Biphenotypic Acute Leukemia Research Articles

Definitive Criteria for Ambiguous Lineage Leukemias

Biphenotypic leukemias are acute leukemias of ambiguous lineage in which there is proliferation of more than one distinct cell type. In this case, both myeloid and lymphoid phenotypes are present in the blast population. As opposed to leukemias in which one specific myeloid or lymphoid population is found, these biphenotypic leukemias present a unique diagnostic challenge in these patients. Treatment is determined by identification of the predominant lineage. This study explores the successful use of the European international scoring system to define biphenotypic acute leukemias. Of nine cases of leukemias of mixed phenotype, determined by flow cytometric analysis, three were scored as biphenotypic by traditional 2001 WHO standards, whereas six were of ambiguous lineage but did not meet the scoring criteria for mixed phenotype. However, on re‐evaluation using the 2008 WHO guidelines, three of these cases were reclassified as biphenotypic based on the increased significance of myeloperoxidase (MPO) and combined CD14/CD64 positivity for myeloid lineage. Considering the worse prognosis of biphenotypic leukemia than AML or ALL, early identification of mixed lineage is critical for appropriate treatment.

Cryptic insertion into 11q23 of MLLT10 not involved in t(1;15;11;10)(p36;q11;q23;q24) in infant acute biphenotypic leukemia

The MLL gene, located on chromosomal band 11q23, is fused to a large number of different partner genes in hematological malignancies. This report describes a case of infant acute biphenotypic leukemia with t(1;15;11;10)(p36;q11;q23;q24). Panhandle polymerase chain reaction (PCR) using cDNA demonstrated the formation of an MLL–MLLT10 fusion transcript, although the 10p12 segment, at which the MLLT10 gene is located, was not involved in the breakpoint of the four-way translocation according to G-banding and spectral karyotyping analyses. Long-distance inverse PCR using genomic DNA revealed that intron 7 of MLL was fused with intron 8 of MLLT10, which was connected with a DNA segment of noncoding region on 15q. In fluorescence in situ hybridization analyses, the duplicated 3′ part of MLLT10 was inserted into the component of chromosome 15 on der(11)(q23). In real-time quantitative PCR with primers that recognized the DNA sequence of the two sites of fusion point, the minimal residual disease (MRD) levels changed in parallel with other clinical markers. Furthermore, the level of MRD had already increased before hematologic relapse. The identification and characterization of MLL rearrangement at the genomic DNA level may be useful for MRD quantification.

Monosomies 7p and 12p and FLT3 internal tandem duplication: possible markers for diagnosis of T/myeloid biphenotypic acute leukemia and its clonal evolution

Biphenotypic acute leukemia co-expressing T-lymphoid and myeloid markers is rare, accounting for less than 1% of acute leukemias. However, several clinical characteristics including male predominance, frequent lymphadenopathy and unfavorable outcome have been identified. Recurrence of monosomies 7p and/or 12p in T/myeloid biphenotypic acute leukemia has been reported. We treated a patient with T/myeloid biphenotypic acute leukemia showing clonal chromosomal and genetic abnormalities including dic(7;12)(p11;p11) and Fms-like tyrosine kinase 3 (FLT3)-internal tandem duplication. Cytogenetic analysis of both bone marrow and lymph node cells disclosed that the patient's lymph node leukemia cells had chromosomal abnormalities in addition to dic(7;12). Our findings suggest that the leukemia cells of systemic lymphadenopathy had evolved as secondary cells from marrow leukemia cells. The patient was successfully treated with induction chemotherapy for acute myeloid leukemia followed by allogeneic bone marrow transplantation.

Infant acute bilineal leukemia

Most cases of acute leukemia can be assigned to the myeloid, B or T lineage. There are rare cases of acute leukemia, which cannot be clearly classified, because either blasts express antigens of more than one lineage (acute biphenotypic leukemias) or distinct blast populations of two lineages co-exist (acute bilineal leukemias, aBLL). We present a 10-month-old infant with de novo aBLL, characterized by blasts of monocytic and B-cell precursor lineages. All leukemic cells harbored identical complex MLL gene rearrangement. Despite poor initial response, both to acute lymphoblastic leukemia (ALL) induction treatment and acute myeloid leukemia induction blocks, the child reached complete clinical remission with minimal residual disease negative status and was transplanted. Unfortunately, 16 months from HSCT the patient experienced BM relapse with all blasts characterized by pro-B-ALL immunophenotype. This case report illustrates that aBLL is a very aggressive type of acute leukemia that should be individually treated and monitored, particularly in children less than 1 year of age.

