Narendra Agrawal

Evaluating New Markers for Minimal Residual Disease Analysis by Flow Cytometry in Precursor B Lymphoblastic Leukemia

Minimal residual disease is currently the most powerful prognostic indicator in Precursor B lymphoblastic leukemia. Multiparameter flow cytometry is the most commonly used modality. Seventy three B ALL cases and 15 normal marrows were evaluated for expression patterns of leukemia markers (CD38, CD58, CD73) in all 73 cases and CD66c, CD86 and CD123 in 23 cases. CD73 was aberrantly expressed in 90.41% cases and CD86 in 60.87% B ALL cases. Thus addition of these markers in MRD panels can increase the sensitivity of the assay.

Incidence and clinical profile of tuberculosis after allogeneic stem cell transplantation

Patients post allogeneic stem cell transplantation (alloSCT) are expected to be at high risk of tuberculosis (TB) owing to underlying immunosuppression. We conducted a retrospective study in patients post alloSCT for clinical features and factors associated with TB.

Haploidentical Stem Cell Transplantation

Allogeneic haematopoietic stem cell transplantation (alloHCT) has emerged as a curative treatment modality for a variety of disorders including haematological malignancies, inherited and acquired bone marrow failure syndromes, congenital immunodeficiencies and errors of metabolism.

Benefits of Pre-harvest Peripheral Blood CD34 Counts Guided Single Dose Therapy with PLERIXAFOR in Autologous Hematopoietic Stem Cell Transplantation: A Retrospective Study at a Tertiary Care Institute in India

Peripheral blood is a convenient source of stem cells for hematopoietic stem cell transplantation. However, in autologous transplants, the harvest failure rates are high because of inadequate mobilization using G-CSF alone. Plerixafor is a potent mobilizer when used with G-CSF. However, its routine use is limited by high cost. This is a retrospective study done at a tertiary care oncology centre in India. All the harvest records were analyzed between Jan 2015 and Nov 2017. May 2016 onwards pre-harvest peripheral blood CD34 count was done in all cases of autologous transplants on day 4 of G-CSF therapy and they were given a single dose of Plerixafor if counts were < 20 cell per cumm. The results were compared amongst various groups. A total of 321 cases were analyzed. 172/321 were allogenic transplant cases of which 5% (n = 7) failed to achieve a target live stem cell dose of > 2 million per kg of the recipient. The overall failure rate in autologous group (n = 149) was 27% (n = 41) (p ≤ 0.001 auto vs. allo). The failure rate was higher (36%, n = 28/77) when no intervention with Plerixafor was done. The overall failure rate in the group treated with pre-harvest 34 count based single dose therapy of Plerixafor was 18% (n = 13/72, p = 0.01). However, within this intervention group, the patients who had pre-harvest peripheral blood CD34 above the desired cutoff had a higher failure rate of 21% (p = 0.13). Pre-harvest CD34 count based intervention with Plerixafor help optimizing the cost.

Mixed Phenotype Acute Leukemia, B/Myeloid with t(9;22): Diagnostic and Therapeutic Challenges

