer-Binding Protein α (***C/EBPα***) mutations**

The *C/EBPα* gene is localized on chromosome 19q13.1. This gene is intronless and it encodes a transcription factor that contains two transactivation domains: a dimerization leucine zipper region and a DNA-binding domain. It recognizes the CCAAT motif in the promoters of target genes [59]. Activity of this protein can modulate the expression of genes involved in cell cycle regulation. *C/EBPα* directly interacts with cyclin-dependent kinase 2 and 4 and arrests cell proliferation by blocking the association of these kinase with cyclins [60]. *C/EBPα* is involved in lineage specification as a transcription factor, it is crucial for the development of myeloid progenitors to the neutrophils. It is exclusively expressed in myelomonocytic cells. *C/EBPα* is specifically upregulated during granulocytic differentiation, and conditional expression of *C/ EBPα* alone is sufficient to trigger neutrophil differentiation in bipotential precursors. In addition, C/EBPα is capable of arrest cell proliferation [61, 62]. C/EBPα regulates the expression of many myeloid genes, including genes encoding growth factor receptors (granulocyte-, macrophage-, and granulocyte-macrophage colony-stimulating factor) and the secondary granule proteins [59, 61]. Numerous studies suggest that *C/EBPα* is a general inhibitor of cell proliferation and a tumor suppressor [63, 64].

When *C/EBPα* gene is altered by mutations in AML, DNA-binding is altered or eliminated. *C/ EBPα* mutation was first described by Pabst and colleagues in 2001 [65]. These mutations are detected in 10%–18% of CN AML patients and are predominantly found in M1 and M2 morphological subtypes of AML [65, 66]. Clinically, *C/EBPα* mutations are associated with lower leukocyte counts and lactate dehydrogenase levels and with aberrant expression of Tcell surface markers such as CD7 at presentation [67].

5%), runt-related transcription factor 1 (*RUNX1*, 3.5%), mixed-lineage leukemia (*MLL*, 2.5%), CCAAT/enhancer binding protein alpha (*C/EBPα*, 1.5%), and core-binding factor (1.5%) [8, 52].

In addition, *FLT3*-ITD mutation status is different approximately in 30% of AML patients at the time of diagnosis and at relapse. *FLT3*-ITD mutations may be present in only a subset of leukemic blasts, consistent with a role in disease progression. This data suggest that *FLT3*-ITD may contribute as the initial transforming event in relapse of AML and it can reflect the selection and outgrowth of a mutant clone or evolution of a new clone harbor‐

Recent studies also show that both the *FLT3* mutations, as well as the collaborating mutations, can have prognostic significance. Recently published data submit that *FLT3*-ITD retains its negative prognostic impact in intermediate-risk AML, even in the context of other genetic

The prevalence and prognostic implications of *FLT3* mutations make them a promising therapeutic target in AML. A number of tyrosine kinase inhibitors (TKI) against *FLT3* are currently in clinical trials, with varying degrees of clinical responses, but even those patients who respond develop resistance to monotherapy [56]. One of the mechanisms of acquired resistance to several *FLT3* TKIs is the selection for *FLT3*-TKD mutations documented in relapsed patients [57]. Moreover, some data reported that *FLT3*-TKD AML blasts do not confer

The *C/EBPα* gene is localized on chromosome 19q13.1. This gene is intronless and it encodes a transcription factor that contains two transactivation domains: a dimerization leucine zipper region and a DNA-binding domain. It recognizes the CCAAT motif in the promoters of target genes [59]. Activity of this protein can modulate the expression of genes involved in cell cycle regulation. *C/EBPα* directly interacts with cyclin-dependent kinase 2 and 4 and arrests cell proliferation by blocking the association of these kinase with cyclins [60]. *C/EBPα* is involved in lineage specification as a transcription factor, it is crucial for the development of myeloid progenitors to the neutrophils. It is exclusively expressed in myelomonocytic cells. *C/EBPα* is specifically upregulated during granulocytic differentiation, and conditional expression of *C/ EBPα* alone is sufficient to trigger neutrophil differentiation in bipotential precursors. In addition, C/EBPα is capable of arrest cell proliferation [61, 62]. C/EBPα regulates the expression of many myeloid genes, including genes encoding growth factor receptors (granulocyte-, macrophage-, and granulocyte-macrophage colony-stimulating factor) and the secondary granule proteins [59, 61]. Numerous studies suggest that *C/EBPα* is a general inhibitor of cell

When *C/EBPα* gene is altered by mutations in AML, DNA-binding is altered or eliminated. *C/ EBPα* mutation was first described by Pabst and colleagues in 2001 [65]. These mutations are detected in 10%–18% of CN AML patients and are predominantly found in M1 and M2

ing this mutation [53, 54].

80 Leukemias - Updates and New Insights

abnormalities, such as *NPM1*, *DNMT3A*, and *TET2* [8, 55].

increased sensitivity to tyrosine kinase inhibition [58].

proliferation and a tumor suppressor [63, 64].