Acute leukemia: Diagnosis improved by flow cytometry in addition to morphology

AbstractAims: Immunophenotyping using flow cytometry (FCM) has become an essential component of acute leukemia (AL) diagnosis. This study evaluated the judicious application of FCM as an adjunct to well‐informed morpho‐cytochemical assessment in patients with acute leukemia.Methods: 100 untreated patients with AL were studied using morpho‐cytochemistry and immunophenotyping through FCM.Results: There were 29 patients with acute myeloid leukemia (AML), 47 with B‐acute lymphoblastic leukemia (ALL), 20 with T‐acute lymphoblastic leukemia (ALL) and four with biphenotypic acute leukemia (BAL). Morpho‐cytochemistry without FCM could provide definite diagnosis only in the AML cases. It failed to provide definite diagnosis in ALL patients. Over half (55%) of ALL patients were given the noncommittal label, AL. The remaining 45% patients were labeled a more definite, probable ALL.Conclusion: FCM thus had a role to play in ALL patients to confirm a definite and a probable diagnosis, to define therapeutically and prognostically groups such as B and T lineage ALL and to distinguish AML – M0 from ALL. FCM helps in diagnosing AML cases as well, but is a less essential mode of investigation in this group purely from the perspective of the therapy regime. But its role in defining different subgroups in AML is its major use. While morpho‐cytochemistry provides a first‐line investigation of great therapeutic value, and more so in AML, it needs to be supplemented by flow cytometry, particularly in ALL.

Invasive Aspergillus flavus sinusitis: case report in a patient with biphenotypic acute leukemia

Here we report a case of invasive pansinusitis with proptosis of the right eye caused by Aspergillus flavus in an immunocompromised patient with acute biphenotypic leukemia without aggressive therapy response.

Autologous hematopoietic stem cell transplantation in adult acute lymphoblastic leukemia: still not out of fashion

The role of autologous hematopoietic stem cell transplantation (autoHSCT) in adult acute lymphoblastic leukemia (ALL) is still unclear. We retrospectively analyzed the results of the autoHSCT and maintenance therapy, with oral 6-mercaptopurine and methotrexate, in comparison to conventional-dose chemotherapy in the consolidation treatment of adult ALL and lymphoblastic lymphoma (LBL). The patients, with HLA identical sibling donor, underwent allogeneic transplantation, while the others were treated with autoHSCT and maintenance therapy with oral 6-mercaptopurine and methotrexate, or by conventional-dose chemotherapy (patient's decision, no autologous hematopoietic stem cells harvest). Sixty consecutive adult patients (median age 35.2 years; range 17.3 to 70.7) with ALL (n = 52), LBL (n = 7), and acute biphenotypic leukemia (n = 1) were treated in our center from 1997 to 2007. Patients treated with chemotherapy alone (n = 35) had a shorter median progression-free survival (PFS) compared to patients who underwent autoHSCT plus maintenance therapy (n = 18), 8.4 and 46.8 months, respectively (p = 0.017). Patients treated with chemotherapy alone had also a shorter median overall survival (OS) compared to patients treated with autoHSCT: 13.0 vs. 46.8 months (p = 0.046). The differences remained statistically significant even after excluding patients with Ph positivity. We can conclude that, in our case, autoHSCT followed by maintenance chemotherapy is a good option for adult patients with ALL and, in standard-risk and high-risk patients, provides more favorable OS and PFS rates compared to patients treated by chemotherapy alone. However, we are aware of the fact that our analysis may have been distorted by the fact that the analysis is retrospective, that treatment with autoHSCT was based on patient's decision, and that chemotherapy may have been administered to negatively selected patients.

Clinico-hematological profile in biphenotypic acute leukemia

We present a clinico-hematological profile and treatment outcome of Biphenotypic Acute Leukemia (BAL). Study incidence and subtypes of BAL, correlate with age, morphology, and cytogenetic findings and correlate the clinico-hematological data with the treatment response. St Jude's and the EGIL's criteria have been compared for their diagnostic and clinical relevance. Diagnosis was based on WHO classification, including clinical details, morphology, cytochemistry, immunophenotyping, and molecular genetics. We included those cases, which fulfilled the European Group for the Immunological Characterization of Acute Leukemia's (EGIL's) scoring system criteria for the diagnosis of BAL, as per recommendation of the WHO classification. There were 32 patients diagnosed with BAL, based on EGIL's criteria. Incidence of BAL was 1.2%. B-Myeloid (14 cases) followed by T-Myeloid BAL (13 cases) were the commonest subtypes. Polymorphous population of blasts (16 cases) was commonly associated with T-Myeloid BAL (10 cases). BCR ABL fusion positivity was a common cytogenetic abnormality (seven cases). Fifteen patients received chemotherapy; eight achieved complete remission (CR) at the end of the induction period. Pediatric BAL and T-B lymphoid BAL have a better prognosis. A comprehensive panel of reagents is required, including cytoplasmic markers; to diagnose BAL. St Jude's criteria is a simple, easy, and cost-effective method to diagnose BAL. The outcome-related prognostic factors include age, HLA-DR, CD34 negativity, and subtype of BAL. BCR-ABL expression is an important prognostic factor, as these cases will be labeled as Chronic myeloid leukemia (CML) in blast crisis with biphenotypic expression and treated accordingly.