Mixed phenotype acute leukemia (MPAL) account for 4% of all cases of acute leukemia. Extensive immunophenotypic analysis is required for evaluation of these neoplasms, as morphologically distinct blast populations may not be evident [1]. The 2008/2016 WHO established strict criteria for diagnosis of MPAL, emphasizing assignment of myeloperoxidase for myeloid lineage, cytoplasmic CD3 for T lineage and CD19 and other B markers for B lineage (Table 1). WHO recognizes 2 distinct categories: MPAL with the t(9;22)(q34;q11)/BCR-ABL1 and MPAL with t(v;11q23)/MLL rearrangement. The remaining cases are designated as MPAL NOS (not otherwise specified). Mixed phenotype acute leukemia (MPAL) with t (9;22) comprises less than 1% of acute leukemias. Chronic Myeloid Leukemia in Mixed Phenotype Blast Crisis (CML-MP-BC) can also be misdiagnosed as MPAL with t (9;22). No specific treatment guidelines are available for its management. The present case highlights the diagnostic and therapeutic challenges involved with MPAL with t(9;22). A 45 year/male with a history of ankylosing spondylitis, presented with complains of generalized weakness and fatigue for 1 month. His hemoglobin had dropped significantly from 112 to 86 g/L and WBC had increased considerably from 8.8 to 16.8 9 10/L l in three months. Examination showed pallor with no lymphadenopathy or organomegaly. Bone marrow aspirate smears demonstrated a hypercellular marrow with 40% blasts of dimorphic morphology (lymphoblast, myeloblasts/promonocytes) (Fig. 1a, b). Flow cytometric immunophenotyping on marrow aspirate revealed 40% blasts gated (R1) on CD45. Blasts were dim CD45 positive with strong expression of CD34, HLA DR (Immaturity). Blasts showed strong intensity for CD19, cyto CD22, cyto CD79a (B-lymphoid) and MPO (Myeloid) (Fig. 1c). Blasts were also positive for CD10, CD13, CD33, CD14 and CD64. Cytogenetics and conventional PCR revealed a t(9;22) BCR-ABL 1. Diagnosis of MPAL (B/Myeloid) with t(9;22) BCR-ABL 1 was rendered. He was treated with Dasatinib and ALL like chemotherapy and achieved molecular CR. He was taken for matched sibling donor allogeneic stem cell transplantation. Post-transplant cyclophosphamides along with tacrolimus were used for GvHD prophylaxis. He developed transplant associated thrombotic microangiopathy on day ?22. He was managed with stopping tacrolimus and starting mycophenolate mofetil and FFP transfusions. He achieved neutrophil and platelet engraftment on day ? 15 and 34 respectively. He started passing loose stools on day ?32 of transplant. After thorough investigations, he was considered to have acute GvHD GI stage-IV, grade-III. He was started on Inj Methyl-Prednisolone 2 mg/kg/day. The symptoms of GvHD worsened and he succumbed to refractory GvHD on day ? 61 of transplant. MPAL is a rare entity, difficult to diagnose and to treat. MPAL is a heterogeneous category in the World Health & Poonam Singh poonamkgmu07@gmail.com

Ibrutinib to Allogenic Stem Cell Transplant in a Case of Refractory Mantle Cell Lymphoma

Mantle Cell Lymphoma (MCL) remains difficult to treat despite of availability of intensive chemo-immunotherapies with number of patients experience relapse or refractory disease. Possible cure in a relapsed/refractory (R/R) case of MCL has been described after allogeneic hematopoietic stem cell transplantation (alloSCT) [1]. Ibrutinib, a bruton’s tyrosine kinase inhibitor, acts by blocking B cell receptor signaling and has been approved by FDA as a single agent in R/R MCL cases for its favorable efficacy and toxicity profile [2–4]. Maintenance of higher PFS rates in these R/R MCL cases on Ibrutinib requires lifelong treatment with good adherence to dosing schedule. Ibrutinib monotherapy is found to be associated with (a) financial burden, (b) possible relapse in the first 2 years of treatment, and (c) acquired resistance after prolonged therapy; demonstrating poor clinical outcomes [4], which calls for different treatment strategies. These pitfalls of Ibrutinib monotherapy could be salvaged with alloSCT as reported in our current case. Here, we report a case of refractory MCL with prior three lines of chemotherapy before successful treatment with Ibrutinib followed by AlloSCT. A 53 year old male presented with jaundice, significant weight loss, hepatosplenomegaly, large lymphnodal masses at porta and peripancreatic region, and obstructive biliopathy. Lymph node histopathology with immunohistochemistry confirmed MCL, while whole body PET–CT showed extensive disease including extranodal sites and bone marrow infiltrates. Patient was planned for 6 cycles chemotherapy with R-CHOP alternating with R-DHAP followed by autologous SCT (ASCT). He received one cycle of R-CHOP and R-DHAP each, to which he developed grade-IV toxicities (abdominal pain, abdominal distension, loose stools) which required prolonged repeated hospitalization. Therefore, treatment was changed to Rituximab–Bendamustine (R–B) chemotherapy regimen which he tolerated well. Post 4 cycles of R–B chemotherapy, patient showed progression in extent of lymphoma. Further, he received bortezomib and lenalidomide based therapy for 2 cycles but again he showed progressive disease. Patient was considered for Ibrutinib monotherapy followed by alloSCT. He tolerated Ibrutinib therapy well and attained complete remission (CR) after 1 month of therapy. Thereafter, he was received reduced intensity conditioning (Fludarabine and Melphalan) and matched sibling donor peripheral blood stem cell transplantation with Methotrexate and Cyclosporine-A (CSA) for GVHD prophylaxis. Neutrophils and platelets were engrafted on day ? 11 post alloSCT. Serial monitoring for CMV DNA was negative. There was no graft versus host disease while the CSA was tapered off by day 180 post transplant. Serial PET–CT at post transplant 90 days and 18 months showed CR. At 25 months of follow up post alloSCT, patient is disease free. Primary refractory MCL is not uncommon and is a challenge to treat. Ibrutinib is a promising agent for such cases of MCL which has shown a response in nearly 2/3rd of R/R MCL patients [2, 5], but its long term utility is limited by high cost, indefinite therapy and poor long-term survival owing to loss of response because of acquired & Rayaz Ahmed rayazhemat@gmail.com

