**4. Results**

In the period of twelve months, between January and December 2008, 77 patients were submitted and tested for acute leukemia at the University Clinic of Hematology-Skopje. Cyto-morphological analyses showed that the average rate of the blast cells in the differential blood counts and in the bone marrow was 54.6%(3-99.0%) and 73,5%(20-98.0%) respectively. In 7 patients blast cells were not detected initially.

### **4.1 Results from the cytochemical analyses**

488 Advances in Cancer Therapy

Sequence

ENR761 ETO, 318–297 (22) 5'–ATC CAC AGG TGA GTC TGG CAT T–3'

ENF803 CBFB, 389–410 (22) 5'–CAT TAG CAC AAC AGG CCT TTG A–3'

ENPr843 CBFB, 434–413 (22) FAM 5'–TCG CGT GTC CTT CTC CGA GCC T–3'

ENF1043 ABL, 467–494 (28) FAM 5'–CCA TTT TTG GTT TGG GCT TCA CAC CAT

T–3' TAMRA

In the period of twelve months, between January and December 2008, 77 patients were submitted and tested for acute leukemia at the University Clinic of Hematology-Skopje. Cyto-morphological analyses showed that the average rate of the blast cells in the differential blood counts and in the bone marrow was 54.6%(3-99.0%) and 73,5%(20-98.0%)

TAMRA ABL ENF1003 ABL, 372–402 (31) 5'-TGGAGATAACACTCTAAGCATAACTAAAGGT–3' ENF1063 ABL, 495–515 (21) 5'–GATGTAGTTGCTTGGGACCCA–3'

ACT CCA–3' TAMRA

5'–CAC CTA CCA CAG AGC CAT CAA A–3'

5'–CCG ATG GCT TCG ACG AGT T–3'

5'–ACC TGG ATG GAC CGC CTA G–3'

5'–TCT TCC TGC CCA ACA GCA A–3'

5'–AGG GCC CGC TTG GAC TT–3'

5'–CCT CGT TAA GCA TCC CTG TGA–3'

5'–CTC TTT CTC CAG CGT CTG CTT AT–3'

5'–GCT TGT AGA TGC GGG GTA GAG–3'

FAM 5'–AGT GCC CAG CCC TCC CTC GC–3' TAMRA

FAM 5'–AAC CTC GAA ATC GTA CTG AGA AGC

Transcript EAC codea Primer/probe

AML1 /ETO

PML /RARα

CBF /MYH11

**4. Results**

localization, 5'–'3' position (size)

ENF701 AML1, 1005–1026 (22)

ENP747 AML1, 1049–295 (30)

ENF905 PML, 1198–1216 (19)

ENF906 PML, 1642–1660 (19)

ENF903 PML, 1690–1708 (19)

ENR962 RARA, 485–465 (21)

ENR942 RARA, 439–458 (20)

ENR862 MYH11, 1952–1936 (17)

ENR863 MYH11, 1237–1217 (21)

ENR865 MYH11, 1038–1016 (23)

aENF = forward primer, ENR = reverse primer, ENP = TaqMan probe

respectively. In 7 patients blast cells were not detected initially.

Table 2. Sequences and positions of the RQ-PCR primers and probes

Results from the cytochemical analyses and the images of peripheral smears with positive examples from different cytochemical staining are presented at Figure 2 and Photo 1 respectively.

B

Fig. 2. Distribution of patients according to reactivity with different cytochemial staining. A:Patient distribution according to MPO reactivity of the blasts cells. B: Patient distribution according to PAS reactivity of the blasts cells. C: Patient distribution according to NSE reactivity of the blasts cells.

Immunophenotyping of the Blast Cells in Correlations with

morphological and cytochemial analyses

detected in 28.5% of the cases.