T(9;22) with 5'ABL1 Deletion and t(6;19) in Biphenotypic Acute Leukemia

Biphenotypic acute leukemia (BAL) is a rare type of leukemia, comprises 4% of all acute leukemias. It is more common in adults and the clinical features, as related to marrow dysfunction, are similar to those found in other patients with acute leukemia. BAL commonly shows a dimorphic blast population with, one resembling lymphoblasts and the other resembling myeloblasts. The majority of BAL patients express B-lymphoid and myeloid markers. BAL can be diagnosed by morphologic studies and by a comprehensive panel of immunological markers, as well as cytogenetic/molecular studies, such as fluorescence in situ hybridization (FISH) and reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, its prognosis is relatively poor. We present here a 27 year-old female patient who showed lymphoblasts and myeloblasts on her marrow studies and these cells were positive for myeloid and B-lymphoid markers on the immunophenotypic studies. Chromosome analysis revealed 46,XX,t(6;19)(p23;p13.1),t(9;22)(q34;q11.2). A major (b3a2) type of BCR-ABL1 mRNA transcript was detected by RT-PCR, and a 5' ABL1 deletion was identified by FISH.

Acute Leukemia of Ambiguous Lineage, Biphenotype, without CD34, TdT or TCR-rearrangement

Biphenotypic acute leukemia (BAL) is a rare entity that comprises 0.5-3% of all acute leukemias and probably arises from multipotent progenitor cells. The optimal approach for BAL therapy is unknown. Thus, it is important to elucidate the origin of the neoplastic cells for determination of the appropriate therapy. We report the case of a 41-year-old man with BAL having myeloid and T-lymphoid lineage phenotypes. Strangely, neither CD34 nor TdT expression nor rearrangement of TCR-alpha/beta, delta/gamma genes were shown. This pattern is rarely encountered and suggests that the blast cells were possibly considered immature with aspects of differentiation indicating myeloid lineage, rather than T-lymphoid lineage.

Clinical Significance of Co-expression of Aberrant Antigens in Acute Leukemia

Background: Acute leukemias co-expressing myeloid and lymphoid antigens but does not meet the criteria for biphenotypic acute leukemia (BAL) is common, however its clinical significance is not fully defined. Methods: In this study, clinical features of 68 co-expressing (myeloid and lymphoid) acute leukemias diagnosed between January 2000 and December 2006 were studied and compared with those of a control group of patients (pure AML or ALL). Results: Age, gender, initial Lactate dehydrogenase (LDH) level and cytogenetics were not different between the co-expressing group and the control group. But, the initial bone marrow blast percent was significantly higher in the co-expressing group (70% vs. 54.5%, P=0.003). Fifty five percent (16/29) of ALL and 30% (52/172) of AML patients showed myeloid and lymphoid markers concomitantly. The lymphoid antigen positive AML (Ly+ AML) patients showed significantly shorter survival rates than pure AML patients (4 year survival rate, 17.6% vs. 45.6%, P=0.002). However hematopoietic stem cell transplantation (HST) abrogated the difference (4 year survival rate, 54.7% vs. 50.6%, P=0.894). In ALL patients, survival rate was not affected by myeloid antigen co-expression (4 year survival rate 26.1% vs. 20%, P=0.954). Conclusion: Co-expression of lymphoid markers in AML should be regarded as a poor prognostic factor and more aggressive treatment such as HST should be considered.

Non-specific interstitial pneumonia as the initial presentation of biphenotypic acute leukemia: a case report

Nonspecific interstitial pneumonia has been linked to numerous etiologies including, most recently, haematologic malignancy. We present a 46-year-old woman with recent-onset rheumatologic illness who developed pulmonary symptoms as the presenting feature of biphenotypic acute leukaemia. Chest radiology demonstrated bilateral infiltrates, and lung biopsy revealed nonspecific interstitial pneumonia. Corticosteroid therapy resulted in resolution of both her pulmonary and rheumatologic symptoms, and her pulmonary symptoms did not recur following treatment of her leukemia. The case highlights the importance of searching for an underlying etiology when confronted with nonspecific interstitial pneumonia.

