**3.1 Images ALL**

*Geriatric Medicine and Gerontology*

**3. Morphologic diagnosis**

single genetic rearrangement is not enough to induce leukemia. Cooperative mutations are necessary for leukemic transformation and include genetic and epigenetic changes in regulatory growth pathways. Candidate genes identified include deletion of the tumor suppressor locus CDKN2A/CDKN2B and NOTCH1 mutations in T cells. The use of single nucleotide polymorphism (SNP) microarrays suggests that genomic instability is not characteristic of most cases. There is a great variation in the number of alterations in different subtypes of leukemia. The infant cases with rearrangements of the MLL gene had less than one copy number alterations (CNA) per case, suggesting that few genetic lesions are required. Conversely, cases with ETV6-RUNX1 [25] and BCR-ABL1 had more than six CNAs, some containing more than 20 lesions, which support the concept that despite the initiating events that may occur in early infancy, additional lesions are required for the subsequent development of ALL. The lymphoid transcription factor PAX5 encodes a protein involved in evolution and fidelity of the B-cell lineage. The second most frequently affected gene was IKZF1, which encodes the protein IKAROS, required for lymphoid differentiation. IKZF1 is absent in most cases with BCR-ABL1. Approximately, half of the patients expressing BCRABL1 also had deletions in CDKN2A/B and PAX5. This finding suggests that alterations in different signaling pathways are needed to induce leukemia [15]. A special role in this disease is played by the presence of the Philadelphia chromosome t (9; 22), which expresses the BCR-ABL fusion gene, and

this has diagnostic, prognostic, and therapeutic implications [3, 6–11].

classification identifies three types of ALL [7, 8, 12].

The bone marrow aspiration test is fundamental to confirm the presence of lymphoblasts (by morphology and/or cytochemistry with special stains that include a negative MPO in 100% of cells, Periodic Acid-Shiff (PAS) (+) in 70–80%, and acid phosphatase (+) in the case of T lymphoblast). The WHO suggests greater than 20% as diagnosis criteria (if the percentage is lower, one must search for extramedullary disease at the nodal level to differentiate from the diagnosis of lymphoblastic lymphoma). The bone marrow aspiration is hypercellular 95–100% of the time; however, in those cases where the aspirate is "dry" (packed bone marrow), which corresponds to 1–2% of the cases, a bone biopsy must be carried out for histopathological confirmation. Based on morphology, the French-American-British (FAB)

The first step to integrate the diagnosis of ALL is the morphological identification of lymphoblasts. For this, it is necessary to perform a bone marrow aspirate and be observed directly under a microscope by an expert in hematology, which can be supported in other tests like special stains, as in the case of myeloperoxidase, which must be negative in all the malignant cells observed; PAS staining, which is considered

**138**

The French-American-British (FAB) classification that was used commonly earlier includes:

	- regular nuclear shape
	- homogeneous chromatin
	- small or absent nucleolus
	- scanty cytoplasm
	- irregular nuclear shape
	- heterogeneous chromatin
	- large nucleolus

In an initial effort, the French-American-British (FAB) was given the task of subclassifying this type of leukemia according to various morphological characteristics in order to try to determine the behavior and prognosis of each type based on its morphology; this is how the FAB morphological classification was born, which subdivides the ALL into three types:


• L3: the least frequent of the three, is reported between 1 and 2% of the time. Its main characteristic is the large number of vacuoles (bubbles) that these cells present in their cytoplasm. The shape of the nucleus may vary.

#### **3.2 Revised version of FAB**

WHO proposed a classification of ALL that was to be the revised version of the FAB classification.

This used the immunophenotypic classification that includes:

	- precursor B acute lymphoblastic leukemia/lymphoma: this has genetic subtypes including t(12,21)(p12,q22) TEL/AML-1, t(1,19)(q23;p13) PBX/ E2A, t(9,22)(q34;q11) ABL/BCR and T(V,11)(V;q23) V/MLL
	- precursor T acute lymphoblastic leukemia/lymphoma

The WHO performed a new categorization of acute lymphoblastic leukemia, based on cytogenetic alterations present in this disease. This classification considered what was previously described in the FAB classification being possible to make an indirect correlation between the morphological findings and the alterations listed in the categories of the WHO classification. In this way, those leukemias that are traditionally classified in the FAB groups L1 and L2 can belong to the group of leukemia of precursors B with alterations such as: t (12; 21) (p12, q22) TEL/AML-1, t (1; 19) (q23; p13) PBX/E2A, t (9; 22) (q34; q11) ABL/BCR, and T (V, 11) (V; q23) V/MLL. Those traditionally classified as FAB L3 correlate with Burkitt's leukemia/ lymphoma; T-cell leukemias are still an independent group and are considered another group where those that meet criteria for two different lineages are included.