**4. CCAAT/Enhancer-Binding Protein α (***C/EBPα***) mutations**

*C/EBPα* mutations can occur across the whole coding region with two main spots frequently involved, one of them affecting the N-terminus, another affecting the C-terminus. Mutations in the amino terminus truncate the full-length protein. N-terminal mutations are nonsense mutations leading to exert dominant-negative effects on the unmutated C/EBPα protein. As the mutant proteins block the binding of wild-type C/EBPα with DNA, occurs transactivation of granulocytes target genes and block of differentiation of myeloid progenitor cells. Nterminal C/EBPα mutations allow the development of committed myeloid progenitors, which represent templates for leukemia-initiating cells. C-terminal mutations are usually located between the basic region and the leucine zipper coding sequence resulting in disturbed DNA binding by the mutant protein as well as altered dimerization with its partner proteins [66]. C-terminus mutations increase the proliferation of premalignant stem cells and block myeloid lineage differentiation when homozygous. The majority of all mutations are homozygous mutations. Combination of both mutations is associated with accelerated disease development [59, 60, 66, 68]. The mechanism of C/EBPα-mutant leukemogenesis has been demonstrated in studies of C/EBPα knockout mice [69].

There is evidence that C/EBPα mutations are early events in the generation of leukemic clones. In contrast to *FLT3* mutation, in C/EBPα mutations, the majority of relapsed patients display the same mutations in both C/EBPα alleles [69]. Schin et al. demonstrated that 91% of *de novo* AML harboring *C/EBPα* mutations at diagnosis retained the identical mutant patterns but frequently changed in the allelic distribution at relapse [70].

Three different *C/EBPα* mutant patterns have been reported in AML patients. One half of patients carry single mutation on one allele (*C/EBPα*-sm), and these patients express wild type of C/EBPα. Second half of patients have double-mutated *C/EBPα* (*C/EBPα*-dm). In these cases, no wild-type *C/EBPα* protein is expressed. Some of *C/EBPα* mutated patients harbor bi-allelic mutations with an N-terminal frame-shift mutation on one allele and a C-terminal in-frame mutation on the other allele [71, 72]. Third variant of aberration is a homozygous *C/EBPα* mutation due to loss of heterozygosity, also no wild-type *C/EBPα* protein is expressed [73].

Expression profiling revealed that *C/EBPα* mutant cases cluster together, suggesting that they share similar gene expression signatures. Moreover, C-terminal *C/EBPα-*sm patients may be less distinct from *C/EBPα*-dm cases than N-terminal *C/EBPα-*sm patients [68, 71, 74]. Recent study suggest that homozygous *C/EBPα* mutations have a similar gene expression signature as *C/EBPα-*dm and thus may be considered as equivalent [75].

Most patients with *C/EBPα* mutations had a normal karyotype. Importantly, *C/EBPα* mutations have not been observed in patients with a favorable karyotype [76]. The association of deletion 9q and *C/EBPα* loss-of-function mutations could suggest that loss of a critical segment of 9q and disruption of *C/EBPα* function possibly cooperate in the pathogenesis of del(9q) AML [77]. Concurrent mutations are significantly less frequent in *C/EBPα*-dm compared with *C/EBPα*sm AML. It is correct for *FLT3*-ITD and in particular for *NPM1*, which are essentially not present among C/EBPα-dm cases [68, 78, 79]. Recently, the mutation in transcription factor *GATA2* was found to have a strong association with *C/EBPα*-dm mutation [75].

The prognostic impact of *C/EBPα* mutations seems to be favorable. The most significant effect of mutation on clinical outcome is its association with better relapse-free survival or OS [80-84]. Recent data show that a favorable outcome is limited to double, not to single, *C/EBPα* mutations [71, 78]. These data suggest that only the *C/EBPα*-dm AML should be definitely designated as AML with the favorable risk of molecular abnormalities [84]. These results have important implications for the application of risk-stratified therapy and require confirmation. Given the evidence of the prognostic value of *C/EBPα* mutations, analysis of a possible interaction between *FLT3-*ITD and *C/EBPα* mutations is of particular interest. There are contradictory studies whether coexisting *FLT3*-ITD adversely affects the favorable prognosis of *C/EBPα* mutations. Some studies showed significantly worsened prognostic outcome in patients with *FLT3-*ITD and *C/EBPα* mutations [82, 85]. In contrast, in other study negative prognostic influence among patients with *C/EBPα* mutations were not found [81]. Obviously, further studies of numerous concurrent mutations analysis are necessary to determine the relationship between these molecular markers.

## **5.** *RUNX1* **mutations**

The runt-related transcription factor 1 (*RUNX1*) *g*ene is located on chromosome 21q22 and it consists of 10 exons. RUNX family proteins were found to have an essential role in the regulation of gene expression by temporal transcriptional repression and epigenetic silencing via chromatin alterations, especially in the context of chromosomal translocations. The protein encoded by *RUNX1* gene represents the alpha subunit of the core binding factor (CBF) and is found to be involved in the development of normal hematopoiesis. CBF is a heterodimeric transcription factor that binds to the core element of many enhancers and promoters [86]. RUNX1 protein consists of runt homology domain, transcription activation domain, and repression domain. The runt homology domain is a highly conserved protein motif, it is responsible for both DNA binding and heterodimerization with the beta-subunit of CBF. The transcription activation domain is responsible for the interaction with a transcription coacti‐ vator of RUNX1 [87]. The *RUNX1* gene is part of the t(8;21) fusion gene in CBF AML and is also affected by recurrent gene mutations in AML. The *RUNX1* gene is one of the most frequently deregulated genes in leukemia.