**4.2 Results from the immunological analyses** 

the Molecular Genetics Analyses for Diagnostic and Clinical Stratification of Patients… 491

observed (X2 with Yates's correction=2.456 p=0.117), expect for AML M4 and M5 entities

Morphology and cytochemistry established myeloid lineage in 57 (74.0%) cases and lymphoid differentiation in 11 (14.3%) cases (Figure 2). Morphology and cytochemistry did not establish lineage involvement in 9 (11.7%) cases. Basic morphological and cytochemical

Fig. 3. Distribution of cases based on lineage assignment of blasts cells detected with basic

Immunological analyses with multi-parameter flow-cytometry were performed in all 77 patients. Further immunological analyses of cases in which lineage could not be assigned based on morphology and cytochemistry established myeloid lineage in 4 patients, (AML-M0) and one case indicated nonhematopoietic malignancy. In this case, immunophenotype of the malignant cells (CD45-/CD56+/CD9+) indicated a neuroectodermal origin of the malignant cells (Bain et al., 2002). Later neuroblastoma was diagnosed in this patient. Immunological analyses change the assigned lineage based on morphology and cytochemistry in 3(3.8%) of the patients from lymphoid to myeloid. The results of our study

Consequently, based on FAB and immunologic criteria of EGIL and BTSH 64 acute leukemias were classified as myeloid. Correlation between the antigen expressions with FAB morphology of AML cases which confirmed the AML diagnosis in 83. 1% of the cases is presented at Table 3. According to FAB criteria, the leukemias were classified as M0 (n=4),

Multivariate Cox-proportional regression analyses showed that in 89.7% of AML cases lineage assignment is defined with the following five markers: CD13, CD33, CD117, HLA-DR and anti-MPO. Most frequently detected maturation myeloid marker which was expressed in 42.9% of AML case was CD15. Twenty three (35.9 %) of AML patients showed expression of lymphoid antigens. Co-expression of two lymphoid markers was detected in eleven (17.1%) cases, and most frequently co-expressed lymphoid marker was CD7 and was

showed that routine immunophenotyping improved diagnosis in 12 (15.5%) cases.

M1 (n=8), M2 (n=20), M3 (n=5), M4 (n=17), M4-Eo (n=1), M5 (n=8), M6 (n=1).

analyses established the lineage assignment of the blasts cells in 68 (88.3%) patients.

which showed statistically significant correlation (p<0, 05) (Figure 2C).

Photo 1. Images from positive reactivity of peripheral blast cells with different cytochemical staining , A: PAS reactivity, B: POX reactivity, C: NSE reactivity

Statistical analyses showed that there is a statistically significant correlation between the AML and MPO positivity (X2 with Yates's correction=16.628 p<0.01)(Figure 2A). According to cross reaction ratio, MPO positivity presents statistically significant risk which improves the chance of AML diagnosis for 39 times (OR=392.1603 (4.177<0R<305.9796, CI 95%)). Also, a statistically significant correlation was noted between the PAS positivity and ALL (X2 with Yates's correction=5.514 p<0.01)( Figure 2B). Strong PAS positivity was registered in 75% of ALL cases. Regarding the cytochemical stain NSE and AML no statistical correlation was

A

B

C Photo 1. Images from positive reactivity of peripheral blast cells with different cytochemical

Statistical analyses showed that there is a statistically significant correlation between the AML and MPO positivity (X2 with Yates's correction=16.628 p<0.01)(Figure 2A). According to cross reaction ratio, MPO positivity presents statistically significant risk which improves the chance of AML diagnosis for 39 times (OR=392.1603 (4.177<0R<305.9796, CI 95%)). Also, a statistically significant correlation was noted between the PAS positivity and ALL (X2 with Yates's correction=5.514 p<0.01)( Figure 2B). Strong PAS positivity was registered in 75% of ALL cases. Regarding the cytochemical stain NSE and AML no statistical correlation was

staining , A: PAS reactivity, B: POX reactivity, C: NSE reactivity

observed (X2 with Yates's correction=2.456 p=0.117), expect for AML M4 and M5 entities which showed statistically significant correlation (p<0, 05) (Figure 2C).