#### **4. Lineage**

The proportion of B-lineage ALL is higher in patients older (75–89%) than 60 years compared to patients younger (59–66%) than 60 years. Accordingly the incidence of T-ALL is lower in older (8–12%) compared to younger (29%) patients [5–7]. A population-based study showed that cytogenetics were less frequently attempted in older (73%) compared with younger (85–91%) patients. The proportion of patients with Philadelphia chromosome positive (Ph+) t(9;22), t(8;14), t(14;18), or complex aberrations increased with age [11]; Ph+ ALL accounted for 24–36% in older patients vs. 15–19% in younger patients. Considering the consequences resulting from diagnostic characterization, it should be self-evident that complete diagnostic characterization is required in all patients with ALL, regardless of age [13, 14].

There are several important differences in the biology of lymphoblastic leukemia in patients over 60 years compared to those under this age, although we know that B-lineage leukemia is the most common in adults, the frequency between both groups can vary reporting a little more frequent in those over 60 years (75–89%/59–66%), another more radical difference is the presentation of leukemia of T lineage, which is

**141**

*Overview and Current News in Acute Lymphoblastic Leukemia*

in randomized trials, and it may improve the outcome [15, 16].

Blasts in pre-B ALL can be initially identified using an SSC vs. a CD45 plot. These blasts have low SSC (many times smaller than normal lymphocytes) and dim

Once the blasts are identified and gated, the following markers are useful in the

**Marker Prevalence** CD10 89% CD13 5% CD19 100% CD20 24% CD22 69% CD33 31% CD34 76%

more common in adults under 60 years (29%) than in elderly patients (12%) [5–7]. Cytogenetic alterations of importance for the prognosis, such as Philadelphia chromosome (Ph+) t (9; 22), t (8; 14), t (14; 18), or complex karyotype are observed more frequently as the patient's age increases [11]. Although the search for cytogenetic alterations is crucial to define the risk and possible response to treatment of acute leukemia, this analysis is not carried out in most elderly patients (73%), contrary to the young patients, who have available cytogenetic studies in up to 91%. The importance of this difference lies in the fact, already mentioned, of the increase in the frequency of highrisk alterations, as an example Ph+ ALL can be found in up to 36% of cases, which have different therapeutic approaches to those that do not suffer from this alteration [13, 14]. As in other B-cell malignancies, monoclonal antibodies to CD20 or CD228 are being tested as adjuncts to chemotherapy in the hope that they will increase remission depth and improve survival without increasing hematologic toxicity. About 60–80% of B-cell ALL patients express these antigens at variable densities, but there is little evidence linking antigen expression to response. CD20 expression may be associated with a worse prognosis, so it is logical to investigate CD20 antibodies

*DOI: http://dx.doi.org/10.5772/intechopen.86662*

**4.1 Immunophenotyping**

classification of pre-B ALL:

to negative CD45.

*Overview and Current News in Acute Lymphoblastic Leukemia DOI: http://dx.doi.org/10.5772/intechopen.86662*

more common in adults under 60 years (29%) than in elderly patients (12%) [5–7]. Cytogenetic alterations of importance for the prognosis, such as Philadelphia chromosome (Ph+) t (9; 22), t (8; 14), t (14; 18), or complex karyotype are observed more frequently as the patient's age increases [11]. Although the search for cytogenetic alterations is crucial to define the risk and possible response to treatment of acute leukemia, this analysis is not carried out in most elderly patients (73%), contrary to the young patients, who have available cytogenetic studies in up to 91%. The importance of this difference lies in the fact, already mentioned, of the increase in the frequency of highrisk alterations, as an example Ph+ ALL can be found in up to 36% of cases, which have different therapeutic approaches to those that do not suffer from this alteration [13, 14].

As in other B-cell malignancies, monoclonal antibodies to CD20 or CD228 are being tested as adjuncts to chemotherapy in the hope that they will increase remission depth and improve survival without increasing hematologic toxicity. About 60–80% of B-cell ALL patients express these antigens at variable densities, but there is little evidence linking antigen expression to response. CD20 expression may be associated with a worse prognosis, so it is logical to investigate CD20 antibodies in randomized trials, and it may improve the outcome [15, 16].

#### **4.1 Immunophenotyping**

*Geriatric Medicine and Gerontology*

**3.2 Revised version of FAB**

types including:

FAB classification.

