Expression Of Lymphoid Antigens Research Articles

CD74 is expressed in a subset of pediatric acute myeloid leukemia patients and is a promising target for therapy: a report from the Children's Oncology Group.

As curative therapies for pediatric AML remain elusive, identifying potential new treatment targets is vital. We assessed the cell surface expression of CD74, also known as the MHC-II invariant chain, by multidimensional flow cytometry in 973 patients enrolled in the Children's Oncology Group AAML1031 clinical trial. 38% of pediatric AML patients expressed CD74 at any level and a comparison to normal hematopoietic cells revealed a subset with increased expression relative to normal myeloid progenitor cells. Pediatric AML patients expressing high intensity CD74 typically had an immature immunophenotype and an increased frequency of lymphoid antigen expression. Increased CD74 expression was associated with older patients with lower WBC and peripheral blood blast counts, and was enriched for t(8;21), trisomy 8, and CEBPA mutations. Overall, high CD74 expression was associated with low-risk status, however 26% of patients were allocated to high-risk protocol status and 5-year event free survival was 53%, indicating that a significant number of high expressing patients had poor outcomes. In vitro pre-clinical studies indicate that anti-CD74 therapy demonstrates efficacy against AML cells but has little impact on normal CD34+ cells. Together, we demonstrate that CD74 is expressed on a subset of pediatric AMLs at increased levels compared to normal hematopoietic cells and is a promising target for therapy in expressing patients. Given that nearly half of patients expressing CD74 at high levels experience an adverse event within 5 years, and the availability of CD74 targeting drugs, this represents a promising line of therapy worthy of additional investigation.

Rare Absence of CD79alpha in B-ALLs is not Associated with Altered Prognosis

Background: The diagnosis of B-cell acute lymphoblastic leukemia (B-ALL) on the basis of the World Health Organization (WHO) recommendations in 2016 requires the expression of CD19 with CD79a (CD79a), cCD22 and CD10. CD79a is an integral B cell antigen expressed at all stages. Its absence in B-ALL has only been rarely reported. In this study, we described three cases of B-ALL showing complete loss or extremely weak expression of CD79a. Methods: Seventy-five patients with B-ALL diagnosed by flow cytometry over a period of 3.5 years were taken out from the records. Cases lacking for CD79a by flow cytometry were further analysed. Results: Out of 75 cases, CD 79a negativity was seen in 4% (3/75). However, every three cases expressed other B cell markers. No aberrant expression of myeloid and lymphoid antigens was seen in any of these three cases. The prognosis of these three cases was similar to CD79a positive cases. Conclusion: Rarely, B cell ALL may lack CD79a. Thus, to avoid the misdiagnosis, a comprehensive antibody panel should be used. There was no prognostic impact of CD79a negativity in this study, however larger studies are needed to confirm these results.

Frequency and Clinical Significance of P-Glycoprotein and Multidrug Resistance-Associated Protein-1 Expression in Leukemic Cells from Acute Myeloid Leukemia

BACKGROUND: Despite advances in the cure rate for acute myeloid leukemia, a considerable number of patients die from the disease due to multidrug resistance (MDR). Overexpression of the transporter proteins P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP) provide resistance to the treatment of these leukemias. OBJECTIVE: To analyze Gpp and MRP1 expression in patients with AML using flow cytometry (FC) and determine the correlation between expression and demographics and clinical and laboratory variables. METHODS: Bone marrow and peripheral blood samples from 346 patients diagnosed with AML were assessed for Pgp and MRP1 expression by FC. RESULTS: Pgp and MRP1 expression was found in 111 (32.1%) and 133 (38.4%) patients, respectively, with greater prevalence in older patients and lower in adolescents, in addition to a high incidence in patients with refractory, recurrent and secondary disease in comparison with cases of de novo AML. With respect to laboratory findings, we observed a statistically significant higher correlation between Pgp and MRP1 expression in AML CD34+ and FAB AML M7, M5A and M2 and lower in the M3 subtype, with no statistically significant correlation between the phenotype MDR and other laboratory data such as hemoglobin, leukocyte count, platelet count, and aberrant lymphoid antigen expression (CD2, CD7 and CD19) and clinical signs related to the disease. CONCLUSIONS: The results showed that detecting MDR phenotype by flow cytometry may be a molecular marker for the independent prognosis of patients diagnosed with AML. DisclosuresNo relevant conflicts of interest to declare.

Clinical and hematological correlates of aberrant immunophenotypic profiles in adult and pediatric acute myeloid leukemia at presentation.