The reported incidence of *RUNX1* mutation in AML varied from 3% to 46% depending on the patient population selected, the regions of *RUNX1* screened, and the methods used [88-90]. The role of *RUNX1* mutation in the leukemogenesis of AML remains to be defined. *RUNX1* mutation, a class II mutation, has been implicated as the initiating event to block differentiation of hematopoietic cells, and the subsequent class I gene mutation would synergistically provide growth advantages of these cells and lead to the development of AML [88]. Most of *RUNX1* mutated patients concurrently had other gene mutations and the majority simultaneously showed class I mutations, most commonly *FLT3/ITD*, *FLT3/TKD*, and *N-RAS*, which might result in hyperactivation of the receptor tyrosine kinase-RAS signaling pathways [88]. Interestingly, Tang et al. reported high coincidence of *RUNX1* mutations with *MLL/PTD*, and both *FLT3/ITD* and *FLT3/TKD* mutations [88].

*RUNX1* mutations in AML are associated with poor outcomes, which contrast with the favorable prognostic effect of gene fusions involving *RUNX1* [88, 90]*.* Differential prognostic value of chromosomal damage and mutation in *RUNX1* consequences the importance of a complete assessment of genetic factors in the pathogenesis of AML.

*RUNX1* mutations are less frequent in cytogenetic high-risk AML and rarely occur in CBF-AML and APL. Among intermediate-risk AML, *RUNX1* mutations are mostly associated with normal karyotype, with trisomy 8, and with trisomy 13 [90, 91]. With regard to the correlation with other molecular makers, in some studies, a higher frequency of coincidence of *FLT3* mutation and *RUNX1* mutations was reported [88, 92]. But data from Dicker et al. did not confirm that [89, 91]. Significant correlation of *RUNX1* mutations with *MLL*-PTD and *IDH* mutations and an inverse correlation with *NPM1* and *CEBPA* mutations was observed in a large cohort of AML patients [90]. Recently, rare coexistence of *RUNX1* and *NPM1* mutations in *de novo* intermediate risk karyotype AML was reported [93, 94]. In the first study, it was found that *RUNX1* mutations in these cases were structurally unusual when compared to *RUNX1* mutations observed in *NPM1* wild-type cases and located outside the *RUNX1* homology domain and were also present in the germline. However, later study did not confirm structurally unusual *RUNX1* mutations in *NPM1* mutated cases. These data could suggest that *FLT3*, *RUNX1*, *MLL*-PTD, and *IDH* mutations contribute to leukemogenesis by other mecha‐ nisms than do *NPM1* and *C/EBPα* mutations.

## **6.** *RAS* **mutations**

present among C/EBPα-dm cases [68, 78, 79]. Recently, the mutation in transcription factor

The prognostic impact of *C/EBPα* mutations seems to be favorable. The most significant effect of mutation on clinical outcome is its association with better relapse-free survival or OS [80-84]. Recent data show that a favorable outcome is limited to double, not to single, *C/EBPα* mutations [71, 78]. These data suggest that only the *C/EBPα*-dm AML should be definitely designated as AML with the favorable risk of molecular abnormalities [84]. These results have important implications for the application of risk-stratified therapy and require confirmation. Given the evidence of the prognostic value of *C/EBPα* mutations, analysis of a possible interaction between *FLT3-*ITD and *C/EBPα* mutations is of particular interest. There are contradictory studies whether coexisting *FLT3*-ITD adversely affects the favorable prognosis of *C/EBPα* mutations. Some studies showed significantly worsened prognostic outcome in patients with *FLT3-*ITD and *C/EBPα* mutations [82, 85]. In contrast, in other study negative prognostic influence among patients with *C/EBPα* mutations were not found [81]. Obviously, further studies of numerous concurrent mutations analysis are necessary to determine the relationship

The runt-related transcription factor 1 (*RUNX1*) *g*ene is located on chromosome 21q22 and it consists of 10 exons. RUNX family proteins were found to have an essential role in the regulation of gene expression by temporal transcriptional repression and epigenetic silencing via chromatin alterations, especially in the context of chromosomal translocations. The protein encoded by *RUNX1* gene represents the alpha subunit of the core binding factor (CBF) and is found to be involved in the development of normal hematopoiesis. CBF is a heterodimeric transcription factor that binds to the core element of many enhancers and promoters [86]. RUNX1 protein consists of runt homology domain, transcription activation domain, and repression domain. The runt homology domain is a highly conserved protein motif, it is responsible for both DNA binding and heterodimerization with the beta-subunit of CBF. The transcription activation domain is responsible for the interaction with a transcription coacti‐ vator of RUNX1 [87]. The *RUNX1* gene is part of the t(8;21) fusion gene in CBF AML and is also affected by recurrent gene mutations in AML. The *RUNX1* gene is one of the most

The reported incidence of *RUNX1* mutation in AML varied from 3% to 46% depending on the patient population selected, the regions of *RUNX1* screened, and the methods used [88-90]. The role of *RUNX1* mutation in the leukemogenesis of AML remains to be defined. *RUNX1* mutation, a class II mutation, has been implicated as the initiating event to block differentiation of hematopoietic cells, and the subsequent class I gene mutation would synergistically provide growth advantages of these cells and lead to the development of AML [88]. Most of *RUNX1* mutated patients concurrently had other gene mutations and the majority simultaneously showed class I mutations, most commonly *FLT3/ITD*, *FLT3/TKD*, and *N-RAS*, which might

*GATA2* was found to have a strong association with *C/EBPα*-dm mutation [75].

between these molecular markers.

frequently deregulated genes in leukemia.