• L3: the least frequent of the three, is reported between 1 and 2% of the time. Its main characteristic is the large number of vacuoles (bubbles) that these cells

WHO proposed a classification of ALL that was to be the revised version of the

• Acute lymphoblastic leukemia/lymphoma or formerly L1 and L2 this has sub-

○ precursor B acute lymphoblastic leukemia/lymphoma: this has genetic subtypes including t(12,21)(p12,q22) TEL/AML-1, t(1,19)(q23;p13) PBX/

The WHO performed a new categorization of acute lymphoblastic leukemia, based on cytogenetic alterations present in this disease. This classification considered what was previously described in the FAB classification being possible to make an indirect correlation between the morphological findings and the alterations listed in the categories of the WHO classification. In this way, those leukemias that are traditionally classified in the FAB groups L1 and L2 can belong to the group of leukemia of precursors B with alterations such as: t (12; 21) (p12, q22) TEL/AML-1, t (1; 19) (q23; p13) PBX/E2A, t (9; 22) (q34; q11) ABL/BCR, and T (V, 11) (V; q23) V/MLL. Those traditionally classified as FAB L3 correlate with Burkitt's leukemia/ lymphoma; T-cell leukemias are still an independent group and are considered another group where those that meet criteria for two different lineages are included.

The proportion of B-lineage ALL is higher in patients older (75–89%) than 60 years compared to patients younger (59–66%) than 60 years. Accordingly the incidence of T-ALL is lower in older (8–12%) compared to younger (29%) patients [5–7]. A population-based study showed that cytogenetics were less frequently attempted in older (73%) compared with younger (85–91%) patients. The proportion of patients with Philadelphia chromosome positive (Ph+) t(9;22), t(8;14), t(14;18), or complex aberrations increased with age [11]; Ph+ ALL accounted for 24–36% in older patients vs. 15–19% in younger patients. Considering the consequences resulting from diagnostic characterization, it should be self-evident that complete diagnostic characterization is required in all patients with ALL, regardless of age [13, 14].

There are several important differences in the biology of lymphoblastic leukemia in patients over 60 years compared to those under this age, although we know that B-lineage leukemia is the most common in adults, the frequency between both groups can vary reporting a little more frequent in those over 60 years (75–89%/59–66%), another more radical difference is the presentation of leukemia of T lineage, which is

E2A, t(9,22)(q34;q11) ABL/BCR and T(V,11)(V;q23) V/MLL

○ precursor T acute lymphoblastic leukemia/lymphoma

• Burkitt's leukemia/lymphoma or formerly L3

• biphenotypic acute leukemia

present in their cytoplasm. The shape of the nucleus may vary.

This used the immunophenotypic classification that includes:

**140**

**4. Lineage**

Blasts in pre-B ALL can be initially identified using an SSC vs. a CD45 plot. These blasts have low SSC (many times smaller than normal lymphocytes) and dim to negative CD45.

Once the blasts are identified and gated, the following markers are useful in the classification of pre-B ALL:



Included are marking prevalences.

The phenotype of the blasts is an independent prognostic parameter. B-ALL is subdivided into following:


#### **4.2 Immunophenotype of T-lineage ALL**

T-cell ALL constitutes approximately 25% of all adult cases of ALL. T-cell markers are CD1a, CD2, CD3 (membrane and cytoplasm), CD4, CD5, CD7, and CD8.

**143**

**4.4 NK cell ALL**

**4.5 Diagnostic cytogenetics**

*Overview and Current News in Acute Lymphoblastic Leukemia*

and expression of at least one myeloid and/or stem cell marker [17].

overall better following ALL rather than AML therapy [7, 16, 18, 19].

CD56, a marker of natural killer (NK) cell differentiation, defines a rare subgroup of about 3% of adult ALL cases, which often display other early T-cell antigens, CD7 CD2 CD5, and sometimes cCD3. True NK ALL is very rare (TdT+, CD56+, other T markers negative, and un-rearranged TCR genes). This diagnosis rely on the demonstration of early NK-specific CD94 or CD161 antigens [18, 19].