Aberrant phenotypes in acute leukemia have been reported with varying frequencies in independent studies and their association with prognostic factors is still a matter of debate. This study aims to identify the frequency of aberrant immunophenotypes in de novo acute myeloid leukemia (AML) and to evaluate their association with initial clinical and hematological features. A total of 181 patients of de novo AML were included during the time (July 2010-June 2012). The immunophenotype of all cases of AML was studied by using flow cytometry. Aberrant lymphoid antigen expression was seen in 43.1% cases. Most frequent aberrant lymphoid antigen was CD7, seen in 26.5% cases. All French-American-British (FAB) subtypes except AML-M3 expressed aberrant lymphoid antigens. The expression was most common in AML-M4 in the current study. CD34 expression in AMLs was significantly associated with the expression of aberrant lymphoid antigens. Lymphoid antigen expression in adult AML was significantly associated with higher white blood cell (WBC) count (>50,000/mm3) and higher number of peripheral blasts (>70%). In summary, CD7 is the most common aberrant lymphoid antigen expressed in AML. FAB subtype AML-M3 is usually not associated with aberrant lymphoid antigen expression. AML cases with CD34 positivity are more likely to express aberrant lymphoid markers. The current study also supports that aberrant lymphoid antigen expression in adult AML is associated with adverse presenting hematological features (WBC count >50,000/mm3, peripheral blasts >70%). Pediatric Ly + AML cases are not associated with adverse presenting clinical and biological features.

Analysis of Heterogeneous Biological Characteristics and Prognosis-Related Factors in AL Patients with Mistranslation-Expressed Lymphoid and Myeloid-Related Antigen

To analyze the heterogeneous biological characteristics of acute leukemia (AL) patients with mistranslation expressed lymphoid and myeloid-related antigens, and it's prognosis-related factors. Two hundred and fourteen AL patiens with mistranslation expressed lymphoid and myeloid-related antigens were grouped according to immunophenotypes, and the heterogeneous biologic charecteristics and prognosis related factors were analyzed, moreover the survival curves were drawn to analyze the survival of patiens. The immunophenotype in 214 cases was mainly cross-expression of myeloid and B lineage antigen (118 cases), followed by cross-expression of myeloid antigen and T lineage (88 cases), while the cross-expression of myeloid, T and B lineages, was less (only 8 cases). In ALL patiens with cross-expression of myeloid antigen, the CD33 was main type; while in AML patients with cross-expression of lymphoid antigen, CD7 was main type of lineage antigen, CD19 was main type of B lineage antigen. Among 214 AL patients, the cross-expression of CD55 and myeloid antigen was found in 30 cases, the cross-expression of CD7, CD19 and CD74 was observed in 6 cases, the cross-expressions of CD7, CD34 and CD56 was detected in 4 cases. Among AML patients with lymphoid antigen expression, the recurrent chromosmal abnormalities were found in 16 cases; among ALL patients with myeloid antigen expression, the recurrent chromosomal abnormalities were observed in 10 cases. The mistranslation antigen expression existed in 26 patients with recurrent chromosomal abnormalities, the mistranslated CD33 and CD13 in ALL patients with myeloid antigen expression was common, while the mistranslated CD2, CD56 and CD19 in AML patients with lymphoid antigen expression was common. As compared with patients without lymphoid antigen expression, the survival rate decreased significantly in patients with mistranslated CD7(+) and CD34(+) (both P<0.05). The logistic regression analysis showed that CD7, CD34 were main influencing factors for prognosis of AL patients (both P<0.05). The AL with mistranslation expressed lymphoid and myeloid antigens is a special kind of leukemia which possesses the heterogencous biological characteristcs and unique prognostic features, thus the immunophemotype of AL patients should be detected by flow cytometry. The existance of mistranlation-expressed differatiation antigens such as CD7 and CD34 is mainly influencing factors for the prognosis of AL patiens.

Clinical and laboratory features of pediatric acute myeloid leukemia with inversion of chromosome 16

