Sumeet Gujral

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.

An integrated genomic profile that includes copy number alterations is highly predictive of minimal residual disease status in childhood precursor B-lineage acute lymphoblastic leukemia

Introduction: Copy number alterations (CNA) have been described in childhood precursor B-lineage acute lymphoblastic leukemia (B-ALL) which in conjunction with chromosomal abnormalities drive leukemogenesis. There is no consensus on the clinical incorporation of CNA in B-ALL. An integrated genomic classification (IGC) has been proposed which includes CNA and cytogenetics. Methods: We correlated this IGC with immunophenotypic minimal residual disease (MRD) as well as other standard criteria for 245 patients of B-ALL such as National Cancer Institute (NCI) risk, D+8 prednisolone response, cytogenetics, and ploidy status. Results: MRD was detectable in 81 patients (33.1%). The most common abnormalities were seen in CDKN2A/B (25.7%) followed by PAX5(20%), ETV6(16.7%), IKZF1(15.5%), Rb1(5.3%), BTG (3.3%), EBF1(2.0%), and PAR1(0.8%). On integrating CNA into the IGC, 170 patients (69.4%) were classified into good genomic risk (GEN-GR) whereas 75 (30.6%) belonged to the poor genomic risk (GEN-PR) category. The IGC showed a significant correlation with MRD and NCI risk. The presence of CNA predicted MRD clearance in intermediate cytogenetics group. Conclusion: These data seem to indicate that in addition to cytogenetics, CNA should be incorporated into routine clinical testing and risk algorithms for B-ALL. The IGC is of prognostic relevance and offers an additional avenue for prognostication and risk-adapted therapy.

Characteristics of BCR-ABL kinase domain mutations in chronic myeloid leukemia from India: not just missense mutations but insertions and deletions are also associated with TKI resistance

Abstract We document the characteristics of BCR-ABL kinase domain mutations (KDM) in the largest study from India comprising of 385 patients and demonstrate that more than half (51.9%) of these patients have detectable abnormalities in the KD both in adult and in pediatric chronic myelogenous leukemia (CML). These comprise singly occurring missense mutations (25.5%), polyclonal/compound point mutations (4.9%), and insertions/deletions (29.6%). Missense mutations were most commonly seen in the imatinib-binding region followed by the P-loop. The commonest mutation in our dataset was T315I. Other common missense mutations were Y253H, M244V, and F317L. A high prevalence of BCR-ABL exon7 deletion (p.R362fs*) was also seen (25.5% of the entire cohort), whereas the 35bpintron-derived insertion/truncation mutation detected in 12 patients. In the pediatric age group, 58.8% of patients harbored missense mutations, polyclonal/compound mutations as well as insertions and deletions. We detected 11 novel mutations (seven missense mutations and four insertions/deletions).

Rapidly Progressive Congenital Rhabdomyosarcoma Presenting with Multiple Cutaneous Lesions: An Uncommon Diagnosis and a Therapeutic Challenge

Congenital rhabdomyosarcomas (RMSs) are rare tumors with variable clinical presentations. A 2 month-old, term male neonate (37 weeks, 4 days), weighing 3.2kg, born to a 24 year-old primigravida, by simple vaginal delivery presented with multiple erythematous papulonodular lesions over his trunk that progressed to his whole body, on the first day of delivery. Prior to conception, his mother was treated for polycystic ovarian disease. On the tenth day, his chest computed tomogram scans revealed multiple, heterogeneously enhancing, bilateral pleural-based soft tissue density nodular lesions, along with multiple soft tissue density lesions, involving skeletal muscles of all his body parts. Microsections from two biopsies (on 10th day and after 2 months) revealed a malignant round cell tumor with cells arranged in a diffuse, solid pattern, comprising embryonal and solid alveolar components. Immunohistochemically, the tumor cells were diffusely positive for desmin, myoD1 and myogenin. Diagnosis of embryonal and alveolar (mixed type) RMS was offered. Further molecular cytogenetic analysis was negative for PAX3-FKHR and PAX7-FKHR. The patient was induced on chemotherapy as per intergroup rhabdomyosarcoma study IV protocol. There was treatment response with near total remission after 8 weeks of treatment. Thereafter, new lesions started appearing that also disappeared after modification of the chemotherapy drugs. However, after 16 months, the baby died of brain metastasis. The present case forms the fourth case report of an aggressive form of a congenital RMS with extensive cutaneous involvement and brain metastasis. A review of previously diagnosed cases of congenital RMSs is discussed herewith.

Extramedullary myeloid cell tumor of the urinary bladder in a patient with myelodysplastic syndrome.

We report a case of extramedullary myeloid cell tumor of the urinary bladder in an elderly male with a three year history of myelodysplastic syndrome (refractory anemia with excess blasts), noninvasive papillary transitional cell carcinoma of the urinary bladder, and in situ transitional cell carcinoma of the left ureter. Light microscopy demonstrated a poorly differentiated neoplasm composed of medium to large cells with eosinophilic cytoplasm. The tumor cells showed immunohistochemical expression of myeloperoxidase, lysozyme, CD15, CD68 and CD43. Bone marrow examination following cystectomy demonstrated refractory anemia with excess blasts (6-10%) and a normal karyotype. Cytogenetics, approximately 1 year after cystectomy, demonstrated a deletion of the short arm of chromosome number 12. Four years after presentation, the patient succumbed to pulmonary aspergillosis.

