Integrative proteome analysis of bone marrow interstitial fluid and serum reveals candidate signature for acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive form of blood cancer and clinically highly heterogeneous characterized by the accumulation of clonally proliferative immature precursors of myeloid lineage leading to bone marrow failure. Although, the current diagnostic methods for AML consist of cytogenetic and molecular assessment, there is a need for new markers that can serve as useful candidates in diagnosis, prognosis and understanding the pathophysiology of the disease. This study involves the investigation of alterations in the bone marrow interstitial fluid and serum proteome of AML patients compared to controls using label-free quantitative proteomic approach. A total of 201 differentially abundant proteins were identified in AML BMIF, while in the case of serum 123 differentially abundant proteins were identified. The bioinformatics analysis performed using IPA revealed several altered pathways including FAK signalling, IL-12 signalling and production of macrophages etc. Verification experiments were performed in a fresh independent cohort of samples using MRM assays led to the identification of a panel of three proteins viz., PPBP, APOH, ENOA which were further validated in a new cohort of serum samples by ELISA. The three-protein panel could be helpful in the diagnosis, prognosis and understanding of the pathophysiology of AML in the future. BIOLOGICAL SIGNIFICANCE: Acute Myeloid Leukemia (AML) is a type haematological malignancy which constitute one third of total leukemias and it is the most common acute leukemia in adults. In the current clinical practice, the evaluation of diagnosis and progression of AML is largely based on morphologic, immunophenotypic, cytogenetic and molecular assessment. There is a need for new markers/signatures which can serve as useful candidates in diagnosis and prognosis. The present study aims to identify and validate candidate biosignature for AML which can be useful in diagnosis, prognosis and understand the pathophysiology of the disease. Here, we identified 201 altered proteins in AML BMIF and 123 in serum. Among these altered proteins, a set of three proteins viz., pro-platelet basic protein (CXCL7), enolase 1 (ENO1) and beta-2-glycoprotein 1 (APOH) were significantly increased in AML BMIF and serum suggest that this panel of proteins could help in future AML disease management and thereby improving the survival expectancy of AML patients.

Rare complications of heart transplant: Autopsy findings.

BACKGROUND: Heart transplantation has evolved as the only treatment option for patients with refractory heart failure. CASE PRESENTATION: We here, report two unusual complications that developed following cardiac transplant to which the recipients succumbed. Post mortem conducted revealed the cause of death as severe antibody mediated rejection in one case and ruptured mycotic aneurysm of ascending aorta in the second recipient. CONCLUSION: Hence, autopsy remains the key procedure that can help establish the cause of death after cardiac transplant. It is also imperative for clinicians to have awareness and high index of suspicion for early detection of the ongoing complications and intervene either surgically or medically to prevent catastrophic events.

Correlation of Thrombosis and Clinicohematological Parameters with JAK2V617F Mutation in Philadelphia-Negative CMPNs: A Study from India.

Objective Philadelphia-negative chronic myeloproliferative neoplasms (CMPNs), which include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are characterized by the presence of JAK2V617F (exon 14) mutation, and this occurs in 90 to 95% cases of PV and 50 to 60% cases of ET and PMF. Still, this is a matter of debate regarding the correlation of this mutation with thrombosis and clinicohematological parameters in CMPNs. So, we conducted this study to ascertain the association of JAK2V617F mutation with thrombotic complications and clinicohematological parameters of these patients. Materials and Methods This prospective and retrospective study was conducted during 2018 to 2019 at the Department of Laboratory Sciences and Molecular Medicine of a tertiary care hospital, and 160 CMPN patients were enrolled. Complete hemogram was done and DNA was extracted, followed by real-time qualitative polymerase chain reaction to check for JAK2V617F mutation. This mutation was then correlated with complications, mainly thrombosis, hematological parameters, and clinical parameters such as age and splenomegaly. Results Among 160 CMPN patients, 60 were females and 100 were males, with male to female ratio of 1:0.6, and age range of 27 to 85 years. Total 91 (56.9%) patients were JAK2V617F positive and the remaining 69 (43.1%) were negative for this mutation. We observed statistically significant correlation of leukocyte count, splenomegaly, and thrombosis in JAK2V617F-mutated patients as compared to unmutated patients. Conclusion This study emphasizes the importance of JAK2V617F mutation in CMPNs and stresses on its association with clinical, hematological phenotype, and thrombotic complications, which may open new horizons in prognostication and management protocol.