Leukemic infiltration of the lung following allogeneic hematopoietic stem cell transplantation

Pulmonary leukemic infiltration (PLI) is more common than generally recognized, but accurate antemortem diagnosis with pathological proof is rarely achieved. We describe herein the clinical courses of two patients with PLI following hematopoietic stem cell transplantation (HSCT). One case is a male patient with acute biphenotypic leukemia, and the other is a female patient with myelodysplastic syndrome. In both cases, hyperleukocytosis did not proceed to PLI. Moreover, the former case presented PLI as the initial manifestation of relapsed leukemia and the latter was accompanied with the fungal pneumonia. High-resolution computed tomography (HRCT) of the chest at onset of PLI showed diffuse small nodular lesions along peribronchovascular bundle, and diagnosis of leukemic infiltration was made based on pathological findings obtained from transbronchial lung biopsy. Biopsy specimens further revealed leukemic infiltration along the lymphatic surrounding the peribronchial or perivascluar regions, which corresponded well with HRCT findings. Thus, radiological and pathological corroborating assessment was important to reach the correct diagnosis.

An Unusual Presentation of Acute Biphenotypic Leukemia without Bone Marrow Involvement

Acute biphenotypic leukemia (ABL) represents a minority of acute leukemia cases (around 5%) in which blasts co-express markers of different lineages (myeloid, T-cell or B-cell). ABL without bone marrow involvement is exceedingly rare. Case report: An 18-year-old man presented with slowly enlarging, non-tender cervical lymph nodes. He had no other symptoms. Family history included Hodgkin's disease in his mother and maternal great aunt, histiocytosis X in his maternal cousin, and non-Hodgkin's lymphoma in his paternal grandfather. Physical exam revealed multiple small cervical and axillary lymph nodes. Laboratory analyses including complete blood count, peripheral blood smear, complete metabolic profile, mononucleosis screen, and hepatitis and HIV serologies were unremarkable. Histopathological examination of the left cervical lymph node revealed rare germinal centers with an interfollicular proliferation of generally medium size cells with a fine blastic chromatin pattern and scant cytoplasm. CD34 was positive in more than half of the cells. Between 30% and 80% of the cells were positive for TdT (Figure 1), CD3, CD4, and CD7. Myeloperoxidase (Figure 2) and lysozyme were positive in about 20% of the cells. Flow cytometry revealed an immature myeloid cell population coexpressing CD33, CD13, CD7, and CD34. Chromosomal analysis revealed a complex karyotype. Bone marrow exam was unremarkable and no evidence of leukemia was observed. Flow cytometry of the bone marrow aspirate revealed only 4% of the cells to be CD34+. Whole body PET scan showed extensive uptake in bilateral axillae, neck, and inguinal regions without any extranodal involvement. The patient received hyper-CVAD regimen (alternating cycles of cyclophosphamide, vincristine, adriamycin, dexamethasone and high dose methotrexate, cytarabine) for 8 cycles and remains in remission five months after the end of hyper-CVAD protocol. To our knowledge, this is the third case reported in the literature with acute biphenotypic leukemia not involving the bone marrow. However, our case cannot accurately be referred to as leukemia since the bone marrow is not involved; neither can it be named as lymphoblastic lymphoma because of its myeloid differentiation. Therefore, we will refer to it as extramedullary granulocytic sarcoma with biphenotypic features. [Display omitted] [Display omitted]

Detection of Twelve Nucleotides Instertion in the BCR-ABL Kinase Domain in an Imatinib-Resistant but Dasatinib-Sensitive Patient with Bi-Phenotypic Acute Leukemia.