Morphology and cytochemistry established myeloid lineage in 57 (74.0%) cases and lymphoid differentiation in 11 (14.3%) cases (Figure 2). Morphology and cytochemistry did not establish lineage involvement in 9 (11.7%) cases. Basic morphological and cytochemical analyses established the lineage assignment of the blasts cells in 68 (88.3%) patients.

Fig. 3. Distribution of cases based on lineage assignment of blasts cells detected with basic morphological and cytochemial analyses

#### **4.2 Results from the immunological analyses**

Immunological analyses with multi-parameter flow-cytometry were performed in all 77 patients. Further immunological analyses of cases in which lineage could not be assigned based on morphology and cytochemistry established myeloid lineage in 4 patients, (AML-M0) and one case indicated nonhematopoietic malignancy. In this case, immunophenotype of the malignant cells (CD45-/CD56+/CD9+) indicated a neuroectodermal origin of the malignant cells (Bain et al., 2002). Later neuroblastoma was diagnosed in this patient. Immunological analyses change the assigned lineage based on morphology and cytochemistry in 3(3.8%) of the patients from lymphoid to myeloid. The results of our study showed that routine immunophenotyping improved diagnosis in 12 (15.5%) cases.

Consequently, based on FAB and immunologic criteria of EGIL and BTSH 64 acute leukemias were classified as myeloid. Correlation between the antigen expressions with FAB morphology of AML cases which confirmed the AML diagnosis in 83. 1% of the cases is presented at Table 3. According to FAB criteria, the leukemias were classified as M0 (n=4), M1 (n=8), M2 (n=20), M3 (n=5), M4 (n=17), M4-Eo (n=1), M5 (n=8), M6 (n=1).

Multivariate Cox-proportional regression analyses showed that in 89.7% of AML cases lineage assignment is defined with the following five markers: CD13, CD33, CD117, HLA-DR and anti-MPO. Most frequently detected maturation myeloid marker which was expressed in 42.9% of AML case was CD15. Twenty three (35.9 %) of AML patients showed expression of lymphoid antigens. Co-expression of two lymphoid markers was detected in eleven (17.1%) cases, and most frequently co-expressed lymphoid marker was CD7 and was detected in 28.5% of the cases.

Immunophenotyping of the Blast Cells in Correlations with

**4.3 Results from the molecular analyses** 

transcript CBF/MYH11 (Figure 4).

group of AML patients;

the Molecular Genetics Analyses for Diagnostic and Clinical Stratification of Patients… 493

Molecular evaluation of AML cases demonstrated presence of the three major recurrent genetic abnormalities as follows: 5 patients were positive for the fusion transcript PML/RAR (Figure 5), 3 patients were positive for AML/ETO1 and 7 for the fusion

Fig. 4. Distribution of molecular abnormalities detected with the RQ-PCR assay in the

Fig. 5. Detection of fusion transcript PML/RAR using the RQ-PCR method


Table 3. Correlation between the antigen expressions with FAB morphology of AML cases

#### **4.3 Results from the molecular analyses**

492 Advances in Cancer Therapy

Table 3. Correlation between the antigen expressions with FAB morphology of AML cases

Molecular evaluation of AML cases demonstrated presence of the three major recurrent genetic abnormalities as follows: 5 patients were positive for the fusion transcript PML/RAR (Figure 5), 3 patients were positive for AML/ETO1 and 7 for the fusion transcript CBF/MYH11 (Figure 4).

Fig. 4. Distribution of molecular abnormalities detected with the RQ-PCR assay in the group of AML patients;

Fig. 5. Detection of fusion transcript PML/RAR using the RQ-PCR method

Immunophenotyping of the Blast Cells in Correlations with

3(3.8%) additional cases.

Goodpasture, 2000).

Swerdlow et al., 2008).

non-hematological malignancy was confirmed.