Real-world experience of anti-D immunoglobulin in immune thrombocytopenia.

In developing countries, anti-D has been used in immune thrombocytopenia (ITP) as a cheaper alternative to human immunoglobulin. We aim to analyze the response and safety profile of anti-D in patients with severe ITP. A retrospective study was conducted at a tertiary care hospital in Northern India. Patients received a single intravenous infusion of 75 µg/kg anti-D. In total, 36 patients (20 females) were included in this study. The median duration from ITP diagnosis to anti-D therapy was 235 days (range 1-1613 days). Four (11.1%) patients received anti-D as an upfront treatment. The patients' platelet counts rose significantly by the end of day three and continued to be significantly high until day 30 of receiving anti-D (p ≤ 0.001). The overall response rate (ORR) by day seven was 88.89%. There was no effect of age, sex, duration of disease, prior therapy, and platelet count on the ORR. Patients were followed up for a median duration of 52 days (longest follow-up: 3080 days). Six (6/36, 16.67%) patients continued to be in remission till the last follow-up. The hemoglobin fall was statistically significant on day three and day seven (p < 0.001 and p = 0.001) and got normalized by day 30. We observed equally good ORR in mixed populations and different phases of ITP along with long-term sustained response. The study demonstrates a quick and high response rate along with good safety profile to anti-D in all forms of ITP.

Real-world Experience of Rituximab in Immune Thrombocytopenia.

Immune thrombocytopenia (ITP) is a relapsing-remitting disease often requiring more than one line of therapy. Rituximab is a recommended second-line therapy, but the real-world data on its efficacy and safety from resource constraint settings is limited. We aimed to analyze the safety and efficacy of rituximab in ITP. This is a single-center, retrospective study. This study was conducted at a tertiary care hospital in Northern India from 2005 to 2019. On audit of medical records, all patients of ITP (n-513) who had received rituximab (n-81) were screened for inclusion. Patients whose response assessment was not possible were excluded. Finally, 66 patients were analyzed using statistical packages of Python v3.7. The cumulative incidence of overall response on day 20 was 30.61%, and day 30 was 51.72%. The median time to response was 28 day (range 21-51 day). Cumulative incidence of complete response was 16.67%, and partial response 37.88%. After a median follow-up of 789 day (range 181-5260 day), the cumulative incidence of relapse was 30.32%, 36.12%, and 56.57% at 1, 2, and 5 years respectively. There was no effect of age, sex, duration of disease, lines of therapy received, and platelet count on either cumulative incidence of overall response or relapse. ANA positivity was significantly related to the better cumulative incidence of overall response (p = 0.012), but not with relapse. Infusion-related reactions were the commonest adverse event noted (n-4, grade ≥ 3 CTCAEv4). Rituximab and its generic version are safe and effective second line agent in ITP with a good overall response and sustained response.

Monoclonal gammopathy of renal significance: Spectrum of diseases and approach to a case.

The occurrence of kidney diseases associated with a monoclonal gammopathy in the absence of symptomatic multiple myeloma is increasingly recognized. When the kidney is involved, the monoclonal etiology of these diseases results in clinical and laboratory features distinct from those of other disease, necessitating the nomenclature monoclonal gammopathy of renal significance (MGRS). The detection of these monoclonal diseases involving the kidney is important since they are poorly responsive to conventional immunosuppression and instead require clone-directed therapy. The new International Kidney and Monoclonal research group consensus definition of MGRS includes all proliferative conditions of B cells and/or plasma cells. Renal lesions due to monoclonal immunoglobulins are quite capable of progression with resulting end-stage renal disease development. Hence, these lesions require therapeutic intervention even if they do not satisfy myeloma criteria or the presence of any myeloma defining event. The spectrum of renal lesions that can be observed in a case of MGRS is wide and mirrors the list that may be seen in a case of any plasma cell neoplasm. This includes Ig light chain, heavy chain, and heavy and light chain amyloidosis; immunotactoid glomerulonephritis (GN); monoclonal immunoglobulin deposition disease including light chain, heavy chain, or heavy and light chain disease; light chain proximal tubulopathy; crystal-storing histiocytosis; proliferative GN with monoclonal immunoglobulin deposits; C3 glomerulopathy with monoclonal gammopathy and cast nephropathy. The initial approach after histological assessment is based on presence or absence of monoclonal immunoglobulin deposits. If monoclonality is evident, it is important to distinguish between conditions with deposition of intact immunoglobulin molecule or light chains only. The treatment of MGRS is directed at the underlying neoplastic B-cell or plasma cell clones.