Cytogenetics represents an important step in ALL classification. Conventional karyotyping can be helpful in the identification of recurrent translocations, as well as gain and loss of gross chromosomal material; however, the major limitation of this technique is that in some cases, leukemic cells fail to enter metaphase. However, fluorescence in situ hybridization (FISH) can enable the detection and direct visualization of virtually all investigated chromosomal abnormalities in ALL, with a sensitivity of around 99%. Finally, array-comparative genomic hybridization (array-CGH, a-CGH) and single nucleotide polymorphisms (SNP) arrays can permit the identification of cryptic and/or submicroscopic changes in the genome. Karyotype changes found in ALL include both numerical and structural alterations, which have profound prognostic significance. With these premises in mind, the

CD2, CD5, and CD7 antigens are markers of the most immature T cells , but none of them is absolutely lineage-specific, so that the unequivocal diagnosis of T-ALL rests on the demonstration of surface/cytoplasmic CD3. In T-ALL, the expression of CD10 is quite common (25%) and not specific; CD34 and myeloid antigens CD13 and/or CD33 can be expressed too. Recognized T-ALL subsets are the following: pro-T EGIL T-I (cCD3+, CD7+), pre-T EGIL T-II (cCD3+, CD7+, and CD5/CD2+), cortical T EGIL T-III (cCD3+, CD1a+, and sCD3+/−), and mature-T EGIL T-IV (cCD3+, sCD3+, and CD1a−). Finally, a novel subgroup that was recently characterized is represented by the so-called ETP-ALL (early-T precursor), which shows characteristic immunophenotypic features, namely lack of CD1a and CD8 expression, weak CD5 expression,

With currently refined diagnostic techniques, the occurrence of acute leukemia of ambiguous cell lineage, i.e., mixed phenotype acute leukemia (MPAL) is relatively rare (<4%) [19]. These cases express one of the following feature: (1) coexistence of two separate blast cell populations (i.e., T- or B-cell ALL plus either myeloid or monocytic blast cells), (2) single leukemic population of blast cells co-expressing B- or T-cell antigens and myeloid antigens, and (3) same plus expression of monocytic antigens. For myelo-monocytic lineage, useful diagnostic antigens are MPO or nonspecific esterase, CD11c, CD14, CD64 and lysozyme; for B-lineage, CD19 plus CD79a, cytoplasmic CD22 and CD10 (one or two of the latter according to staining intensity of CD19); and for T-lineage, cytoplasmic or surface CD3. Recognized entities include Ph+ MPAL (B/myeloid or rarely T/ myeloid), t(v;11q23); MLL rearranged MPAL, and genetically uncharacterized B or T/myeloid MPAL. Very rare cases express trilineage involvement (B/T/myeloid). Lack of lineage-specific antigens (MPO, cCD3, cCD22) is observed in the ultrarare acute undifferentiated leukemia. In a recent review of 100 such cases, 59% were B/ myeloid, 35% T/myeloid, 4% B/T lymphoid, and 2% B/T/myeloid. Outcome was

*DOI: http://dx.doi.org/10.5772/intechopen.86662*

**4.3 Mixed phenotype acute leukemia**

*Overview and Current News in Acute Lymphoblastic Leukemia DOI: http://dx.doi.org/10.5772/intechopen.86662*

*Geriatric Medicine and Gerontology*

Included are marking prevalences.

• *Early Pre-B ALL*: TdT+, CD19+, CD10-

• *Common ALL*: CD19+, CD10+/CALLA+

**4.2 Immunophenotype of T-lineage ALL**

• *Pre-B ALL*: CD10+/−, CD19+, HLA DR+, cytoplasmic IgM+

• *Mature B ALL*: CD10+, CD19+, CD20+, CD22+, surface IgM+

subdivided into following:

The phenotype of the blasts is an independent prognostic parameter. B-ALL is

**Marker Prevalence** CD45 (bright) 2% CD45 (moderate) 33% CD45 (dim) 36% CD45 (negative) 29% CD56 36% CD79a 88% CD117 0% Cytoplasmic IgM 22% HLA Dr 98% TdT 91%

T-cell ALL constitutes approximately 25% of all adult cases of ALL. T-cell markers are CD1a, CD2, CD3 (membrane and cytoplasm), CD4, CD5, CD7, and CD8.

**142**

CD2, CD5, and CD7 antigens are markers of the most immature T cells , but none of them is absolutely lineage-specific, so that the unequivocal diagnosis of T-ALL rests on the demonstration of surface/cytoplasmic CD3. In T-ALL, the expression of CD10 is quite common (25%) and not specific; CD34 and myeloid antigens CD13 and/or CD33 can be expressed too. Recognized T-ALL subsets are the following: pro-T EGIL T-I (cCD3+, CD7+), pre-T EGIL T-II (cCD3+, CD7+, and CD5/CD2+), cortical T EGIL T-III (cCD3+, CD1a+, and sCD3+/−), and mature-T EGIL T-IV (cCD3+, sCD3+, and CD1a−). Finally, a novel subgroup that was recently characterized is represented by the so-called ETP-ALL (early-T precursor), which shows characteristic immunophenotypic features, namely lack of CD1a and CD8 expression, weak CD5 expression, and expression of at least one myeloid and/or stem cell marker [17].

