Pg Subramanian

BCR-ABL1 kinase domain mutations: Methodology and clinical evaluation

The introduction of tyrosine kinase inhibitors (TKIs), starting with imatinib and followed by second and third generation TKIs, has significantly changed the clinical management of patients with chronic myeloid leukemia (CML). Despite their unprecedented clinical success, a proportion of patients fail to achieve complete cytogenetic remission by 12 months of treatment (primary resistance) while others experience progressive resistance after an initial response (secondary resistance). BCR‐ABL1 kinase domain (KD) mutations have been detected in a proportion of patients at the time of treatment failure, and therefore their identification and monitoring plays an important role in therapeutic decisions particularly when switching TKIs. When monitoring KD mutations in a clinical laboratory, the choice of method should take into account turnaround time, cost, sensitivity, specificity, and ability to accurately quantify the size of the mutant clone. In this article, we describe in a “manual” style the methods most widely used in our laboratory to monitor KD mutations in patients with CML including direct sequencing, D‐HPLC, and pyrosequencing. Advantages, disadvantages, interpretation of results, and their clinical applications are reviewed for each method. Am. J. Hematol., 2012.

Activity-based costing methodology as tool for costing in hematopathology laboratory

BACKGROUND Cost analysis in laboratories represents a necessary phase in their scientific progression. AIM To calculate indirect cost and thus total cost per sample of various tests at Hematopathology laboratory (HPL). SETTINGS AND DESIGN Activity-based costing (ABC) method is used to calculate per cost test of the hematopathology laboratory. MATERIAL AND METHODS Information is collected from registers, purchase orders, annual maintenance contracts (AMCs), payrolls, account books, hospital bills and registers along with informal interviews with hospital staff. RESULTS Cost per test decreases as total number of samples increases. Maximum annual expense at the HPL is on reagents and consumables followed by manpower. Cost per test is higher for specialized tests which interpret morphological or flow data and are done by a pathologist. CONCLUSIONS Despite several limitations and assumptions, this was an attempt to understand how the resources are consumed in a large size government-run laboratory. The rate structure needs to be revised for most of the tests, mainly for complete blood counts (CBC), bone marrow examination, coagulation tests and Immunophenotyping. This costing exercise is laboratory specific and each laboratory needs to do its own costing. Such an exercise may help a laboratory redesign its costing structure or at least understand the economics involved in the laboratory management.

Immunophenotyping of mature B-cell non Hodgkin lymphoma involving bone marrow and peripheral blood: critical analysis and insights gained at a tertiary care cancer hospital.

We evaluated the diagnostic utility of flow cytometry immunophenotyping in bone marrow aspirates and peripheral blood, in the assessment of mature B-cell non-Hodgkin lymphoma (MBNHL). We analyzed 356 cases of MBNHL received for immunophenotyping over a 4 year period. All cases were reviewed, correlated with biopsy specimen (lymph node and splenectomy). Discrepant cases were re-evaluated. Common subtypes included chronic lymphocytic leukemia (CLL) (243 cases, 68.5%), follicular lymphoma (30 cases, 8.5%), mantle cell lymphoma (20 cases, 5.5%), splenic marginal zone lymphoma (18 cases, 5%), hairy cell leukemia (18 cases, 5%). CD5+/CD23+ had a high positive predictive value (PPV) for diagnosing CLL whereas CD5+/CD23− had a high negative predictive value (NPV) for diagnosing mantle-cell lymphoma (MCL). Limited panel of 9 antibodies mainly CD19, CD5, CD23, CD10, FMC7, kappa, lambda, CD3 and CD20 help diagnose more than 92% of cases of MBNHL. Minimal diagnostic panels become important in countries with limited resources.

Immunophenotypic profile of plasma cell leukemia: a retrospective study in a reference cancer center in India and review of literature.