To evaluate the clinical and laboratory features of pediatric inv(16) acute myeloid leukemia (AML) retrospectively. Dual color fluorescence in situ hybridization (D-FISH) using a LSI CBFβ inv(16) break apart probe labeled by Spectrum red and Spectrum green was performed in 15 acute myeloid leukemia cases, including 13 cases with or without abnormal eosinophils but with positive core binding factor β (CBFβ)-MYH11 fusion transcript detected by RT-PCR, and 2 cases with trisomy 8 (+8). The results were compared with the morphology, immunophenotype, karyotype and RT-PCR. Morphologically, 12 cases were diagnosed as M(4)EO, 2 as M(4), and 1 as M(2a). Immunophenotypically, all 13 AML cases with inv(16) showed positive expression of CD(13) and CD(33), but without the expression of any lymphoid lineage antigens. Karyotyping analysis with G-banding detected inv(16) in 10 AML cases, including 9 M(4)EO cases and 1 M(2a), but only 5 positive cases were detected using R-banding technique. Among them, 2 cases had simultaneous +8 and trisomy22 (+22), one had +22 only in addition to inv(16). D-FISH revealed a CBFβ-MYH11 rearrangement in 13 cases of AML with positive RT-PCR results, and the mean positive rate of cell detection was 55.15% (range 37.0% - 86.0%). The complete remission rate (CR) and median survival period in this series of inv(16) AML were 81.5%and 11 months, respectively, of whom, 8 cases were still in CR. Relapse and karyotypic evolution were seen in case 5 with +8, +22 in addition to inv(16). AML with inv(16) is a special subtype. Most cases belong to M(4)EO. Its prognosis is good in general, but it seems to be an unfavorable feature for AML with inv(16) and +8, +22 simultaneously, especially with karyotypic evolution. For detection of inv(16), G-banding technique is evidently superior to R-banding technique. D-FISH combined with RT-PCR are more sensitive and reliable than chromosome banding analysis.

Association between P-glycoprotein and lymphoid antigen expression on myeloblasts versus therapy response and survival in de novo acute myeloid leukemia: long-term follow-up results

P-glycoprotein (PGP) over-expression on malignant cells is associated with poor prognosis and treatment outcome due to the development of a multidrug resistance phenotype. In this study, we analyzed the correlation between expression of PGP and lymphoid antigens (Ly) on leukemic myeloblasts versus response to therapy and survival in acute myeloid leukemia (AML). Fifty-one consecutive patients, aged 16-75 (median age 44.6 years), diagnosed with de novo AML between 1997 and 2000, and who received at least one induction chemotherapy course, were enrolled in the study. Expression of PGP on ≥ 10% of the myeloblasts (PGP(+)AML) at the time of diagnosis was observed in 21 patients (41%). The complete remission rate did not differ between PGP(+) (13/21) and PGP(-) (20/30) patients (62 vs. 67%). Twelve of the 51 patients (24%) were still alive after a median follow-up time of 11.5 years (range 10.7-13.1). The Ly(+)AML patients showed significantly better overall survival compared with Ly(-)AML patients (8/18 vs. 4/33 patients alive at the last follow-up, P = 0.003). The subgroup of patients with co-expression of PGP and Ly also showed better overall survival compared with PGP(+)AML patients without Ly expression (4/8 vs. 0/13 patients alive at the last follow-up; P = 0.04). Our results suggest that expression of lymphoid antigens on PGP(+) myeloblasts in AML can positively affect survival in AML patients, mainly due to a decreased relapse risk and better survival. Although the small number of patient may be perceived as a limitation of the study, the long follow-up period strengthens its value. Further prospective trials are needed to obtain more information concerning the association between PGP and lymphoid antigens in AML, which would put our results in their ultimate proper context.

Flow cytometric scoring system (FCMSS) assisted diagnosis of myelodysplastic syndromes (MDS) and the biological significance of FCMSS‐based immunophenotypes

In the myelodysplastic syndromes (MDS), the haematopoietic cells show various levels of abnormal maturation and differentiation, which can be detected by flow cytometry. Testing the anomalies of stage- or lineage-specific surface antigens in CD34+ blasts can distinguish MDS from non-clonal cytopenic diseases, and also reflect the pathological characteristics of MDS as a class of clonal diseases for providing new clues to basic research. The present study established a flow cytometric scoring system (FCMSS) based on theproportion and antigenic co-expression of CD34+ blasts. This FCMSS showed good sensitivity and specificity (77.8% and 100%) in the assisted diagnosis of low-risk MDS without chromosome anomalies, ringed sideroblasts and excess marrow blasts. Moreover, we explored and reported different modes of abnormal expression of CD34+ blasts antigens in different disease stages and analyzed the biological significance of the immunotypes for the first time. We found expression of mature myeloid antigens and lymphoid antigens gradually decreased, and early functional antigens gradually increased from low-risk MDS with normal karytype to low-risk MDS with abnormal karyotype then to high-risk MDS. The patients with higher FCM scores were generally accompanied with HLA-DR15 allele or hypocellular marrow. Evolution of clones and immunological factors might have influence on expression of antigens in CD34+ blasts.