Although targeted inhibition of BCR-ABL with imatinib (IM) is an effective therapy for patients with Philadelphia chromosome-positive leukemia, a minority of patients (most of them in advanced phase) acquire mutations in the BCR-ABL kinase domain (KD) and relapse. The spectrum of the mutations described to date consists in-almost exclusively-single nucleotide substitutions affecting IM binding or impairing the conformational changes of the BCR-ABL KD. Recently, rare cases of intron/truncation mutations of 35 nucleotides, presumably inserted by alternative splicing events but inducing no conservation of the open reading frame (ORF), have been described in CML patients undergoing IM therapy (Laudadio et al. J Mol Diagn. 2008; 10:177–180). Here, we describe a novel mutation involving the BCR-ABL KD, acquired at the moment of the IM resistance, consisting in an insertion of 12 nucleotides leading to the conservation of the ORF. The 57-year old female patient was diagnosed in July 2006 with bi-phenotypic acute leukemia based on peripheral blood and bone marrow findings (hyperleukocytosis at 48 G/l with 47% of blasts exhibiting myeloid and lymphoid features (CD13+, CD33+, CD19+, CD10+ and CD22+), on karyotypic analysis found a t(9;22)(q34;q11) in all mitosis and on molecular analysis detected M BCR-ABL transcript. There was no sibling donor identified. Induction chemotherapy (Hyper C-VAD) and IM at 800mg/day were given. She achieved a complete remission (CR), a complete cytogenetic response and the BCR-ABL/ABL ratio was 2.3% (RT-RQ PCR performed according to the ELN recommendations with ICF 0.51). Consolidation therapy with alternating high dose methotrexate plus cytarabine and Hyper C-VAD plus IM was given and the BCR-ABL/ABL ratio dropped to 0.06%. After a successful PBSC collection with G-CSF alone, she was autografted in April 2007. IM was re-introduced at 600mg/day leading to a sustained drop in transcript level to 0.035% 6 months later. Then the transcript ratio rose rapidly within 3 months (0.7%;2.4%;8.1%) and a mutation screen performed in April 2008 revealed the insertion of 12 nucleotides in 100% of the BCR-ABL transcripts inducing the insertion of 4 amino acid residues (A, F, G and P) between A293 and K294 in the BCR-ABL KD. This insertion into exon 5 of ABL in the BCR-ABL transcript sequences did not seem related to alternative splicing events or to a constitutional polymorphism because this insertion was present at the genomic level in DNA extracted from leukemic cells but was missing in genomic DNA extracted from the patient's epithelial mouth cells. Moreover, retrospective analysis with a fluorescent probe and allele specific oligonucleotide performed by RQ-PCR on the cDNA from patient at diagnosis did not show the insertion at diagnosis, suggesting that the mutational mechanism was induced or selected by IM therapy. The increase of IM up to 800mg/day during six weeks led to the loss of hematologic response. Dasatinib was introduced at 70mg twice daily in may 2008 and molecular monitoring performed monthly showed a strong reduction of the BCRABL/ABL ratio to 0.7%; 0.2% and 0.07% respectively. The mutant ratios performed on the latest follow-up did not show any change in the proportion (100%) of the mutated BCR-ABL transcripts among total BCR-ABL transcripts suggesting no resurgence of the wild-type BCR-ABL clone. Therefore dasatinib seems to efficiently target the mutated clone as well as the wild-type leukemic clone and appears to be highly effective on this kind of mutation.

The Molecular Screening Using Multiplex Reverse Transcription Polymerase Chain Reaction and the Comparison with Cytogenetic Analysis in the Prognostic Stratification of Acute Leukemia