Haplo-identical Hematopoietic Stem Cell Transplant is a Possible Cure in a Patient with Relapsed Hemophagocytic Lymphohistiocytosis Syndrome

Hemophagocytic lymphohistiocytosis (HLH) is a lifethreatening hyperinflammatory syndrome of uncontrolled immune responses resulting in hypercytokinemia because of underlying primary or acquired immune defect. Currently, the only cure for primary or relapsed HLH is allogenic stem cell transplantation (AlloSCT) [1]. Our report concerns a 13-year-old boy with a relapsed distinctively blurred HLH [2], who achieved prolonged complete remission (CR) after haplo-identical stem cell transplant (Haplo-SCT) following salvage chemotherapy. A previously healthy 11-year-old boy, the youngest son of a healthy nonconsanguineous parents without family history of HLH/similar disorders in first/second-degree relatives was hospitalised at a local hospital with complaints of 106 F fever and hepatosplenomegaly and found to have Brucella infection and histiocyte proliferation in bone marrow (BM). On further investigations, he had raised serum triglyceride, serum ferritin, and low fibrinogen levels, on the basis of which he was diagnosed with HLH. Cerebrospinal fluid was normal. The genetic mutations of HLH were not tested for parents did not give consent for same. He received chemotherapy using HLH2004 protocol and achieved CR. On routine follow up, he was found to have relapse of HLH without any underlying infective stimulus, after 25 months of initial therapy for which he received etoposide-based salvage chemotherapy and attained a second complete remission. Thereafter he was referred to us for alloSCT. He had no HLA matched donor in the family, but had 3/6 HLA match with his mother with negative donor specific anti-HLA antibodies screen. Considering the relapsed nature of HLH and in need of alloSCT lacking a matched HLA donor from family, he received a Haplo-SCT using T cell replete peripheral blood stem cell (PBSC) from his mother with a CD34 cell dose of 6.06 9 10 cells/kg after Fludarabine, cyclophosphamide and TBI (FluCyTBI) conditioning. For GvHD prophylaxis, cyclophosphamide at day ? 3 and ? 4 post SCT was employed. ANC and Platelet Engraftment was achieved at day ? 16 and ? 21 of SCT respectively, and was further confirmed by 100% donor karyotype on FISH for XX analysis of peripheral blood at day ? 30. Acute GvHD (aGvHD), overall grade II with skin and gut involvement, became apparent at day ? 16 which was managed with corticosteroids. By day ? 120, he developed aGvHD of liver, which progressed to grade IV despite being on corticosteroids, etanercept and basiliximab and was controlled after Anti thymocyte globulin (ATG) therapy. He had no major infections except for chronic otitis media and UTI with Enterococcus fecalis. Currently, he has mild chronic GvHD involving oral mucosa and eyes requiring minimal immunosuppression and is disease free with maintenance of complete chimerism at day ? 840 of Haplo-SCT. Use of alloSCT for HLH has remarkably improved the survival and prognosis of patients. Henter et al. [3] demonstrated 62% survival rate at 3 years and 10% of graft failure (GF) among 65 HLH patients treated with alloSCT. Ouachée-Chardin et al. [4] reported a series of 48 primary HLH patients who received alloSCT out of which 60% received haplo-SCT, was found associated with increased treatment related toxicity, poor prognosis and 22% GF rate. Uppuluri et al. [5] reported a case of 2 young children with & Rayaz Ahmed rayazhemat@gmail.com