Common misdiagnoses in lymphomas and avoidance strategies

Lymphoma diagnosis integrates clinical, morphological, immunophenotypical, and molecular genetic features, as shown in WHO classifications of lymphoid malignancies. Diagnosis of lymphoma is challenging. Reactive lesions such as Kikuchi lymphadenitis, infectious mononucleosis, autoimmune lymphoproliferative syndrome, and immunoglobulin G4-related sclerosing disease can be misdiagnosed as lymphomas. Anaplastic large-cell lymphoma variants that are positive for anaplastic lymphoma kinase, classical Hodgkin's lymphoma variants, and infarcted lymphomas might be misdiagnosed as reactive disorders. Difficulties with classification of lymphomas are also encountered, such as the distinction of classical Hodgkin's lymphoma from anaplastic large-cell lymphoma that is negative for anaplastic lymphoma kinase. Interpretation of immunophenotyping results is complicated in some cases by aberrant or cross-lineage expression of lymphoid antigens on lymphomas, and the occasional lymphoid antigen expression on non-lymphoid malignancies. Molecular analysis can help to define clonality and lineage, but can be affected by the sensitivity and specificity of tests and cross-lineage gene rearrangement and pseudoclonality. To resolve these issues, a close collaboration between the clinician, histopathologist, and molecular biologist is needed. The aim of this review is to provide pathologists and clinicians with a concise account of these pitfalls and avoidance strategies.

Flow Cytometry Analysis of Peripheral Blood CD34+ Cells in Patients with Primary Myelofibrosis

Primary myelofibrosis (PMF) is a clonal chronic myeloproliferative disorder characterized by the accumulation of megakaryocytes in the bone marrow (BM), variable degrees of BM fibrosis, tear-drop erythrocytes, increased numbers of CD34+ hematopoietic progenitors in the peripheral blood (PB), and extramedullary hematopoiesis. Since the antigenic properties of the circulating CD34 cells may yield clues to disease pathogenesis and have not been extensively studied, we used five-color flow cytometry to examine these cells from 20 well characterized patients with PMF and 10 normal controls. Bone marrow biopsies, molecular and cytogenetic studies were also reviewed. As expected, the percentages of peripheral-blood CD34 cells were significantly higher in the PMF patients (mean 1.4%, range, range 0.065–7.15) compared to the controls (mean 0.05%, range 0.01–0.57). The mean fluorescence intensity (MFI) values related to HLA-DR expression were increased (more than 3 fold) on the CD34+ cells in 12/20 (60%) PMF patients relative to normal control levels, while increased levels of CD13 were seen in 5/20 (25%) of PMF patients. CD33 and CD117 expression were similar on the CD34+ cells in both groups. Aberrant expression of lymphoid antigens was observed in 6/20 (30%) with CD7, 6/20 (30%) with CD4, and 3/20 (15%) with CD56 on CD34 positive cells in PMF. In the18 cases also studied with antibodies against CD45RA and CD123, the majority of CD34+ CD38 + cells phenotypically resembled megakaryocyte-erythroid precursors (CD45RA−, CD123−) in 5 cases, common myeloid progenitors (CD45RA−, CD123+) in 12 cases, and granulocyte-macrophage progenitors (CD45RA+, CD123 +) in 1 case. JAK2-V617F mutations were detected in 9 of 20 cases, but were present in only 1 of 5 cases showing predominately megakaryocyte-erythroid precursors. The percentage of CD34+ cells also expressing CXCR4 (CD184) appears to be increased in some patients relative to normal controls in contrast to other reported studies. In conclusion, the peripheral blood CD34+, progenitor cells in PMF patients are heterogeneous phenotypically resembling megakaryocyte-erythroid precursors in approximately 30% of cases, and common myeloid progenitors in approximately 70% of cases. In addition, these cells often show phenotypic abnormalities (increased intensity of HLA-DR and CD13 expression) that can be detected with flow cytometry relative to normal peripheral blood CD34+ cells. Patterns of antigen expression in PMF also appear to differ from those reported for CD34 positive cells in other myeloproliferative disorders which may help in early diagnosis and/or monitoring treatment responses.