Immunogenetics of chronic lymphocytic leukemia

Introduction: Cytogenetic aberrations as well as presence of IGVH mutations are the underlying reason for clinical heterogeneity in Chronic Lymphocytic Leukemia (CLL). The presence of IGVH mutations as well as the predominant gene usage shows geographical variations. However, there is no study from India addressing immunogenetics of CLL. In a first Indian study we document the immunogenetics of CLL in a large tertiary hospital. Methods: We analyzed IGVH mutation status, VH gene usage, cytogenetic abnormalities using FISH, immunophenotyping data and correlated them with standard clinical variables in 84 patients of CLL. Results: Advanced Rai stage (Stage 3/4) was seen in 45% of our patients, where as 13q deletion was the commonest clonal cytogenetic abnormality detected in 48.4% of the cases. IGVH unmutated cases (55.2%) showed higher proportion expressing CD38 and CD49d, a preferential usage for VH1 and VH3 families (55.2%), presentation at an advanced Rai stage (52.8%) as well as more frequent presence of p53 deletions. As compared to the IGVH mutated cases greater proportion of IGVH unmutated patients (70%) required treatment. However, there was no significant difference in the time to treatment between mutated and unmutated cases which can be attributed to relatively short median follow up of 10 months. Conclusion: To summarize, we have seen a higher proportion of IGVH unmutated patients in our cohort (55.2%). The commonly used VH genes in the Indian population are IGVH 2-5, IGVH 1-2 and IGVH 1-69. Longer clinical follow up and a larger cohort is necessary to confirm the prognostic value of IGVH mutation analysis in Indian Patients with CLL.

A rare extramedullary and extralymphoid presentation of mixed phenotypic blastic hematolymphoid neoplasm: A study of two cases

Mixed phenotype acute leukemia (MPAL) is a rare hematolymphoid neoplasm, representing only 3%–5% of acute leukemia. Although MPAL has been sufficiently described in the literature, its extramedullary presentation as a solitary lesion without leukemic (bone marrow [BM]) involvement is rarely described. We are presenting two cases of mixed phenotypic blastic hematolymphoid neoplasms without leukemic involvement at disease presentation in 8-year-old female and 21-year-old male patients. Both the cases had extralymphatic bone involvement in the form of solitary bone lesion. Initially, there was no leukemic involvement in both the cases, but the second case progressed to acute leukemia during the course of the disease. On immunophenotypic evaluation, both the cases revealed blasts showing unequivocal evidence of myeloid and B-lymphoid lineage commitment. These cases were difficult to categorize either into MPAL as the BM was not involved or into lymphoblastic lymphoma due to coexpression of myeloid differentiation. Therefore, we chose to classify them as a bi/mixed phenotypic blastic hematolymphoid neoplasm. Detailed immunophenotypic analysis either by immunohistochemistry or flow cytometric immunophenotyping is important for the diagnosis of such cases as they have a poor prognosis.

Clinical Relevance of Multicolour Flow Cytometry in Plasma Cell Disorders

Multicolor flow cytometric (MFC) immunophenotyping is one of the basic test that is needed in the evaluation of hematolymphoid malignancies. Previously, there has been some reluctance in the use of MFC in plasma cell disorders (PCD). It was mainly due tolack of standardization, inadequate experience and detection of the lower number of plasma cells by MFC as compared to morphology. However, MFC has gone through many technological advancements in the last few years and a wide variety of reagents are now commercially available which worldwide allowed the establishment of standardized sensitive MFC-based immunophenotypic assay for PCD. Various studies have proven that MFC has a high clinical relevance in the diagnosis and risk stratification of multiple myeloma, its precursor conditions and other PCDs. Moreover, recent studies have shown that MFC is a highly sensitive and reliable technique for the monitoring of clinical response in the era of novel therapies. In this review, we have discussed the various applications of MFC in the management of PCD and their clinical relevance.

CD19 negative precursor B acute lymphoblastic leukemia (B-ALL)—Immunophenotypic challenges in diagnosis and monitoring: A study of three cases

CD19 is a B‐cell specific marker, expressed on all stages of B‐lymphocytes including plasma cells. It is widely used in the flow cytometric immunophenotyping (FCI) of B‐cell and plasma cell malignancies. The analysis approach of FCI for the diagnosis and monitoring of B‐cell acute lymphoblastic leukemia (B‐ALL) is totally based on the CD19‐based primary gating strategy and it would be challenging to study B‐ALL without CD19 expression. Since CD19 negative B‐ALL are extremely rare, we report three cases of B‐ALL with negative expression of CD19 and discussed its implication in the diagnosis, residual disease monitoring and future targeted therapy.