INTRODUCTION: The detection of chromosomal aberrations is essential for the diagnosis and prognostic stratification of acute leukemia. Classical cytogenetic analysis has the advantage of comprehensive screening of all types of abnormalities such as numerical aberrations and deletions as well as translocations. However, this technique is time-consuming and labor-intensive, and cannot detect cryptic translocations. Recently, a multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) has emerged as a tool for overcoming disadvantages of cytogenetic analysis with some degree of comprehensiveness. We designed multiplex RT-PCR system for acute leukemia based on sets of chromosomal translocations rationally selected with cost-effectiveness and clinical relevance. Herein, we tried to use molecular screening of acute leukemia with a multiplex RT-PCR system we designed, and compared with the results of cytogenetic analysis in terms of prognostic stratification.MATERIALS AND METHODS: A total of 121 consecutive patients with acute leukemia were included from November 2006 to July 2007. Multiplex RT-PCR system (Seegene, Korea) for molecular screening was carried out to detect 16 fusion transcripts: BCR-ABL, PML-RARα, PLZF-RARα, AML1-ETO, CBFβ-MYH11, DEK-CAN, E2APBX1, TEL-AML1, and MLL rearrangements (MLL-AF4, MLL-AF6, MLL-AF9, MLL-AF10, MLL-ELL, MLL-ENL, MLL-AF17, MLL-PTD). Cytogenetic analysis was performed by standard GTL-banding technique. The discordant findings were validated by fluorescence in situ hybridization (FISH) or conventional single RT-PCR.RESULTS: Eighty-nine (73.6%) adults and 32 (26.4%) children were included in this study. A total of 77 (63.6%) were diagnosed as acute myeloid leukemia (AML) and the remaining 44 (36.4%) were acute lymphoblastic leukemia (ALL) including 7 biphenotypic acute leukemia. Cytogenetic analysis was available in 118 (97.5%) of the patients. Sixty-four cases of fusion transcripts were detected in 60 patients (49.6%). Within the AML group, the following fusion transcripts were detected: 14 AML1-ETO, 8 PML-RARα, 3 CBFβ-MYH11, 1 DEK-CAN, 1 BCR-ABL, 6 MLL-PTD, 4 MLL-AF6, 3 MLL-AF9, 1 MLL-ELL. Within the ALL group, the following fusion transcripts were detected: 12 BCR-ABL, 4 TEL-AML1, 2 E2A-PBX1, 3 MLL-AF4, 1 MLL-AF9, 1 MLLPTD. The concordance rate between two tests was 88.3%. In 14 cases, the results of two methods did not agree. Seven cytogenetically unrevealed abnormalities were detected with the multiplex RT-PCR: 3 TEL-AML1, 2 MLL-AF9, 1 MLL-AF6, 1 PML-RARα. The frequency of cryptic translocation was 5.8%. MLL-PTD which is impossible to be observed by cytogenetic analysis was detected in remaining seven patients. On the other hand, the corresponding fusion transcripts were not detected by multiplex RT-PCR in three cases of inv(16), t(4;11), and MLL rearrangement suggested by FISH, respectively. Molecular screening could preferentially assign 32.5% of AML patients to favorable-risk group, and did 34.1% of ALL patients to adverse-risk group. Although the concordance rate between two methods as a prognostic factor was 89.5% in risk-determined group by multiplex RT-PCR, six patients (3 AML and 3 ALL) disclosed different risk group by molecular screening from cytogenetic analysis.CONCLUSIONS: We conclude that molecular screening using multiplex RT-PCR was useful in providing informations about genetic abnormalities more rapidly and giving appropriate therapeutic decision in acute leukemia patients. It was also useful in the detection of cytogenetically cryptic translocations. Although the multiplex RT-PCR system could not be a whole substitute for cytogenetic analysis, we expect that two methods are complementary at the time of diagnosis of acute leukemia, by which more genetic aberrations would be detected efficiently.

BCR-ABL Protein Detection by Flow Cytometry in Acute Lymphoblastic Leukemia Samples

The development of agents directed specifically against the BCR-ABL tyrosine kinase has opened therapeutic avenues that have profoundly modified the management of diseases which harbor the genetic abnormality that leads to the production of the BCRABL transcript. While the role of tyrosine kinase inhibitors of 1st and 2nd generation in chronic myeloid leukemia (CML) patients is well established, important clinical responses have been documented also in BCR-ABL+ acute lymphoblastic leukemia (ALL), the most relevant genetic ALL subgroup which accounts for about 25% of adult cases and is associated with a dismal prognosis. Following the administration of tyrosine kinase inhibitors alone as induction treatment, complete hematologic remissions have in fact been recorded in virtually all cases. Thus, the possibility of identifying the presence of the BCR-ABL transcript in the shortest possible time and with a simple and broadly utilizable technique would translate into the timely implementation of a targeted therapy. The availability of a method capable of highlighting the presence of the BCR-ABL protein has important implications because it can document the effective transduction of the molecular transcript; since tyrosine kinase inhibitors are effective on the protein, this would unequivocally support the use of such specific agents in these cases. For this purpose, we have utilized the BCR-ABL Protein Kit**Research Use Only, not for diagnostics or therapeutic procedures. (BD Biosciences, San José, CA) which consists of an immunoassay that identifies qualitatively the presence of the BCR-ABL protein in the lysates of leukemic cell populations. The BCR-ABL fusion protein is captured and detected on Cytometric Bead Array (CBA) and analyzed on a FACSCanto (BD Biosciences) flow cytometer. Lysates from normal mononuclear cells and from the K562 cell line were used as negative and positive controls, respectively. The presence of the BCR-ABL protein was investigated on fresh bone marrow or peripheral blood samples from 56 consecutive cases of acute leukemia aged between 11 and 81 years referred to our Institution. The BCR-ABL protein could be documented in 11 of the 56 cases analyzed. By conventional immunophenotypic analysis they proved to be a B-lineage ALL in 9 cases and a B lymphoid blast crisis of CML in 2. In all 11 cases, the presence of the BCR-ABL transcript was subsequently documented by molecular biology. In the other 45 cases the diagnosis was: B-lineage ALL in 26 (2 of which were ALL/AF4+), T-ALL in 2, acute myeloid leukemia in 22 (5 of which were PML-RARα+) and acute biphenotypic leukemia in 1. None of these latter 45 cases harbored the BCRABL transcript. The assay utilized was successfully completed within a maximum of 4 hours from the marrow or blood collection on 20 × 106 cells containing at least 20% of leukemic cells. The correlation between the presence/absence of the BCR-ABL protein evaluated using the BCR-ABL Protein Kit* and the positivity/negativity of the transcript assessed according to conventional genetic techniques has been absolute. In our laboratory, the assay has shown to be reliable, reproducible and of simple execution. It allows in a very short time to identify the presence of the BCR-ABL protein – both p190 and p210 – through a flow cytometric analysis and thus to reliably offer proteomic information to investigators researching targeted anti-tyrosine kinase treatment.