Proteomic Alterations in Multiple Myeloma: A Comprehensive Study Using Bone Marrow Interstitial Fluid and Serum Samples.

Multiple myeloma (MM) is a plasma cell-associated cancer and exists as the second most common hematological malignancy worldwide. Although researchers have been working on MM, a comprehensive quantitative Bone Marrow Interstitial Fluid (BMIF) and serum proteomic analysis from the same patients' samples is not yet reported. The present study involves the investigation of alterations in the BMIF and serum proteome of MM patients compared to controls using multipronged quantitative proteomic approaches viz., 2D-DIGE, iTRAQ, and SWATH-MS. A total of 279 non-redundant statistically significant differentially abundant proteins were identified by the combination of three proteomic approaches in MM BMIF, while in the case of serum 116 such differentially abundant proteins were identified. The biological context of these dysregulated proteins was deciphered using various bioinformatic tools. Verification experiments were performed in a fresh independent cohort of samples using immunoblotting and mass spectrometry based SRM assays. Thorough data evaluation led to the identification of a panel of five proteins viz., haptoglobin, kininogen 1, transferrin, and apolipoprotein A1 along with albumin that was validated using ELISA in a larger cohort of serum samples. This panel of proteins could serve as a useful tool in the diagnosis and understanding of the pathophysiology of MM in the future.

Highlights of Abstracts on Plasma Cell Dyscrasia in the Annual Conference ISHBT (Kochi).

Since last few years plasma cell dyscrasias have been in the hit list of research especially pertaining to its therapy and pathophysiology. India has taken a great leap as far as research in plasma cell dyscrasias is concerned. The year started with myeloma conference in New Delhi conducted by Army Hospital (R&R) and ended at Haematocon Kochi organised by team from CMC Vellore (Haematocon 2018). Haematocon 2018 provided an exceptional platform for the research on various aspects of plasma cell dyscrasias both from the country and abroad. A total of 23 abstracts were presented pertaining to plasma cell disorders; 13 original research papers, 08 case reports and 02 case series. There were a wide spectrum of papers which ranged from the discussion about morphological aspects, mimickers, clinical spectrum, life style in the setting of the disease, diagnostic modalities to management aspects and finally to progress and survival of these patients. Through this review we will summarize and analyze the key findings of the original research abstracts on diagnosis, risk stratification, clinical profile and treatment aspects of plasma cell disorders presented at the Haematocon 2018.

Current Understanding of the Potential of Proteomics and Metabolomics Approaches in Cancer Chemoresistance: A Focus on Multiple Myeloma.

Chemoresistance is one of the major hurdles in cancer treatment leading to recurrence of cancer and affects the overall survival of patients. Cancer chemoresistance can be associated with various phenomena including modulation of vital cellular pathways. Unrevealing these alterations could provide a better understanding of chemoresistance and assist in the identification of new targets to overcome it. Recent advances in the field of proteomics and metabolomics have substantially helped in the identification of potential targets for chemoresistance in various cancers. This review highlights the potential of proteomics and metabolomics research to explore the putative targets associated with cancer chemoresistance with a special focus on Multiple Myeloma (MM). MM is a type of hematological malignancy which constitutes about 13% of all blood cell cancers. The therapeutic advancements for MM have increased the median overall survival rate to over 3-fold in the last one and half decade. Although in recent times, significant improvements in the overall survival rate of MM are achieved, MM remains an incurable disease with unpredictable refractory mechanisms. In spite of therapeutic advances, chemoresistance thrives to be a major hurdle in the treatment of multiple myeloma which demands a better understanding of chemoresistance. In this review, we have attempted to highlight the potential applications of proteomics and metabolomics research in the understanding of chemoresistance in MM.

Study of associated genetic variants in Indian subjects reveals the basis of ethnicity related differences in susceptibility to venous thromboembolism.