Bi-Phenotypic Leukemic Cells as Transient Intermediates Between Leukemic Stem Cells with Lymphoid Characteristics and the Leukemic Myeloid Bulk: Insights from a Murine Model of CALM/AF10 Positive Acute Myeloid Leukemia

Co-expression of lymphoid and myeloid surface markers is a common phenomenon in human acute myeloid leukemia (AML). One explanation for this could be the aberrantly expression of lymphoid antigens on malignant myeloid cells. Another intriguing possibility is that these leukemic bi-phenotypic cells emerge from a leukemic stem cell (LSC) with lymphoid characteristics. In this scenario co-expression of lymphoid and myeloid markers on a substantial proportion of the leukemic cell population would rather reflect the nature of the LSC in these AML cases than deregulated antigen expression of a malignant transformed cell. We have recently characterized the LSCs in a murine model of CALM/AF10 positive AML. In each leukemic mouse we could demonstrate the presence of three distinct populations, a minor B220+/myeloid marker (MM)− population which showed the highest leukemogenicity in vitro and in vivo (candidate LSCs), a bi-phenotypic population which was MM+/B220+, and the leukemic bulk population which expressed only myeloid markers without the expression of the lymphoid associated marker B220 (MM+/B220−) (Deshpande et al. Cancer Cell 2006). We now sought to analyze the relationship between these three leukemic populations. Single cell-sorted LSC candidates (B220+/MM−) could give rise to the other leukemic populations in vitro. Real time RT-PCR for 5 lymphoid-associated and 5 myeloid-associated transcripts showed that most lymphoid-associated genes were highly expressed in the B220+/MM− cells and their expression levels were decreased or not detectable in the myeloid bulk. The converse was true with the myeloid associated transcripts confirming that the LSCs were more lymphoid-like than the myeloid bulk that they generate. The expression of lymphoid-associated genes such as Rag2, Pax5, IL7Ra and Ebf1 showed a progressive downregulation and that of myeloid associated transcripts such as c-fms, GM-CSFR, GCSF-R showed a progressive upregulation upon differentiation of single B220 positive cell to the myeloid lineage. The expression of most (7 out of 10) of these lineage-associated genes was intermediate in the B+/MM+ cells suggesting a transient lympho-myeloid stage of differentiation. We then performed a DNA microarray analysis of these sub-populations by using a chip with 20,172 PCR-amplified, sequence-verified, gene-specific DNA fragments (LION Biosciences). Correlation based principal component analysis (PCA) was conducted on the normalized expression values of genes showing differential expression in the three cell populations. Two statistically significant principal components were extracted, explaining more than 95% of the variation, where the genes were clustered into three classes based on their loadings. These clusters were overlaid with the loadings of the three cell populations to see whether the clusters match to a certain cell type. We observed that the three cell populations actually corresponded to the three clusters of genes and interestingly the B220+/MM+ bi-phenotypic cells lie between the B220+/MM− LSCs and the B220−/MM+ bulk. These data demonstrate that the AML in the CALM/AF10 murine model is arranged as a hierarchy of progressively differentiated cells with the B220+/MM+ bi-phenotypic cells as transient intermediates between the B220+/MM− LSCs and the myeloid B220−/MM+ bulk. These data support a model in which myelo-lymphoid cells observed in cases of human AML could arise as intermediates during the differentiation process of LSCs with lymphoid characteristics into leukemia with predominantly myeloid features. These observations encourage studies which test the hypothesis that AML subtypes characterized by co-expression of lymphoid and myeloid surface markers are originating from a leukemia propagating cell with lymphoid characteristics. The presence of lymphoid markers on LSCs in certain AML sub-sets could pave the way for a more effective anti-leukemic therapy.

Induction of acute lymphocytic leukemia differentiation by maintenance therapy

Despite extensive study in many malignancies, maintenance therapy has clinically benefited only two diseases: acute lymphocytic leukemia (ALL) and acute promyelocytic leukemia (APL). ALL maintenance therapy utilizes low-dose 6-mercaptopurine (6MP) and methotrexate (MTX), while maintenance in APL primarily consists of all-trans-retinoic acid (ATRA). 6MP and MTX as used in ALL are also now usually added to maintenance ATRA for APL, based on data suggesting an improved disease-free survival. Although the mechanism of action of MTX and 6MP as maintenance is unknown, low-dose cytotoxic agents are potent inducers of differentiation in vitro. Thus, we studied whether maintenance therapy in ALL, like ATRA in APL, may be inducing terminal differentiation of ALL progenitors. The APL cell line NB4, the ALL cell lines REH and RS4;11, and patients' ALL blasts were incubated with ATRA, 6MP, and MTX in vitro. All three drugs inhibited the clonogenic growth of the APL and ALL cell lines without inducing immediate apoptosis, but associated with induction of phenotypic differentiation. The three drugs similarly upregulated lymphoid antigen expression, while decreasing CD34 expression, on patients' ALL blasts. These data suggest that induction of leukemia progenitor differentiation plays an important role in the mechanism of action of maintenance therapy in ALL.

The Clinical Significance of Biphenotypia in Blastic Phase CML.