Immunophenotypic Profile of Acute Leukemia: Critical Analysis and Insights Gained at a Tertiary Care Center in India

The purpose of this retrospective study, the largest series in our country, was to illustrate the spectrum of subtypes and the immunophenotypic features of cases referred as acute leukemia (AL). Morphological diagnosis was correlated with cytochemical stains, immunophenotyping, and molecular genetic studies including fluorescent in-situ hybridization (FISH) and polymerase chain reaction (PCR). Two thousand five hundred and eleven consecutive new referral cases of Acute Leukemia (AL) between January 1, 2003 and December 31, 2006 were evaluated based on WHO classification. It included 1464 cases (58.3%) of Acute Lymphoblastic Leukemia (ALL), 962 cases (38.3%) of Acute Myeloid Leukemia (AML), 45 cases (1.8%) of Chronic Myelogenous Leukemia in blast crisis (CMLBC), 37 cases (1.5%) of biphenotypic acute leukemia (BAL), 1 case of Triphenotypic AL and 2 cases of Acute Undifferentiated leukemia (AUL). Common subtypes of ALL were B-cell ALL (76%) which comprised of intermediate stage/CALLA positive (73%), early precursor/Pro BALL (3%). T-cell ALL constituted 24% (348 cases) of ALL. Common subtypes of AML included AMLM2 (27%), AMLM5 (15%), AMLM0 (12%), AMLM1 (12%), APML (11%), AML t(8;21) (9%). CMLBC included myeloid blast crisis (40 cases), lymphoid blast crisis (2 cases), and biphenotypic leukemia (3 cases). CD13 was most sensitive and CD117 most specific for determining myeloid lineage. An immunoprofile of CD34+, HLADR+, CD14+ and CD33− is unlikely to be APML. CD117 is not a sensitive marker for APML and shows negative to weak continuous expression on promyelocytes. CD19 was expressed in 45% of FAB AMLM2 with t(8;21)(q22;q22). At least five cases of hepatosplenic gamma delta T-cell lymphomas were misdiagnosed as T-ALL based on a minimal selective panel where blasts expressed surface CD3. These cases were correctly diagnosed at relapse. Overall incidence of AML in adults is low (53% only). T-ALL is common in adolescent males. We recommend at least ten antibodies mainly CD13, CD33, CD117, CD10, CD19, HLADR, CD7, CD5 (or CD2), CD45 and CD34 as a primary minimal panel for a case of AL. Tdt is invaluable in work-up of pediatric T-cell neoplasm, to differentiate T-ALL from hepatosplenic gamma delta T cell lymphoma. Only 8% cases required additional markers (including intracytoplasmic markers) in the secondary panel for a lineage assignment. cCD22 is more sensitive than cCD79a for B-cell lineage assignment. Drawbacks of this study included referral bias, no clinical correlation, and three color immunophenotyping with FSC/SSC gating. Our approach for immunophenotyping was semi directed, a primary minimal panel followed by additional antibodies, as and when required. Cytoplasmic lineage specific markers (used in additional panel) for a lineage assignment were required only in 8% cases (n=199). We used only 14–20 antibodies per case (mean = 16), a number much less then many other laboratories. WHO classification has made it mandatory to do FISH/PCR to diagnose various leukemias which otherwise could be diagnosed with reasonable accuracy based on morphology, cytochemistry and flow cytometry like CML, APML, AMLM4Eo etc. Significant number of our FAB AMLM2 and AMLM4E0 cases did not show associated translocations. Few patients were partially treated by referring physician (steroids, blood transfusion, heavy metals etc), a category not addressed in WHO classification. Cytogenetics made a major difference in therapeutic decision in 0.8% cases only. Though flow cytometry is extremely popular amongst hematopathology laboratories, cytogenetics is available at few selected centers in countries with limited resources.