#### **4.3 Mixed phenotype acute leukemia**

With currently refined diagnostic techniques, the occurrence of acute leukemia of ambiguous cell lineage, i.e., mixed phenotype acute leukemia (MPAL) is relatively rare (<4%) [19]. These cases express one of the following feature: (1) coexistence of two separate blast cell populations (i.e., T- or B-cell ALL plus either myeloid or monocytic blast cells), (2) single leukemic population of blast cells co-expressing B- or T-cell antigens and myeloid antigens, and (3) same plus expression of monocytic antigens. For myelo-monocytic lineage, useful diagnostic antigens are MPO or nonspecific esterase, CD11c, CD14, CD64 and lysozyme; for B-lineage, CD19 plus CD79a, cytoplasmic CD22 and CD10 (one or two of the latter according to staining intensity of CD19); and for T-lineage, cytoplasmic or surface CD3. Recognized entities include Ph+ MPAL (B/myeloid or rarely T/ myeloid), t(v;11q23); MLL rearranged MPAL, and genetically uncharacterized B or T/myeloid MPAL. Very rare cases express trilineage involvement (B/T/myeloid). Lack of lineage-specific antigens (MPO, cCD3, cCD22) is observed in the ultrarare acute undifferentiated leukemia. In a recent review of 100 such cases, 59% were B/ myeloid, 35% T/myeloid, 4% B/T lymphoid, and 2% B/T/myeloid. Outcome was overall better following ALL rather than AML therapy [7, 16, 18, 19].

#### **4.4 NK cell ALL**

CD56, a marker of natural killer (NK) cell differentiation, defines a rare subgroup of about 3% of adult ALL cases, which often display other early T-cell antigens, CD7 CD2 CD5, and sometimes cCD3. True NK ALL is very rare (TdT+, CD56+, other T markers negative, and un-rearranged TCR genes). This diagnosis rely on the demonstration of early NK-specific CD94 or CD161 antigens [18, 19].

#### **4.5 Diagnostic cytogenetics**

Cytogenetics represents an important step in ALL classification. Conventional karyotyping can be helpful in the identification of recurrent translocations, as well as gain and loss of gross chromosomal material; however, the major limitation of this technique is that in some cases, leukemic cells fail to enter metaphase. However, fluorescence in situ hybridization (FISH) can enable the detection and direct visualization of virtually all investigated chromosomal abnormalities in ALL, with a sensitivity of around 99%. Finally, array-comparative genomic hybridization (array-CGH, a-CGH) and single nucleotide polymorphisms (SNP) arrays can permit the identification of cryptic and/or submicroscopic changes in the genome. Karyotype changes found in ALL include both numerical and structural alterations, which have profound prognostic significance. With these premises in mind, the

karyotype changes that occur in ALL can be roughly subdivided in those associated, respectively, with a relatively good, intermediate, and poor prognosis. However, it must be kept in mind that the incidence of certain aberrations is very low, and that for some of them, the prognostic impact can be strongly affected by the type and intensiveness of therapy administered [8, 20].

#### **5. Clinical status**

Features associated with large tumor mass or rapid progression, such as high white blood cell count, mediastinal tumors, or other organ involvement, appear to be less common in older patients. Even "smoldering" ALL is observed in some cases. Most studies report a lower proportion of males among older ALL patients. Secondary ALL after myelodysplastic syndromes or other malignant disease may become increasingly important, particularly in older patients; so far, very limited data are available. Performance status often deteriorates in older patients with onset of disease. In two studies, 30–43% of patients older than age 60 years vs. 18–22% of younger patients had a performance status of 2 or more. Therefore, it is important not only to consider the current general condition in newly admitted older ALL patients but also to discern their status before the onset of leukemia-associated symptoms [17, 21].

The determination of the clinical status at the moment of making the diagnosis provides us with information about the global state of the patient, so that we can make better decisions. This varies in comparison with the younger groups in questions such as the low initial presentation of large tumor mass, identified by the elevated white blood cells count in the peripheral blood, the rare extranodal affection and even in some cases being observed, apparently "benign" clinical presentation with low tumor burden. A smaller proportion of male patients in this group have also been observed as compared with younger groups. Secondary leukemia, which we define as that which occurs after a premalignant pathology, most frequently myelodysplastic syndrome, or after treatment of nonhematological neoplasms, is a condition that has been observed more and more frequently in recent years. However, there is little data to help us determine its nature. It is important to assess these patients comprehensively in order to determine their physical and health status prior to the onset of symptoms related to leukemia [17, 21].