**5.** *RUNX1* **mutations**

82 Leukemias - Updates and New Insights

The *RAS* oncogene family was the first human oncogene discovered in human cancer and has been extensively studied over the last 3 decades. RAS gene is named for "rat sarcoma." The *RAS* gene family is comprised of three homologues, *HRAS* (11p15.5), *KRAS* (12p12.1), and *NRAS* (1p13.2). The members of the RAS family are tyrosine kinase receptors that are important participants of many signaling pathways connected with functional control of a large variety of cellular effects including cell cycle progression, growth, migration, cytoskeletal changes, apoptosis, and senescence. The crosstalk between these multiple signaling pathways and others controlled by different sets of signaling molecules creates molecular networks whose balance is crucial to determine the final outcome of cellular responses in the cell [95, 96]. RAS proteins function as a conduit for signals received from RTK on the cell surface through downstream cell signaling partners to nuclear transcription factors regulating cell growth and cell-cycling proteins [97]. Under physiologic conditions, RAS activation is initiated by binding with ligand that induces RTK autophosphorylation, dimerization, and activation [98].

Mutations in *RAS* genes are frequent in AML and exemplify mutation Class I, initiating key downstream hyperproliferative signal transduction pathways. *NRAS* mutations are the most common. *NRAS* and *KRAS* mutations are present in about 25% and 15% of AML patients [52]. Constitutive activation of RAS originates from mutations in *RAS* itself or from mutation or overexpression of related RTK such as *FLT3* or *KIT*. Activated NRAS signals get through the several pathways to mediate oncogenic effects, especially the MAPK, PI3K–AKT, and Ral– GDS pathways [97, 98]. In contrast to other gene mutations frequently involved in AML, *NRAS* mutations are present much more often in patients with myelodysplastic syndromes (MDS) and secondary AML (sAML) arising from MDS. An analysis of samples from MDS patients and sAML identified only a modest increase in the frequency of *NRAS* mutations in the sAML cohort compared with the MDS group, suggesting that *NRAS* mutations may be an early event in MDS [89].

Despite being initially described almost 30 years ago, the prognostic implications of RAS mutations remain controversial. Several studies indicate that *RAS* mutation did not impact prognosis in CN AML patients [99, 100]. In contrast, *RAS* mutations have been linked to an inferior outcome in AML by some researchers [101]. In childhood AML, activating *NRAS* mutations commonly in cooperation with *NPM1* mutations occur frequently in the favorable risk population [102].

No association *NRAS* mutations with cytogenetic alterations have been identified. *NRAS* are similarly distributed among the major cytogenetic groups [102]. *NRAS* has been previously found to correlate with abnormalities of chromosomes 3 and 16 [100, 103]. However, this was not confirmed in the next study [102]. *RAS* mutations tend to occur together with *NPM1* mutations, while coexistence of *RAS* mutations with *FLT3-ITD*, *CEBPA*, or *WT1* appears to be less common [99, 102].

## **7.** *KIT* **(CD117) mutations**

The *KIT* gene is located on chromosome 4q12 and encodes transmembrane glycoprotein that belongs to a family of the type III RTK. The structure of RTK consists of five immunoglobulinlike domains in the extracellular portion of the receptor, a transmembrane and juxtamembrane domain, and an intracellular kinase domain [104]. The KIT protein is found in the cell mem‐ brane and binds with ligand. This binding activates the KIT protein, which then activates other proteins inside the cell by adding a phosphate group at specific positions. This phosphoryla‐ tion leads to the activation of a series of proteins in multiple signaling pathways. The signaling pathways stimulated by the KIT control cell growth, proliferation, survival, and migration of cells [104, 105]. The majority of stem cells in the bone marrow express CD117. KIT expression and intensity on normal blast cells in the bone marrow decrease during maturation as a strong negative regulation during hematopoiesis. *KIT* is expressed on the surface of leukemic blasts in 80% of AML patients.

Ligand-independent activation of *KIT* can be caused by gain-of-function mutations that have been reported in core binding factor (CBF) AML [106, 107]. In cytogenetically favorable CBF-AML, which is associated with t(8;21)(q22;q22) and inv(16)/t(16;16)(q13;q22), *KIT* mutation is found most frequently within exon 17, which encodes the *KIT* activation loop in the kinase domain, and in exon 8, which encodes a region in the extracellular portion of the *KIT* receptor [104, 108, 109]. Mutations of *KIT* occur in 20%–25% of t(8;21) and in approximately 30% of inv(16) cases [106].

The clinical significance of *KIT* mutations in CBF-AML has been intensively studied. The clinical significance of c-*KIT* mutations in CBF-AML is potentially related to mutation type, patient age, and type of chromosomal translocation. Paschka et al. reported that *KIT* mutations confer higher relapse risk and adverse OS in AML with inv(16) and t(8;21) AML [106, 110]. Contrary to most published studies, in a single CBF AML group no association between c-KIT mutations and prognosis of AML was found [107]. Various further studies confirmed that *C-KIT* mutations linked to adverse outcome in patients with t(8;21) but not in inv(16)/t(16;16) AML [108, 111-113]. National Comprehensive Cancer Network (NCCN) guidelines have defined t(8;21) and inv(16) AML with *KIT* mutations as intermediate risk guidelines, whereas ELN has provided no further recommendation for those with a *KIT* mutation [9, 114].