The characteristics and clinical outcomes of CML blast phase (CML-BP) patients whose blasts express antigens from more than one lineage are currently unknown. We systemically assessed lineage fidelity among patients with CML-BP using the definition of biphenotypic leukemia as proposed by the European Group for the Immunological Characterization of Leukemias (EGIL; Leukemia 1995; 9:1783). Briefly, individual antigens are assigned scores based on known specificity for one of three lineages (myeloid, B-lymphoid or T-lymphoid); biphenotypic leukemia is defined by a score of >2 points for two lineages. Between 02/97 and 01/06, 163 patients were treated for CML-BP at the MD Anderson Cancer Center, of which 120 had adequate flow cytometric assessment for calculation of EGIL score. The number of patients by lineages was: 89 myeloid (74%), 21 lymphoid (17.5%; B 14%, T 3.5%), 9 biphenotypic (7.5%), 1 unclassifiable (<1%). Four of nine biphenotypic BP patients had a history of antecedent myeloid BP (n=3) or lymphoid BP (n=1). Lymphoid BP patients had lower mean white cell count (p=0.04), lower mean platelets (p=0.007) and higher mean bone marow blast percentage (p<0.001) than myeloid BP patients; those with biphenotypic BP had values between the lymphoid and myeloid groups. Seven imatinib-naïve biphenotypic patients received imatinib as initial therapy for CML-BP, with six regaining a second chronic phase; however, median time to failure was only 2 months and, with the exception of one patient censored for allogeneic transplantation, all patients progressed within 4 months. The patient who underwent allogeneic transplantation died of transplant-related complications. An additional patient who had previously failed imatinib and nilotinib in chronic phase had no response to dasatinib in biphenotypic BP. The final patient developed biphenotypia during resistant relapse of previous B-lymphoid BP, and received supportive therapy only. Overall survival for lymphoid, myeloid and biphenotypic BP were 13, 8 and 6 months respectively (p=0.04). In order to determine the significance of minor degrees of lineage aberrancy, we examined the survival of myeloid and lymphoid BP patients with EGIL score 1.0 – 2.0 in a second lineage. For lymphoid BP patients, minor expression of myeloid antigens did not affect survival; in contradistinction, myeloid BP patients with minor expression of lymphoid antigens had a trend to inferior survival. Biphenotypic BP may either be de-novo or may emerge from previous unilineage CML-BP, highlighting the trend for lineal infidelity in CML-BP. Responses to tyrosine kinase inhibitors are short-lived, and survival using current therapeutic strategies is very poor. [Display omitted]

The AML1-ETO Fusion Gene and the FLT3 Length Mutation Collaborate in Inducing Acute Leukemia in a Murine Bone Marrow Transplantation Model.

Experimental data have shown that two of the most frequent genetic alterations in AML, the AML1-ETO (A/E) fusion gene and the FLT3 length mutation (FLT3-LM) are both mostly insufficient on their own to induce leukemia. These findings support the model that collaboration of two classes of genetic alterations, altering proliferation or differentiation, is necessary for leukemogenesis. When we first analyzed 135 patients with A/E positive AML, additional mutations affecting signal transduction were found in 38 % of all cases (FLT3-LM 10.3 %, KIT 8.1 % and NRAS 9.6 %). In contrast, none of the patient with A/E positive leukemia had alterations associated with transcriptional regulation such as MLL-PTD. To test the hypothesis that A/E collaborates with FLT3-LM in inducing acute leukemia, we transplanted mice with bone marrow (BM) cells retrovirally expressing A/E, FLT3-LM or both alterations. Mice transplanted with BM cells expressing A/E or FLT3-LM alone did not develop any disease. In contrast, mice (n=11) transplanted with BM cells expressing both alterations succumbed to an aggressive acute leukemia. Intriguingly, developing leukemias differed with regard to their phenotype with 7 animals developing AML and 4 animals developing ALL. Furthermore, the majority of AML cases showed simultaneous expression of lymphoid antigens as described in patients with A/E positive AML. The collaboration of A/E with FLT3-LM was depending on DNA binding activity of the fusion gene as the L148D point mutation in the Runx1 domain of the construct abrogated collaboration of A/E with the FLT3-LM in the CFU-S assay. Furthermore, inactivation of the kinase activity of the FLT3-LM (FLT3-LM K672R mutant) resulted in the complete loss of collaboration with the A/E fusion. Treatment of cells co-infected with A/E and FLT3-LM with the kinase inhibitor PKC412 resulted in a 62 % reduction of the CFU-S frequency. To further explore a possible contribution of retroviral insertional mutagenesis to the transformation process in this model, 10 retroviral integration sites were subcloned and sequenced from 4 leukemic mice: all 10 sites were unique with no indication of a common integration site associated with the leukemic transformation. Moreover, 5 sites were intergenic or not linked to known genes. The remaining sites were in introns in a 5′ to 3′ orientation most likely to lead to gene knockdown rather than activation. These data provide direct functional evidence for the oncogenic collaboration between A/E with a class of activating mutations, recurrently found in patients with t(8;21), and add experimental data to the clinical observation which demonstrated a significant inferior treatment outcome in patients with AML1-ETO and additional mutations of receptor tyrosine kinases.