**5.1 Immunophenotyping in modern diagnosis of AML** 

the Molecular Genetics Analyses for Diagnostic and Clinical Stratification of Patients… 495

Assignment of lineage is critical in the diagnostic evaluation of acute leukemia, as treatment for AML and ALL markedly differs. Myeloid and lymphoid lineage may be distinguished based on cellular morphology, cytochemical staining, and expression of lineage-specific antigens (Döhner et al., 2010). Analyses of diagnostic evaluation of acute leukemia in our study showed that immunophenotyping was necessary for lineage assignment in 4 (8.9%) cases that were morphologically and cytochemically undifferentiated, and also for correction of the lineage that was assigned based on morphology and cytochemistry in

Flow cytometric immunophenotyping is a powerful technological tool that aids in the diagnosis, classification and monitoring of hematological malignancies. It is essential in the diagnosis of AML, as it demonstrates a particular lineage involvement and has a prognostic significance in the majority of AML cases. We used the panel based on the recommendation of the EGIL group and BTSH. Our analyses comprised of a two step process with the first panel of markers being applied to all cases of acute leukemia and the second only in patients with AML that did not demonstrate a clear myeloid commitment. We also evaluated further lymphoid antigen positive AML cases by using the second panel of McAb (Stelzer &

The second panel of McAb was aimed at identifying uncommon types of AML, such as those with megakaryocytic or elytroid differentiation and the exclusion or conformation of diagnosis of non-hematological malignancy. In our study, we applied the second AML panel in 27 cases; 4 which did not demonstrate clear myeloid commitment and 23 cases with lymphoid antigen positive AML cases. With our primary McAb we were able to differentiate AML form ALL in 96.8% of cases. Only in one patient (AML-M6-1.5%) lineage differentiation was assigned after staining with the secondary McAB panel and one case of

Immunophenotyping was crucial in all cases of poorly differentiated myeloid leukemia (AML-M0), megakaryoblatstic leukemia (AML-M7) and in some case of monoblastic leukemia (AML-M5) and those with primitive erythroid cells as predominant leukemic cells (AML-M6). This is also important for recognizing an AML case that co-expresses lymphoidassociated antigens. In addition, immunophenotyping enables recognition of unusual forms of acute leukemia: designated acute biphenotypic or acute mixed lineage leukemia. The leukemia associated immunophenotype (LAIP) of the blast cells is a useful tool for detection of minimal residual disease in AML cases (Döhner et al., 2010; Stelzer & Goodpasture, 2000;

In order to classify AML cases in the different AML entities we correlated the immunological data from immunophenotyping with the FAB morphological and cytochemical classification. One of the difficulties in knotting the flow cytometric data with traditional morphology is the lack of routine flow cytometric data analyses that would ensure correlation of the immunophenotyping data to abnormal morphologic counterpart. Our approach was based on the fact that complete eight-part differential of the myeloid lineage in the normal bone marrow could be done with correlations of the expression of CD45 expression versus light side scatter (SSC) characteristics of the cells. Extension of this technique to the analyses of leukemias allows abnormal cell to be recognized as independent clusters in CD45/SSC histograms with pattern of CD45 and SSC expression that correlate to the same pattern of the morphologically similar cells in normal bone marrow. Flow-cytometric analyses by using CD45 gating strategy reveled that leukemic

In 5 of the AML case PML/RAR fusion transcript was detected. Four of those patients had morphology and immunophenotype that correlate with AML-M3 diagnosis. The five patients were first diagnosed as AML-M2 and only after the positive result for PML/RAR the diagnosis was revised as AML-M3. In all those five patients target therapy with ATRA was initiated.

RT-PCR analysis also detected molecular abnormalities in the Core binding factor (CBF) in 10 AML patients; the presence of the AML/ETO1 fusion gene was confirmed in 3 patient and CBF/MYH11 in 7 patients.

Molecular analyses enabled 23.7% of the cases from our study to be classified in the adequate genetic entities of AML with different prognosis requiring different therapeutic approach.