## **8.** *TET2* **mutations**

Constitutive activation of RAS originates from mutations in *RAS* itself or from mutation or overexpression of related RTK such as *FLT3* or *KIT*. Activated NRAS signals get through the several pathways to mediate oncogenic effects, especially the MAPK, PI3K–AKT, and Ral– GDS pathways [97, 98]. In contrast to other gene mutations frequently involved in AML, *NRAS* mutations are present much more often in patients with myelodysplastic syndromes (MDS) and secondary AML (sAML) arising from MDS. An analysis of samples from MDS patients and sAML identified only a modest increase in the frequency of *NRAS* mutations in the sAML cohort compared with the MDS group, suggesting that *NRAS* mutations may be an early event

Despite being initially described almost 30 years ago, the prognostic implications of RAS mutations remain controversial. Several studies indicate that *RAS* mutation did not impact prognosis in CN AML patients [99, 100]. In contrast, *RAS* mutations have been linked to an inferior outcome in AML by some researchers [101]. In childhood AML, activating *NRAS* mutations commonly in cooperation with *NPM1* mutations occur frequently in the favorable

No association *NRAS* mutations with cytogenetic alterations have been identified. *NRAS* are similarly distributed among the major cytogenetic groups [102]. *NRAS* has been previously found to correlate with abnormalities of chromosomes 3 and 16 [100, 103]. However, this was not confirmed in the next study [102]. *RAS* mutations tend to occur together with *NPM1* mutations, while coexistence of *RAS* mutations with *FLT3-ITD*, *CEBPA*, or *WT1* appears to be

The *KIT* gene is located on chromosome 4q12 and encodes transmembrane glycoprotein that belongs to a family of the type III RTK. The structure of RTK consists of five immunoglobulinlike domains in the extracellular portion of the receptor, a transmembrane and juxtamembrane domain, and an intracellular kinase domain [104]. The KIT protein is found in the cell mem‐ brane and binds with ligand. This binding activates the KIT protein, which then activates other proteins inside the cell by adding a phosphate group at specific positions. This phosphoryla‐ tion leads to the activation of a series of proteins in multiple signaling pathways. The signaling pathways stimulated by the KIT control cell growth, proliferation, survival, and migration of cells [104, 105]. The majority of stem cells in the bone marrow express CD117. KIT expression and intensity on normal blast cells in the bone marrow decrease during maturation as a strong negative regulation during hematopoiesis. *KIT* is expressed on the surface of leukemic blasts

Ligand-independent activation of *KIT* can be caused by gain-of-function mutations that have been reported in core binding factor (CBF) AML [106, 107]. In cytogenetically favorable CBF-AML, which is associated with t(8;21)(q22;q22) and inv(16)/t(16;16)(q13;q22), *KIT* mutation is found most frequently within exon 17, which encodes the *KIT* activation loop in the kinase domain, and in exon 8, which encodes a region in the extracellular portion of the *KIT* receptor

in MDS [89].

risk population [102].

84 Leukemias - Updates and New Insights

less common [99, 102].

in 80% of AML patients.

**7.** *KIT* **(CD117) mutations**

The TET (ten–eleven translocation) protein family includes three members (TET1, TET2, and TET3) and is involved in the epigenetic regulation, in particular, responsible for demethylation. *TET2* gene located on chromosome 4q24 and catalytic activity converts 5-methylcytosine to 5 hydroxymethylcytosine in an α-ketoglutarate-dependent reaction [115]. TET proteins further oxidize 5-hydroxymethylcytosine to formylcytosine and carboxylcytosine, which are replaced by unmodified cytosines through the DNA repair machinery [115]. The data published suggests a role for the TETs in the regulation of gene expression through modification of chromatin at promoter regions [116]. The *TET* family members have two highly conserved regions, an *N*-terminal cysteine-rich domain followed by a 2-oxoglutarate -Fe(II) oxygenase characteristic double-stranded b-helix [117]. Somatic loss-of-function mutations in *TET2* gene occur in a significant proportion of patients with myeloid malignancies. In AML, TET2 mutations affect 7%–10% of the adult and 1.5%–4% of pediatric patients [118-121].

*TET2* mutations show loss-of-function phenotype [118, 122] and are anticipated to result in hypermethylation [123]. It was shown that *TET2* mutation samples display low levels of 5 hydroxymethylcytosine compared with normal controls, supporting a functional relevance of *TET2* mutation in leukemogenesis. *TET2* mutant AML displays increased promoter methyla‐ tion [123]. In addition, it was shown that *TET2* mutants do not suppress the function of the wild-type protein and hence do not show dominant negative traits [118].

Several studies based on mouse model suggested that *TET2* mutation occurs in progenitor cells, which creates a predisposition to the development of myeloid malignancy. These studies confirm the role of *TET2* mutations in the pathogenesis of myeloid malignancies. Therefore, *TET2* mutation may exist as an early event, and in cooperation with secondary mutations, drives the phenotype of the disease [124, 125].

*TET2* mutations were spread over all cytogenetic subgroups. It was reported that *TET2* alterations are associated with *NPM1* and *FLT3-*ITD mutations [119, 121, 126]. Recent data observed that *TET2* and *IDH1/2* mutations were mutually exclusive in a large, genetically annotated *de novo* AML cohort, suggesting that these lesions may be biologically redundant [123]. The high incidence of *DNMT3A* mutations in both of these groups was reported [118].

This could indicate of a cooperative mechanism through which mutations impairing DNA hydroxymethylation and DNA methylation contribute to leukemogenesis [118].

The prognostic relevance of *TET2* mutation is still not well established and remains contro‐ versial. In some studies no prognostic impact of *TET2* mutations on clinical outcome as well as in CN AML subtype was observed [121, 126]. A study of Cancer and Leukemia Group B Study with a large cohort of AML patients reported an adverse prognostic impact in the molecular favorable-risk cytogenetically CN AML group whereas there was no impact of mutation in the intermediate risk I group [120]. In a study by Chou et al., shorter OS was observed in patients with intermediate-risk cytogenetic [119]. An integrated genetic analysis revealed that mutations of *TET2* gene are associated with poor OS in intermediate risk patients, regardless of the presence of the *FLT3-ITD* mutation. Weissmann et al. also showed a negative impact of *TET2* mutations on survival in favorable risk AML patients with normal cytogenetics [127]. Most recently, the negative effect of *TET2* mutation on OS was confirmed in various risk groups in adult AML patients less than 60 years of age [118].