Blastic Plasmacytoid Dendritic Cell Neoplasm: Still an Enigma

Dear Sir, Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare aggressive malignancy representing less than 1% of all acute hematologic cancers with no ethnic predilection [1]. It arises from precursor of the plasmacytoid dendritic cells and functionally belongs to a group of interferon alpha-secreting cells responsible for the detection of viral nucleic acid fragments as well as bacterial antigens via the Toll-like receptors [2, 3]. Here we report a case series of three BPDCN patients which show marked heterogeneity in terms of their clinical presentation, skin or bone marrow involvement, immunophenotypic expression and treatment strategies applied as shown in Table 1 and Figs. 1, 2. BPDCN is known to be frequently seen in elderly (median age at presentation: 67 years). However, it can occur in any age group with slight male predominance [1, 4]. In this case series, all the three patients were B 25 years of age with an M:F ratio of 2:1. BPDCN is associated with high frequency of primary cutaneous involvement (64–100% of cases) attributable to expression of antigens such as CLA and CD56 that favor skin migration and chemokine receptors expressed by neoplastic plasmacytoid dendritic cells (pDC) [5]. Cutaneous lesions may occur in the form of non-pruritic asymptomatic single or multiple skin lesions like plaques, nodules or brown to violaceous bruise-like skin lesions. Patients with skin involvement at diagnosis are considered to have better prognosis [6]. BPDCN without skin lesions is typically seen in young patients with peripheral involvement [7] as seen in Case I. In Case II, trucut biopsy from the left arm mass exhibited a cellular tumor with focal areas of necrosis and comprised of sheets of small to intermediate sized atypical cells with hyperchromatic nuclei, irregular nuclear membrane and scant to absent cytoplasm. On IHC, tumor cells expressed LCA (diffuse and strong), CD99, Tdt (focal) and were negative for CD3, CD5, CD7, CD10, CD22, SOX11, EMA, ALK, CD30, MPO, MIC-2, CD34 and PAX-5. In the absence of any lineage specific markers and after ruling out the commoner lymphomas with cutaneous presentation such as anaplastic large cell lymphomas, peripheral T cell lymphoma etc., it was decided to rule out a distant possibility of BPDCN. An extended panel of markers comprising of CD4, CD43, CD56 and CD123 was applied which came out to be strongly positive. Diagnosis of BPDCN is based on expression of CD4, CD56, CD123 and HLA-DR by flowcytometry or immunohistochemistry along with other pDC associated antigens such as TC1, BDCA-2 and CLA and negative expression of B, T or myeloid lineagespecific markers. In about 8% cases of BPDCN, CD4 or CD56 is negative. Differential diagnosis includes other hematologic neoplasms such as extranodal NK/T cell leukemia/lymphoma, cutaneous T cell lymphoma, leukemia cutis etc. which may express CD56 with or without CD4, necessitating an exhaustive IHC panel for a definitive diagnosis. In such overlapping cases, presence of additional dendritic cell markers such as CD303/BDCA-2 or TCL-1 may be useful. Here, it should be made clear that CD56 negative staining does not rule out BPDCN if CD4, CD123 and TCL-1 are positive [8–13]. All the three patients in this case series had positive expression of CD4/CD56/CD123, with or without bone marrow involvement. Myeloid & Neha Singh drnehasingh123@gmail.com