Immunological classification of acute myeloblastic leukemias: relevance to patient outcome.

Immunophenotyping is a major tool to assign acute leukemia blast cells to the myeloid lineage. However, because of the large heterogeneity of myeloid-related lineages, no clinically relevant immunological classification of acute myeloblastic leukemia (AML) has been devised so far. To attempt at formulating such a classification, we analyzed the pattern of expression of selected antigens, on blast cells collected at AML diagnosis. Patients were eligible if they had a first diagnosis of de novo AML and a sufficient number of blast cells for proper immunophenotyping. The relative expression of CD7, CD13, CD14, CD15, CD33, CD34, CD35, CD36, CD65, CD117, and HLA-DR were analyzed by cytometry in a test series of 176 consecutive AML cases. Statistical tools of clusterization allowed to remove antigens with overlapping distribution, leading us to propose an AML classification that was validated in a second AML cohort of 733 patients. We identified five AML subsets (MA to ME) based on the expression of seven antigens within four groups (CD13/CD33/CD117, CD7, CD35/CD36, CD15).-MA and MB-AML have exclusively myeloid features with seldom extramedullary disease and rare expression of lymphoid antigens. No cases of acute promyelocytic leukemia (APL) were observed within MB AML. MC AML have either myeloid or erythroblastic features. MD AML have more frequently high WBC counts than other subsets, which were related to the expression of CD35/CD36 and CD14 and to monoblastic differentiation. ME AML lack CD13, CD33, and CD117 but display signs of terminal myeloid differentiation. Specific independent prognostic factors were related to poor overall survival in each immunological subset: CD34+ (P<3 x 10(-4)) in MA AML, CD7+ in MB AML, non-APL cases (P<0.03) in MC AML, CD34+ (P<0.002) and CD14+ (P<0.03) in MD AML, CD14+ in ME AML (P<0.01). The inclusion of seven key markers in the immunophenotyping of AML allows a stratification into clinically relevant subsets with individual prognostic factors, which should be considered to define high-risk AML populations.

Haematologic and immunophenotypic profile of acute myeloid leukemia : an experience of Tata Memorial Hospital

To study the hematologic and immunophenotypic profile of 260 cases of acute myeloid leukemia at diagnosis. This is a retrospective analysis of 260 cases of AML diagnosed at our institution between 1998 and 2000. Diagnosis was based on peripheral blood and bone marrow examination for morphology cytochemistry and immunophenotypic studies. SPSS software package, version 10, was used for statistical analysis. Seventy-six percent of our cases were adults. The age of the patients ranged from one year to 78 years with a median age of 27.2 years. There were 187 males and 73 females. The commonest FAB subtype, in both children and adults, was AML-M2. The highest WBC counts were seen in AML-M1 and the lowest in AML-M3 (10-97 x 10(9)/L, mean 53.8 x 10(9)/L). The mean values and range for hemoglobin was 6.8 gm/l (1.8 gm/l to 9.2 gm/l), platelet count 63.3 x 10(9)/L (32-83 x 10(9)/L), peripheral blood blasts 41.4% (5 to 77%) and bone marrow blasts 57.6% (34-96%). Myeloperoxidase positivity was highest in the M1, M2 and M3 subtypes. CD13 and CD33 were the most useful markers in the diagnosis of AML. CD14 and CD36 were most often seen in monocytic (38%) and myelomonocytic (44%) leukemias. Lymphoid antigen expression was seen in 15% of cases. CD7 expression was the commonest (11%). AML accounted for 39.8% of all acute leukemias at this institution. The most common subtype was AML-M2. Myeloperoxidase stain was a useful tool in the diagnosis of myeloid leukemias. CD13 and CD33 were the most diagnostic myeloid markers.

Lymph node infarction. An immunohistochemical study of 11 cases.