BACKGROUND Plasma cell leukemia (PCL) is a rare but aggressive subtype of plasma cell dyscrasia. It is known to present with highly variable morphological features and may mimic with other lymphoid neoplasms. Multicolor flow cytometry (MFC) with availability of newer markers is highly useful in the diagnosis of the plasma cell leukemia. We present an immunophenotypic profile in ten cases of PCL along with their clinical and laboratory findings. MATERIALS AND METHODS We retrospectively studied immunophenotypic profile of 10 cases of plasma cell leukemia (out of 4615 cases of hematolymphoid neoplasms) using five parameter, three color flow cytometric analysis. We also studied their clinical presentation and other laboratory findings. RESULTS Common clinical features at presentation were weakness, bone pain, anemia, thrombocytopenia and osteolytic lesions. Plasma cell population was identified on strong expression of CD38 and co-expression of CD38 and CD138. CD56 was expressed in 44% cases. CD19 and CD20 were negative in all cases. Surface light chain restriction was seen in 50% cases and in remaining 50% cases revealed cytoplasmic light chain restriction. CD117 was expressed in one out of two cases studied. CONCLUSIONS MFC immunophenotyping is highly useful to differentiate Plasma cell leukemia from other chronic lymphoproliferative disorders with plasmacytoid morphology as well as from non-neoplastic reactive PC and co-expression of CD38 and CD138 is a best combination to identify the plasma cells by MFC.

Immunophenotypic profile of acute leukemia: Critical analysis and insights gained at a tertiary care center in India

To analyze the spectrum of various types and subtypes of acute leukemia.

MYD88 mutant lymphoplasmacytic lymphoma/Waldenström macroglobulinemia has distinct clinical and pathological features as compared to its mutation negative counterpart

Abstract In a first series from India, we report 32 cases of lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM) over 7 years. Here, we analyzed 32 patients with LPL/WM for MYD88 L265P mutation and correlated mutation staus with hematological and biochemical parameters and also with the International Prognostic Scoring System (ISSWM) and treatment response. Twenty-seven out of 32 cases of LPL/WM (84.3%) harbored the MYD88 L265P mutation. MYD88 wild-type WM was associated with a lower number of tumor cells (p < 0.01) and older age (p = 0.02) and a lower ISSWM score at presentation (p = 0.03) as compared to mutated LPL/WM. On evaluation of response (n = 23), 44.4% of patients with MYD88 mutated LPL/WM had progressive disease, whereas no patient in the MYD88 unmutated group changed their baseline status. We confirm the high frequency of MYD88 mutations in LPL/WM. Although the number of MYD88 wild-type cases was limited, our data indicate that MYD88 may represent an adverse prognostic marker for LPL/WM.

Clinico-pathological profile of Hairy cell leukemia: Critical insights gained at a tertiary care cancer hospital

CONTEXT Hairy cell leukemia (HCL) is a rare, low grade, B-cell neoplasm with a characteristic morphologic and immunophenotypic profile. It has to be distinguished from chronic lymphoproliferative disorders because of different treatment protocol and clinical course. AIMS To evaluate clinicopathological features including immunophenotypic analysis of cases diagnosed as HCL. MATERIALS AND METHODS The present study included 28 cases diagnosed over a period of nine years (2002-2010). Clinical presentation, complete blood count, bone marrow aspirate, and flow cytometric analysis of cases were reviewed. Treatment and follow-up details (ranging from 3-90 months) were noted. RESULTS This study revealed 28 cases (referrals-7, indoor-21), aged 26-69 years with a median age of 47 years, with a male predominance (M:F=6:1). The presenting complaints were weakness (80%) followed by fever (56%) and abdominal pain. Physical examination revealed splenomegaly in most patients (92%) and hepatomegaly in a minority (28%). The common laboratory features were anemia in 23 cases, pancytopenia in 14 cases, while two patients had leukocytosis and three patients had normal WBC count. Dry tap was observed in 84% of the cases where hairy cells constituted 16-97% of non-erythroid nucleated cells. Tartarte resistant acid phosphate staining was positive in all the eight cases where it was done. CD5 was negative in all the cases, while CD10 was expressed in three cases (13%) and CD23 in five cases (19%). CONCLUSIONS Though pancytopenia is common, occasional patient can present with normal blood counts or leukocytosis. Few unusual findings include presence of lymphadenopathy, absence of palpable splenomegaly, and expression of CD23 and CD10 by the leukemic cells.

Study of the morphological patterns and association of Epstein-Barr virus and human herpes virus 8 in acquired immunodeficiency deficiency syndrome-related reactive lymphadenopathy