In addition, recently demonstrated low levels of *TET2* expression as a poor prognostic marker for patients without *TET2* or *IDH1* mutations can suggest that both loss of function mutations and low expression of *TET2* are markers of poor prognosis in AML [118]. The development of target therapies could be beneficial for these patients [118].

## **9.** *IHD1* **and** *IDH2* **mutations**

Isocitrate dehydrogenases (IDH) 1 and 2 are NADP-dependent enzymes of the citrate cycle that convert isocitrate to α-ketoglutarate. *IDH1* and *IDH2* genes encode cytoplasmic/peroxi‐ somal isocitrate dehydrogenase 1 and mitochondrial isocitrate dehydrogenase 2, respectively. These are homodimeric, NADP+-dependent enzymes that catalyze the oxidative decarboxy‐ lation of isocitrate to α-ketoglutarate (α-KG), generating NADPH from NADP+. NADPH is an important source of synthetic reducing power and has key functions in cellular detoxifica‐ tion processes. Both genes function in a crossroads of cellular metabolism, cellular defense against oxidative stress, oxidative respiration, and oxygen-sensing signal transduction [128].

Somatic mutations in *IDH1* and *IDH2* occur frequently (50%–80%) in adult glioma [129]. In *de novo* CN AML, *IDH1/2* mutations occur in 15% and in 20% in sAML [24, 123, 130]. The frequency is higher in CN AML and in elderly patients. In contrast with glioma, in AML, *IDH2* mutations occur more frequently than *IDH1* mutations, with *IDH2-*R140Q as the most common mutation [131, 132].

The typical *IDH1* mutation affects the evolutionary conserved arginine residue 132 (*IDH1* R132) and the analogous amino acids 172 (*IDH2* R172) and 140 (*IDH2* R140) of the *IDH2* gene [133]. A great variety of *IDH1/2* mutants were reported (*IDH1-*R132, *IDH2-*R140, *IDH2-*R172 and their variants). All of them possess varying enzymatic properties. The most common *IDH1/2* mutants in AML, *IDH2* R140Q, and another mutant *IDH1-*R132H are weak *D*-2HG producers, as compared with other variants. Different mutant, *IDH1* R132C, produces relatively high levels of *D*-2HG [129]. It has been hypothesized that in oncogenesis, the intracellular *D*-2HG concentration that gives the largest growth advantage varies depending on the tumor's cell type of origin. This could explain why each type of cancer has a specific *IDH1/2* mutation. In addition, an *IDH1/2* mutation and the subsequent high *D*-2HG levels may affect which specific type of cancer is being formed [129, 134]. Acquired somatic mutations of *IDH1* and *IDH2* genes contribute to abnormal metabolic processes. Despite these effects of 2- HG on DNA and histone methylation, there is a growing consensus that, while obviously important, IDH1/2 mutations are insufficient to drive neoplasia [134]. *IDH1* and *IDH2* missense mutations were infrequent in patients with preleukemic disorders, particularly in MDS, Paroxysmal nocturnal hemoglobinuria (PNH), and Aplastic anemia (AA) patients, suggesting that these genetic alterations may not be essential in the development and transformation of preleukemic disorders to AML [135].

In AML, *IDH1* mutations correlate with worse patient survival, whereas *IDH2-*R140Q mutations are associated with a moderately prolonged survival [136, 137]. Prognostic effect is known to depend on certain biological factors as well as a combination of cytogenetics and other mutations such as those in *FLT3* and *NPM1* [24, 130-132].

*IDH* mutations in AML are predominantly associated with CN AML and *NPM1* mutations.

*IDH1* and *IDH2* mutations are mutually exclusive of each other, or with *TET2* mutations, which suggests functional redundancy [123].

## **10.** *DNMT3A* **mutations**

observed that *TET2* and *IDH1/2* mutations were mutually exclusive in a large, genetically annotated *de novo* AML cohort, suggesting that these lesions may be biologically redundant [123]. The high incidence of *DNMT3A* mutations in both of these groups was reported [118]. This could indicate of a cooperative mechanism through which mutations impairing DNA

The prognostic relevance of *TET2* mutation is still not well established and remains contro‐ versial. In some studies no prognostic impact of *TET2* mutations on clinical outcome as well as in CN AML subtype was observed [121, 126]. A study of Cancer and Leukemia Group B Study with a large cohort of AML patients reported an adverse prognostic impact in the molecular favorable-risk cytogenetically CN AML group whereas there was no impact of mutation in the intermediate risk I group [120]. In a study by Chou et al., shorter OS was observed in patients with intermediate-risk cytogenetic [119]. An integrated genetic analysis revealed that mutations of *TET2* gene are associated with poor OS in intermediate risk patients, regardless of the presence of the *FLT3-ITD* mutation. Weissmann et al. also showed a negative impact of *TET2* mutations on survival in favorable risk AML patients with normal cytogenetics [127]. Most recently, the negative effect of *TET2* mutation on OS was confirmed in various risk