Context The etiology of lymph node infarction may be difficult or impossible to determine by histologic examination. Lymph node infarction is followed by malignant lymphoma in some but not all patients. The role of immunohistochemistry in the evaluation of lymph node infarction is not well defined. Although it is widely believed that necrotic tissue is not suitable for immunohistochemical study, this view may be inaccurate. Objective To determine whether lymphoid antigens are preserved in infarcted lymph nodes and to determine the utility of immunohistochemical staining in the evaluation of lymph node infarction. Design Retrospective immunohistochemical study of infarcted lymph nodes using archival formalin-fixed, paraffin-embedded tissue. Setting Academic medical center. Patients Eleven adult patients with lymph node infarction retrieved from pathology files. Main outcomes measures Results of immunohistochemistry, diagnosis of lymphoma. Results Preservation of lymphoid antigens was observed in 4 of 6 cases of lymph node infarction associated with malignant lymphoma, including 3 of 5 cases of diffuse large B-cell lymphoma and 1 case of peripheral T-cell lymphoma. Nonspecific staining was not encountered. In 1 case, in which an infarcted lymph node showed a benign pattern of lymphoid antigen expression, lymphoma has not developed after 5 years. Conclusion Lymphoid antigens are frequently preserved in cases of lymph node infarction, and immunohistochemical study of infarcted lymph nodes may provide clinically useful information.

Immunophenotypic variability of B-cell non-Hodgkin lymphoma: a retrospective study of cases analyzed by flow cytometry.

Flow cytometric analysis is important in the diagnosis, classification, and follow-up of non-Hodgkin lymphoma. It is assumed that the lymphoma phenotype for each patient remains unchanged over time and is consistent from one specimen to another. To determine the variability in expression of lymphoid antigens, we reviewed 211 flow cytometry specimens of malignant lymphoma from 81 patients. Some antigens showed a stable pattern of expression such as CD5, CD10, CD19, CD20, and HLA-DR. In contrast, CD21, CD22, CD23, and CD25 showed more variability from one specimen to another. We believe several factors affect the stability of antigen expression. True differences in expression most probably are related to the biology and function of the different antigens. For instance, CD19 and CD20 are essential in cell maturation and function and, therefore, are present on the majority of cells. In contrast, CD22 has a role during B-cell activation and, therefore, is more variable. Lack of standardization inflow cytometry procedures also is responsible for some variability. Instrument settings for adequate compensation and the criteria used to determine when an antigen is reported as positive are important considerations when evaluating flow cytometry histograms.

Prognostic relevance of the expression of Tdt and CD7 in 335 cases of acute myeloid leukemia.

We have analyzed the expression of Tdt and CD7 in 335 cases of unequivocal acute myeloid leukemia (AML). Tdt was expressed in 80 (25%) of 321 evaluable cases. Twenty-six of 77 (34%) Tdt+ patients assessable for response, entered complete remission (CR) vs 121 of 209 (58%) Tdt- cases (P < 0.001). CD7 was expressed in 102 of 332 (30%) evaluable cases; 37 of 93 assessable (40%) CD7+ patients attained a CR as compared to 114/204 (56%) CD7- (P = 0.013). Duration of survival was significantly shorter for patients with CD7+ or Tdt+ AML (P = 0.006 and 0.001, respectively). In a multivariate analysis, Tdt was found to significantly adverse achievement of CR (P = 0.018), while CD7 affected duration of CR (P = 0.037). Overall the expression of either Tdt or CD7 correlated with a relatively high expression of CD34 (P < 0.001), GP-170 (P = 0.003), lymphoid antigens (LyAg) (P < 0.001), t(9;22) or anomalies of chromosome 5/7 (P < 0.001). Finally, we pooled the patients into four phenotypic classes, according to the presence of Tdt, CD7 or both: [Tdt-CD7-], [Tdt+CD7-], [Tdt-CD7+] and [Tdt+CD7+]. The category [Tdt+CD7+] was characterized by a more unfavorable outcome as suggested by a lower rate of CR (P < 0.001) and a shorter duration of survival as compared to cases [Tdt-CD7-], [Tdt+CD7-] and [Tdt-CD7+] (P = 0.002). This figure is consistent with the frequent convergence in the subset [Tdt+CD7+] of GP-170 positivity (P = 0.003), translocation t(9;22), anomalies of chromosome 5 and/or 7 (P < 0.001) and signs of lineage infidelity (deviant expression of lymphoid antigens) (P < 0.001). We conclude that the expression of Tdt or CD7 is associated with an unfavorable outcome and that the combination of both defines a clinical subset with a poorer prognosis due to the significantly higher association with MDR phenotype, and 'poor prognostic' chromosomal abnormalities.

Classification of acute leukaemia: the need to incorporate cytogenetic and molecular genetic information.

The purpose of any classification of leukaemia is to identify distinct biological entities which diVer in aetiology, mechanisms of leukaemogenesis, clinicopathological features, and prognosis. Since the optimal treatment for such...