CD38—Negative Myeloma with Anaplastic Morphology at Presentation: A Case Report

CD38 is a 46-kDa type II trans-membrane glycoprotein involved in receptor-mediated cell adhesion, signal transduction and intracellular calcium metabolism. Normal plasma cells show higher expression of CD38 than the hematopoietic stem cells, T and B lymphocytes. However, malignant plasma cells over-express CD38 at all stages of maturation. In fact, high and consistent expression of CD38 by myeloma cells together with its significance in cell signaling pathways have suggested the role of CD38 as a potential target for the treatment of multiple myeloma (MM). Daratumumab is a humanized CD38 monoclonal antibody that has emerged as a breakthrough targeted therapy in myeloma patients, used both as a monotherapy and in combination with other agents. Recently published studies have documented loss of CD38 in relapsed or refractory myeloma [1]. Here, we report a case of newly diagnosed MM with CD38 negative plasma cells and anaplastic morphology as evaluated by immunohistochemistry that posed a diagnostic challenge due to such rare association. A 32-year old male presented with complaints of dyspnea for last 1–2 months. Physical examination was unremarkable. Complete hemogram showed HB = 7.9 g/dl, TLC = 14.76 9 10/L with a differential count of N:44 L:30 M:15 Atypical cells? Blasts:07 My:03 E:01; nRBC:7/ 100 WBCs and platelet count = 11 9 10/L. Peripheral smear showed leuco-erythroblastic picture with thrombocytopenia and 07% large atypical cells. A provisional diagnosis of acute leukemia/lymphoma was made and further investigations advised. Serum albumin was 3.9 g/dl and serum LDH was markedly elevated (2444 IU/ml). 24 h urinary protein was 4.38 gm/24 h. Serum creatinine (4.8 mg/dl) and serum calcium (13.68 mg/dl) levels were raised. Viral serology for HIV, Hepatitis Band C was negative. Serum total protein was 6.1 g/dl with A:G ratio of 1.7:1 (Albumin: 3.9 g/dl; Globulin: 2.2 g/dl). MRI showed multiple partial vertebral collapses with biconcave wedged appearance of most of mid/lower dorsal vertebral segment with bulging posterior vertebral outline from D9 to L2 vertebral segments, suggestive of marrow infiltrative disorder. PET-Scan revealed metabolically active extensive bony lesions with pancreatic involvement and mild hepatosplenomegaly. Bone marrow imprint smears showed 57% large atypical cells with marked nuclear convolutions which were negative for myeloperoxidase stain. Flowcytometry could not be performed due to dry tap. Hypercellular bone marrow biopsy sections showed near-total replacement of normal hematopoeitic components with undifferentiated large atypical cells with pleomorphic nuclei, conspicuous nucleoli and moderate amount of cytoplasm. The first line of immunohistochemical markers comprising of cytokeratin, desmin, S100 and LCA was negative. A possibility of LCA negative hematopoeitic neoplasm was strongly considered and expanded panel of IHC markers was applied. However, CD34/CD117/TDT/ CD3/MPO/CD79a/PAX5/CD4/CD61/CD20/CD30/CD38 were found to be negative. Before labeling the case as Undifferentiated /poorly differentiated malignancy, a review of the case was done. Positive clinical findings included bicytopenia, renal insufficiency, hypercalcemia and raised LDH. No M band was found on serum protein & Neha Singh drnehasingh123@gmail.com

Florid Plasmacytosis in Angioimmunoblastic T Cell Lymphoma: A Diagnostic Conundrum

Angio-immunoblastic T-cell lymphoma (AITL) is a rare subtype of non-Hodgkins lymphoma (NHL), accounting for about 1–2% of all NHLs and 15–27% of peripheral T-cell lymphomas [1]. Most patients present with systemic lymphadenopathy, constitutional symptoms or abnormal immunological features. Abnormal laboratory test findings include cytopenias, positive Coomb’s test, polyclonal hypergammaglobulinemia, elevated LDH or presence of autoimmune antibodies. Circulating plasma cells and plasmacytoid lymphocytes have been occasionally detected in AITL patients. However, plasma cells[2 9 10 cells/ll mimicking plasma cell leukemia or exuberant reactive plasmacytosis in bone marrow in the order of [50% plasma cells including atypical forms is infrequently reported in AITL [2, 3]. We reviewed twelve cases of AITL reported in last 5 years at our institution. Bone marrow involvement by AITL was seen in one case (1/ 12 = 8.1%) while two cases (2/12 = 16.6%) revealed extensive bone marrow involvement by plasmacytes along with peripheral blood spillage, that led us to investigate for a coexisting myeloma or plasma cell leukemia. Case 1