The genetic variants linked with the susceptibility of individuals to VTE are well known; however, the studies explaining the ethnicity based difference in susceptibility to VTE are limited. Present study assesses mutations in six candidate genes contributing to the etiology of VTE in Indian subjects. The study comprised 93 VTE patients and 102 healthy controls. A PCR-RFLP based analysis was performed for nine mutations in the following genes associated with VTE: favtor V Leiden (FVL), prothrombin, tissue factor pathway inhibitor (TFPI), fibrinogen-beta, plasminogen activator inhibitor 1 (PAI-1), and methylene tetrahydrofolatereductase (MTHFR). All the subjects were found to be monomorphic for FVL 1691G/A, prothrombin 20210G/A and TFPI -536C/T mutations. The mutation in the MTHFR gene (677C/T) was observed only in patients. Contrarily, higher frequency of mutation in the PAI-1 -844G/A and the fibrinogen-ß -455G/A was observed in controls in comparison to the patients. This study suggests that the PAI-1 -844G/A and fibrinogen-ß -455G/A could be protective variants against VTE in Indians. While MTHFR 677C/T mutation was found to be associated, in contrast to other populations, the established genetic variants FVL 1691G/A, prothrombin 20210G/A, and TFPI -536C/T may not be associated with VTE in Indians thus revealing the basis of ethnicity related differences in susceptibility of Indians to VTE.

Burkittts lymphoma revisited: series of three cases with varied clinical presentation.

Burkitt's lymphoma is a form of non-Hodgkin's B-cell lymphoma with more than one identifiable variant. This tumour was first noted in Africans. The sporadic form most commonly presents with abdominal lymph node involvement. This tumour predominently affects children and is probably the fastest growing tumours in humans, with exuberant proliferation. We here in report on three patients from our experience both adult and children who presented with varied clinical features.

Rare case of acquired haemophilia and lupus anticoagulant.

Acquired haemophilia or factor VIII (FVIII) deficiency, caused by FVIII inhibitor antibodies, is a very rare condition that commonly results in severe haemorrhagic complications. We report a case of acquired haemophilia presenting with multiple bluish patches affecting face, neck, upper & lower limbs, history of gum bleeding and left knee haemarthrosis. The patient was found to have acquired FVIII inhibitor and lupus anticoagulant (LAC). The simultaneous presence of LAC and FVIII inhibitor is exceedingly rare. The differentiation between these two conditions is crucial, because both result in a prolongation of the activated partial thromboplastin time test, which does not correct when mixed with the plasma of a normal control; however, the clinical manifestations range from thrombosis in the presence of LAC to massive haemorrhage with FVIII inhibitors.

Altered expression of platelet proteins and calpain activity mediate hypoxia-induced prothrombotic phenotype.

Oxygen-compromised environments, such as high altitude, air travel, and sports, and pathological conditions, such as solid tumors, have been suggested to be prothrombotic. Despite the indispensable role of platelets in thrombus formation, the studies linking hypoxia, platelet reactivity, and thrombus formation are limited. In the present study, platelet proteome/reactivity was analyzed to elucidate the acute hypoxia-induced prothrombotic phenotype. Rats exposed to acute simulated hypoxia (282 torr/8% oxygen) demonstrated a decreased bleeding propensity and increased platelet reactivity. Proteomic analysis of hypoxic platelets revealed 27 differentially expressed proteins, including those involved in coagulation. Among these proteins, calpain small subunit 1, a 28-kDa regulatory component for calpain function, was significantly upregulated under hypoxic conditions. Moreover, intraplatelet Ca(2+) level and platelet calpain activity were also found to be in accordance with calpain small subunit 1 expression. The inhibition of calpain activity demonstrated reversal of hypoxia-induced platelet hyperreactivity. The prothrombotic role for calpain was further confirmed by an in vivo model of hypoxia-induced thrombosis. Interestingly, patients who developed thrombosis while at extreme altitude had elevated plasma calpain activities and increased soluble P-selectin level. In summary, this study suggests that augmented calpain activity is associated with increased incidence of thrombosis under hypoxic environments.

A rare case of hilar lymph node schwannoma.

Schwannoma arising in a lymph node is an extremely rare primary mesenchymal tumor of the lymph node. Although intranodal schwannoma is benign, this lesion in a lymph node may be initially alarming and it may suggest a diagnosis of intranodal metastasis from a spindle cell tumor. Microscopic examination of tumor shows Antoni A areas, which are moderate to highly cellular areas of elongated cells arranged in fascicles, and Antoni B areas, which are hypocellular areas with myxoid and microcystic change. Tumor cell morphology is similar in both areas with elongated shape and regular oval nuclei. Immunohistochemistry report shows strong and diffuse positivity for S-100 which is consistent with Schwann cell origin. We report a rare case of intranodal schwannoma arising in the hilar lymph node with its clinical, light microscopic, and immunohistochemical findings.