In addition, recently demonstrated low levels of *TET2* expression as a poor prognostic marker for patients without *TET2* or *IDH1* mutations can suggest that both loss of function mutations and low expression of *TET2* are markers of poor prognosis in AML [118]. The development of

Isocitrate dehydrogenases (IDH) 1 and 2 are NADP-dependent enzymes of the citrate cycle that convert isocitrate to α-ketoglutarate. *IDH1* and *IDH2* genes encode cytoplasmic/peroxi‐ somal isocitrate dehydrogenase 1 and mitochondrial isocitrate dehydrogenase 2, respectively. These are homodimeric, NADP+-dependent enzymes that catalyze the oxidative decarboxy‐ lation of isocitrate to α-ketoglutarate (α-KG), generating NADPH from NADP+. NADPH is an important source of synthetic reducing power and has key functions in cellular detoxifica‐ tion processes. Both genes function in a crossroads of cellular metabolism, cellular defense against oxidative stress, oxidative respiration, and oxygen-sensing signal transduction [128]. Somatic mutations in *IDH1* and *IDH2* occur frequently (50%–80%) in adult glioma [129]. In *de novo* CN AML, *IDH1/2* mutations occur in 15% and in 20% in sAML [24, 123, 130]. The frequency is higher in CN AML and in elderly patients. In contrast with glioma, in AML, *IDH2* mutations occur more frequently than *IDH1* mutations, with *IDH2-*R140Q as the most common

The typical *IDH1* mutation affects the evolutionary conserved arginine residue 132 (*IDH1* R132) and the analogous amino acids 172 (*IDH2* R172) and 140 (*IDH2* R140) of the *IDH2* gene [133]. A great variety of *IDH1/2* mutants were reported (*IDH1-*R132, *IDH2-*R140, *IDH2-*R172

hydroxymethylation and DNA methylation contribute to leukemogenesis [118].

groups in adult AML patients less than 60 years of age [118].

target therapies could be beneficial for these patients [118].

**9.** *IHD1* **and** *IDH2* **mutations**

86 Leukemias - Updates and New Insights

mutation [131, 132].

Somatic mutations in the *DNMT3A* have been reported approximately in 22% of *de novo* AML and 36% of CN AML [138]. Mutations in *DNMT3A* were first described by Ley et al. using whole genome sequencing [139]. *DNMT3A* belongs to the mammalian methyltransferase gene family which is responsible for tissue-specific gene expression [140]. DNA methyltransferases are the key enzymes for genome methylation, which plays an important role in epigenetically regulated gene expression and repression. *DNMT3A* together with other methyltransferase conducts *de novo* methylation of cytosine residues in CpG islands by the enzymatic addition of methyl residues from S-adenosyl-L-methionine to the 5-carbon atom of the cytosine ring. CpG islands are often located proximate to gene promoters thereby regulating their activation. Actively transcribed genes display unmethylated CpG islands that supports the euchromatin structure whereas methylated CpG islands are associated with untranscribed genes stabilizing the heterochromatin structure [141, 142]. Cancer genomes are most commonly characterized by global DNA hypomethylation. However, cancer cells also typically exhibit distinct regions of DNA hypermethylation, which are particularly well characterized in the CpG islands of promoter regions of tumor-suppressor genes. *DNMT3A* mutations are typically heterogenous. More common, mutations affect residual R882 within the methyltransferase domain [139, 143]. The biology of *DNMT3A* is not fully understood. Holz-Schietinger et al. reported that muta‐ tions in *DNMT3A* could retard its function by multiple mechanisms as changes in the catalytic properties, its processivity, and the disruption of interaction with binding partners [144]. Furthermore, Russler-Germain et al. found that mutations in the position R882 inhibit the formation of active tetramers of *DNMT3A* [145]. The impaired function of mutated *DNMT3A* leads to a hypomethylated genome of myeloid cells possibly promoting leukemogenesis and influencing disease outcome [146]. *DNMT3A* mutations are typically heterozygous. Continued expression of both mutated and wild-type *DNMT3A* in heterozygous cells, observed *in vitro*, suggests a dominant negative or possibly a neomorphic gain of function role for *DNMT3A* mutations in AML [147].

Since the *DNMT3A* mutations are present in the early preleukemic cells, this alteration seems to be a "founder" mutation, which can be implicated as functional components of AML evolution [148, 149]. *DNMT3A* mutations are highly associated with *NPM1*, *FLT3*, *IDH1*, and *IDH2* [150, 151]. Patients with *DNMT3A* mutations are typically older than average, have a higher white cell count, and are more likely to have monocytic or myelomonocytic leukemia (FAB M4/M5) [8].

Several studies reported a negative prognostic impact of *DNMT3A* mutations [150-154]. Prognostic effect is known to depend on certain biological factors as well as a combination of cytogenetics and other mutations such as those in *FLT3* and *NPM1*.

Some authors have found stability of *DNMT3A* mutations during the course of disease; therefore those aberrations could be a potential marker for minimal residual disease (MRD). Furthermore, the presence of *DNMT3A* mutations seems to be associated with the incidence of *FLT3-*ITD-positive clones at relapse possibly influencing the responsiveness of *FLT3* positive cases to chemotherapy [155, 156]. In contrast, Hou et al. reported persistence of *DNMT3A* mutations at CR in AML patients, which later achieved relapse and died of disease progression. These data could relate the persistence of *DNMT3A* mutations and high risk of relapse [156]. Recently, Pløen et al. have identified persistence of *DNMT3A* mutations in longterm remission of patients with AML that received cytoreduction or palliative therapy [157]. Using cell-sorting, the authors showed that *DNMT3A* mutations were present in T-cells and B-cells at diagnosis in some patients, and also in T-cells several years after diagnosis. The presence of *DNMT3A* in both B-cells and T-cells could lead to assumption that mutation had occurred in an early pre-leukemic stem cell prior to the acquisition of other genetic events, and could be resistant to chemotherapy [157]. Therefore, further exploration of the role of *DNMT3A* R882H mutations for the progression of AML disease is needed.