Expression of the Cell Polarity Protein PTK7 in Acute Myeloid Leukaemia.

The planar cell polarity pathway plays a major role in embryogenesis and tissue organisation. Recent genetic studies have highlighted the role of novel receptors and signaling molecules implicated in this pathway. Amongst the receptors, the pseudo tyrosine kinase receptor 7 (PTK7) is an orphean tyrosine kinase receptor with kinase-dead activity. Knock-out of PTK7 in mice strongly affects embryonic development leading to a major neural tube defect. Presence of PTK7 was previously investigated in epithelial and endothelial cells that both express the receptor. In normal donors, we found no expression of PTK7 in peripheral blood (n=5) whereas PTK7 expression was found with low levels in PBPC after G-CSF stimulation (n=3) and high levels in normal myeloid progenitors and CD34+ CD38− bone marrow cells (n=3). Overexpression of PTK7 was already described in solid tumors including breast, lung and pancreatic cancers. We decided to study the potential implication of PTK7 in haematological malignancies. We performed a wide range multicolour immunophenotyping screen on more than 240 patient samples treated at Institut Paoli-Calmettes and 10 leukemia cell lines. In hematologic malignancies, we demonstrated that PTK7 was widely expressed in AML (136 of 195 patients) and in the most immature subsets of Acute lymphoblastic (5 of 20 patients) or biphenotypic leukaemia (3 of 3 patients). We found no expression of PTK7 in chronic disorder such low grade NHL (n=7), CLL (n=6) or Chronic Myelomonocytic Leukemia (n=3). In AML, we demonstrated that PTK7 expression mostly correlates with granulocytic lineage differentiation and that it could be partially expressed in AML 4 or 5 subsets. Flow cytometry analysis confirmed the co-expression of PTK7 with granulocytic lineage markers and that PTK7 expression in myelomonocytic leukaemia was limited to the myeloid subset of blasts. The strongest immunophenotyping correlation was found with CD117/c-Kit expression (p<0.001) and PTK7 Mean Fluorescence Intensity directly correlates with c-Kit MFI (p=0.001). Interestingly, stimulation of cultured TF1 cells (that endogenously express c-Kit and PTK7) with SCF triggered an increased expression of PTK7. Correlation between PTK7 expression and biological or clinical features was also evaluated. We demonstrated that PTK7 expression clustered with some cytogenetic subsets (high levels in CBF AML (n=19) or APL (n=13), low levels in FLT3 mutated AML(n=17) or complex karyotype (n=20)). We also found that PTK7 expression was associated with a lower WBC count at diagnosis (p=0.001) and a lower frequency of extramedullary disease (p<0.001) in whole population and in both AML1–3 and AML 4–5 subgroups. We report here novel findings that potentially implicate ptk7, a PCP gene, in hematopoiesis and AML. In vitro, we showed that ectopic expression of PTK7 promotes cell migration, cell survival and resistance to anthracyclin-induced apoptosis. Ongoing works are currently investigating which molecular mechanisms regulate PTK7 functions in normal and pathological situations.

Biological features and outcome of biphenotypic acute leukemia: a case series

Biphenotypic acute leukemia (BAL) is a distinct entity that is immunophenotypically defined by the European Group for the Immunological Classification of Leukemia (EGIL) scoring system and accounts for less than 5% of all acute leukemia cases. Since it is a rare and heterogeneous form of acute leukemia with an allegedly poor outcome, there is no consensus on the best treatment approach in these patients. Our objective was to analyze the biological features and outcome of patients diagnosed with BAL in our institution. Using the EGIL system, we identified 21 cases (3.9%) of BAL from 535 newly diagnosed acute leukemia patients in an 11-year period. There were ten cases of myeloid+B-lymphoid leukemia, eight cases of myeloid+T-lymphoid, one case of B+T-lymphoid and two cases of trilineage (myeloid+B+T-lymphoid leukemia). The complete remission (CR) rate with high-dose chemotherapy was 72% and overall survival at 5 years was 21%. Patients that received acute lymphoblastic leukemia-oriented chemotherapy had a higher CR rate compared with those who received acute myeloid leukemia-oriented chemotherapy (100% vs. 60%, P = .007). The white blood cell count at diagnosis was found to have statistically significant impact on survival. Despite the progress in the treatment of acute leukemia, the prognosis of BAL remains poor and treatment protocols devised explicitly for this entity should be investigated in prospective collaborative studies.