AIMS Study of the morphological patterns of acquired immunodeficiency syndrome (AIDS)-related lymphadenopathy. SETTINGS AND DESIGN We retrospectively selected cases of AIDS-related benign lymphadenopathy. Cases with lymphomas, frank granulomas and necrosis were excluded. We analyzed different morphological patterns and correlated these with immunophenotypic markers along with viral markers human herpesvirus 8-latency-associated nuclear antigen (HHV8-LANA), and Epstein-Barr virus-encoded ribonucleic acid (EBER) studies via in situ hybridization (EBER-ISH). MATERIALS AND METHODS We present the morphological patterns of 13 cases of human immunodeficiency virus (HIV)-reactive lymph nodes and their clinical, hematological, biochemical and radiological parameters with special emphasis on the presence or absence of viral markers, including HHV8 and EBV. RESULTS Common patterns included follicular hyperplasia only (five cases), mixed pattern of follicular hyperplasia with burnt-out germinal centres (four cases), completely atretic follicle (two cases), folliculolysis (11 cases), dumbbell-shaped follicles (three each), progressive transformation of germinal centers (four cases), T-zone expansion (two cases), Reed Sternberg (RS) cells like immunoblasts (two cases), Castlemans-like features with lollipop-like follicles (three cases) and a spindle cell prominence (one case). CD8+ T-cells were predominant in 12 cases. CD8+ T-cells were prominent in germinal centers (eight cases). Plasmablasts were seen in four cases within the perigerminal center area. Immunohistochemistry for HHV8, i.e. HHV8-LANA were negative in all cases while EBER was detected in 11 cases in the centrocyte-like B cells. Two cases of multicentric Castlemans disease expressed EBER; however, they did not express HHV8. CONCLUSION The wide spectrum of histological changes in HIV-associated lymphadenopathy requires recognition. The histological changes can mimic those of other infective lymphadenitis, follicular lymphoma, Castlemans disease, progressive transformation of germinal center, Hodgkins disease and spindle cell neoplasms. Presence of EBV is common while HHV8 was not seen.

Evaluation of new markers for minimal residual disease monitoring in B-cell precursor acute lymphoblastic leukemia: CD73 and CD86 are the most relevant new markers to increase the efficacy of MRD 2016; 00B: 000–000

Multiparametric flow cytometry (MFC) is a popular technique for minimal residual disease (MRD) analysis. However, its applicability is still limited to 90% of B‐cell precursor acute lymphoblastic leukemia (BCPALL) due to two major issues, i.e. a proportion of cases do not express adequate leukemia associated immunophenotype (LAIPs) with currently used markers and drug‐induced antigen modulation. Hence, the incorporation of additional reliable markers is required for the further improvement of MFC‐based MRD evaluation. We studied the utility of new markers in improvising MFC‐based MRD detection in BCPALL.

A novel and easy FxCycle™ violet based flow cytometric method for simultaneous assessment of DNA ploidy and six-color immunophenotyping

Abnormal DNA ploidy is a valuable prognostic factor in many neoplasms, especially in hematological neoplasms like B‐cell acute lymphoblastic leukemia (B‐ALL) and multiple myeloma (MM). Current methods of flow‐cytometric (FC) DNA‐ploidy evaluation are either technically difficult or limited to three‐ to four‐color immunophenotyping and hence, challenging to evaluate DNA‐ploidy in minute tumor population with background rich of its normal counterpart cells and other hematopoietic cells. We standardized a novel sensitive and easy method of simultaneous evaluation of six‐ to seven‐color immunophenotyping and DNA‐ploidy using a dye–FxCycle Violet (FCV). Linearity, resolution, and coefficient of variation (CV) for FCV were studied using chicken erythrocyte nuclei. Ploidy results of FCV were compared with Propidium iodide (PI) in 20 samples and intra‐assay variation for FCV was studied. Using this six‐color immunophenotyping & FCV‐protocol DNA‐ploidy was determined in bone‐marrow samples from 124 B‐ALL & 50 MM patients. Dilution experiment was also conducted to determine the sensitivity in detection of aneuploidy in minute tumor population. FCV revealed high linearity and resolution in 450/50 channel. On comparison with PI, CV of Go/G1‐peak with FCV (mean‐CV 4.1%) was slightly higher than PI (mean‐CV 2.9%) but had complete agreement in ploidy results. Dilution experiment showed that aneuploidy could be accurately detected up to the limit of 0.01% tumor cells. Intra‐assay variation was very low with CV of 0.005%. In B‐ALL, hypodiploidy was noted in 4%, hyperdiploidy in 24%, near‐hyperdiploidy in 13% and remaining 59% were diploid. In MM, hypodiploidy was in 2%, hyperdiploidy in 58%, near‐hyperdiploidy in 8% and remaining 30% were diploid. FCV‐based DNA‐ploidy method is a sensitive and easy method for simultaneous evaluation of six‐color immunophenotyping and DNA analysis. It is useful in DNA‐ploidy evaluation of minute tumor population in cases like minimal residual disease and MM precursor conditions.