Recent discoveries utilizing whole-exome sequencing in a large cohort of persons unselected for cancer or hematologic phenotypes have demonstrated somatic mutations in significant proportion of persons particularly older than 65 years. Moreover, *DNMT3A* gene together with *TET2*, *ASXL1* (additional sex combs like transcriptional regulator 1), and *PPM1D* (Protein phosphatase 1D) had disproportionately high numbers of somatic mutations [5, 158]. The data suggest that mutations in pre-leukemic cells could precede leukemia. Furthermore, *DNMT3A* mutations could drive clonal expansions.

## **11. Conclusion**

The biology of *DNMT3A* is not fully understood. Holz-Schietinger et al. reported that muta‐ tions in *DNMT3A* could retard its function by multiple mechanisms as changes in the catalytic properties, its processivity, and the disruption of interaction with binding partners [144]. Furthermore, Russler-Germain et al. found that mutations in the position R882 inhibit the formation of active tetramers of *DNMT3A* [145]. The impaired function of mutated *DNMT3A* leads to a hypomethylated genome of myeloid cells possibly promoting leukemogenesis and influencing disease outcome [146]. *DNMT3A* mutations are typically heterozygous. Continued expression of both mutated and wild-type *DNMT3A* in heterozygous cells, observed *in vitro*, suggests a dominant negative or possibly a neomorphic gain of function role for *DNMT3A*

Since the *DNMT3A* mutations are present in the early preleukemic cells, this alteration seems to be a "founder" mutation, which can be implicated as functional components of AML evolution [148, 149]. *DNMT3A* mutations are highly associated with *NPM1*, *FLT3*, *IDH1*, and *IDH2* [150, 151]. Patients with *DNMT3A* mutations are typically older than average, have a higher white cell count, and are more likely to have monocytic or myelomonocytic leukemia

Several studies reported a negative prognostic impact of *DNMT3A* mutations [150-154]. Prognostic effect is known to depend on certain biological factors as well as a combination of

Some authors have found stability of *DNMT3A* mutations during the course of disease; therefore those aberrations could be a potential marker for minimal residual disease (MRD). Furthermore, the presence of *DNMT3A* mutations seems to be associated with the incidence of *FLT3-*ITD-positive clones at relapse possibly influencing the responsiveness of *FLT3* positive cases to chemotherapy [155, 156]. In contrast, Hou et al. reported persistence of *DNMT3A* mutations at CR in AML patients, which later achieved relapse and died of disease progression. These data could relate the persistence of *DNMT3A* mutations and high risk of relapse [156]. Recently, Pløen et al. have identified persistence of *DNMT3A* mutations in longterm remission of patients with AML that received cytoreduction or palliative therapy [157]. Using cell-sorting, the authors showed that *DNMT3A* mutations were present in T-cells and B-cells at diagnosis in some patients, and also in T-cells several years after diagnosis. The presence of *DNMT3A* in both B-cells and T-cells could lead to assumption that mutation had occurred in an early pre-leukemic stem cell prior to the acquisition of other genetic events, and could be resistant to chemotherapy [157]. Therefore, further exploration of the role of *DNMT3A*

Recent discoveries utilizing whole-exome sequencing in a large cohort of persons unselected for cancer or hematologic phenotypes have demonstrated somatic mutations in significant proportion of persons particularly older than 65 years. Moreover, *DNMT3A* gene together with *TET2*, *ASXL1* (additional sex combs like transcriptional regulator 1), and *PPM1D* (Protein phosphatase 1D) had disproportionately high numbers of somatic mutations [5, 158]. The data suggest that mutations in pre-leukemic cells could precede leukemia. Furthermore, *DNMT3A*

cytogenetics and other mutations such as those in *FLT3* and *NPM1*.

R882H mutations for the progression of AML disease is needed.

mutations could drive clonal expansions.

mutations in AML [147].

88 Leukemias - Updates and New Insights

(FAB M4/M5) [8].

The whole gene analysis has revealed that leukemic cell carry hundreds of mutated genes. Most of them are "passenger" mutations, which do not provide a selective advantage, and a less number of mutations are "driver" mutations. The latter can cause the tumor. The simul‐ taneous presence of genetic alterations with different functional effects on hematopoietic progenitors led to the concept of leukemogenesis as a multi-step process that ultimately gives rise to malignant transformation. Evidence from many murine models confirmed that a single genetic change is not sufficient for the occurrence of AML. Moreover, two modern studies have demonstrated that somatic mutations that drive clonal expansion of blood cells were a common finding in the elderly and most frequently involved *DNMT3A*, *TET2*, or *ASXL1*. The agerelated clonal hematopoiesis is a common premalignant condition that is also associated with increased overall mortality [5, 158]. Overall this knowledge has provided useful elements to stratify AML patients into different subgroups, resulting in better prognosis and therapy.

## **Author details**

Olga Blau\*

Address all correspondence to: olga.blau@charite.de

Charité Universitätsmedizin Berlin, Department of Hematology, Oncology and Tumourimmunology, Berlin, Germany

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**Chapter 5**