Chitosan encapsulated quantum dots platform for leukemia detection.

We report results of the studies relating to electrophoretic deposition of nanostructured composite of chitosan (CS)-cadmium-telluride quantum dots (CdTe-QDs) onto indium-tin-oxide coated glass substrate. The high resolution transmission electron microscopic studies of the nanocomposite reveal molecular level coating of the CdTe-QDs with CS molecules in the colloidal dispersion medium. This novel composite platform has been explored to fabricate an electrochemical DNA biosensor for detection of chronic myelogenous leukemia (CML) by immobilizing amine terminated oligonucleotide probe sequence containing 22 base pairs, identified from BCR-ABL fusion gene. The results of differential pulse voltammetry reveal that this nucleic acid sensor can detect as low as 2.56 pM concentration of complementary target DNA with a response time of 60s. Further, the response characteristics show that this fabricated bioelectrode has a shelf life of about 6 weeks and can be used for about 5-6 times. The results of experiments conducted using clinical patient samples reveal that this sensor can be used to distinguish CML positive and the negative control samples.

Nanopatterned cadmium selenide Langmuir-Blodgett platform for leukemia detection.

We present results of the studies relating to preparation of Langmuir-Blodgett (LB) monolayers of tri-n-octylphosphine oxide-capped cadmium selenide quantum dots (QCdSe) onto indium-tin oxide (ITO) coated glass substrate. The monolayer behavior has been studied at the air-water interface under various subphase conditions. This nanopatterned platform has been explored to fabricate an electrochemical DNA biosensor for detection of chronic myelogenous leukemia (CML) by covalently immobilizing the thiol-terminated oligonucleotide probe sequence via a displacement reaction. The results of electrochemical response studies reveal that this biosensor can detect target DNA in the range of 10(-6) to 10(-14) M within 120 s, has a shelf life of 2 months, and can be used about 8 times. Further, this nucleic acid sensor has been found to distinguish the CML-positive and the control negative clinical patient samples.

Disseminated intravascular coagulation in acute leukemia at presentation and during induction therapy.

Between January 2001 and December 2003, 67 patients with acute leukemia were evaluated prospectively for hemostatic abnormality at presentation, of which 43 (64.2%) had acute lymphoblastic leukemia and 24 (35.8%) had acute myelogenous leukemia. At presentation, 27 patients (40.3%) had bleeding manifestations. Thrombocytopenia was present in 57 patients (85%), and 33(49.3%) had some abnormality of global coagulation markers. Disseminated intravascular coagulation was defined by International Society of Thrombosis and Hemostasis criteria. Disseminated intravascular coagulation was more often associated with bleeding manifestations in acute myelogenous leukemia cases than in acute lymphoblastic leukemia cases. Two patients presented disseminated intravascular coagulation on day 7 of chemotherapy, without any bleeding manifestations. Four of 15 evaluated cases who had a bleeding or infection complication after day 7 of induction therapy also had disseminated intravascular coagulation. It is recommended that all patients with leukemia be investigated for disseminated intravascular coagulation at presentation.

Philadelphia-chromosome positive thrombocythemia in a child.

Philadelphia-chromosome positive thrombocythemia without features of chronic myeloid leukemia in peripheral blood has been described in adults. It is rare in children. We present a case of Philadelphia positive thrombocythemia in a child who was managed with a combination of imatinib and hydroxyurea. Although a reduction in the BCR-ABL transcript was documented, the thrombocytosis was refractory to imatinib alone and required the addition of hydroxyurea.

Hb SE disease: a clinico-hematological profile.

Hb S and Hb E are globally common hemoglobinopathies. However, Hb SE double heterozygous state is uncommon, with only 25 cases reported so far in literature. We present two more cases. One presented with gallstones, and the other was asymptomatic. This type of disorder was previously described as a relatively asymptomatic condition compared to HbSS. A review of the 25 reported cases in literature shows that 40.7% (11/27) of these cases are symptomatic. Gender, hematological parameters and levels of Hb S, E or F do not predict clinical severity.