Vivi M. Srivastava

Long-Term Cultured Human Term Placenta-Derived Mesenchymal Stem Cells of Maternal Origin Displays Plasticity

Mesenchymal stem cells (MSCs) are an alluring therapeutic resource because of their plasticity, immunoregulatory capacity and ease of availability. Human BM-derived MSCs have limited proliferative capability, consequently, it is challenging to use in tissue engineering and regenerative medicine applications. Hence, placental MSCs of maternal origin, which is one of richest sources of MSCs were chosen to establish long-term culture from the cotyledons of full-term human placenta. Flow analysis established bonafied MSCs phenotypic characteristics, staining positively for CD29, CD73, CD90, CD105 and negatively for CD14, CD34, CD45 markers. Pluripotency of the cultured MSCs was assessed by in vitro differentiation towards not only intralineage cells like adipocytes, osteocytes, chondrocytes, and myotubules cells but also translineage differentiated towards pancreatic progenitor cells, neural cells, and retinal cells displaying plasticity. These cells did not significantly alter cell cycle or apoptosis pattern while maintaining the normal karyotype; they also have limited expression of MHC-II antigens and are Naive for stimulatory factors CD80 and CD 86. Further soft agar assays revealed that placental MSCs do not have the ability to form invasive colonies. Taking together all these characteristics into consideration, it indicates that placental MSCs could serve as good candidates for development and progress of stem-cell based therapeutics.

Role of minimal residual disease monitoring in acute promyelocytic leukemia treated with arsenic trioxide in frontline therapy

Data on minimal residual disease (MRD) monitoring in acute promyelocytic leukemia (APL) are available only in the context of conventional all-trans retinoic acid plus chemotherapy regimens. It is recognized that the kinetics of leukemia clearance is different with the use of arsenic trioxide (ATO) in the treatment of APL. We undertook a prospective peripheral blood RT-PCR-based MRD monitoring study on patients with APL treated with a single agent ATO regimen. A total of 151 patients were enrolled in this study. A positive RT-PCR reading at the end of induction therapy was significantly associated on a multivariate analysis with an increased risk of relapse (relative risk = 4.9; P = .034). None of the good risk patients who were RT-PCR negative at the end of induction relapsed. The majority of the relapses (91%) happened within 3 years of completion of treatment. After achievement of molecular remission, the current MRD monitoring strategy was able to predict relapse in 60% of cases with an overall sensitivity and specificity of 60% and 93.2%, respectively. High-risk group patients and those that remain RT-PCR positive at the end of induction are likely to benefit from serial MRD monitoring by RT-PCR for a period of 3 years from completion of therapy.

Comparison of Newly Diagnosed and Relapsed Patients with Acute Promyelocytic Leukemia Treated with Arsenic Trioxide: Insight into Mechanisms of Resistance

There is limited data on the clinical, cellular and molecular changes in relapsed acute promyeloytic leukemia (RAPL) in comparison with newly diagnosed cases (NAPL). We undertook a prospective study to compare NAPL and RAPL patients treated with arsenic trioxide (ATO) based regimens. 98 NAPL and 28 RAPL were enrolled in this study. RAPL patients had a significantly lower WBC count and higher platelet count at diagnosis. IC bleeds was significantly lower in RAPL cases (P=0.022). The ability of malignant promyelocytes to concentrate ATO intracellularly and their in-vitro IC50 to ATO was not significantly different between the two groups. Targeted NGS revealed PML B2 domain mutations in 4 (15.38%) of the RAPL subset and none were associated with secondary resistance to ATO. A microarray GEP revealed 1744 genes were 2 fold and above differentially expressed between the two groups. The most prominent differentially regulated pathways were cell adhesion (n=92), cell survival (n=50), immune regulation (n=74) and stem cell regulation (n=51). Consistent with the GEP data, immunophenotyping revealed significantly increased CD34 expression (P=0.001) in RAPL cases and there was in-vitro evidence of significant microenvironment mediated innate resistance (EM-DR) to ATO. Resistance and relapse following treatment with ATO is probably multi-factorial, mutations in PML B2 domain while seen only in RAPL may not be the major clinically relevant cause of subsequent relapses. In RAPL additional factors such as expansion of the leukemia initiating compartment along with EM-DR may contribute significantly to relapse following treatment with ATO based regimens.

Acute myeloid leukaemia: challenges and real world data from India

The management of acute myeloid leukaemia (AML) in India remains a challenge. In a two‐year prospective study at our centre there were 380 newly diagnosed AML (excluding acute promyelocytic leukaemia, AML‐M3) patients. The median age of newly diagnosed patients was 40 years (range: 1–79; 12·3% were ≤ 15 years, 16·3% were ≥ 60 years old) and there were 244 (64·2%) males. The median duration of symptoms prior to first presentation at our hospital was 4 weeks (range: 1–52). The median distance from home to hospital was 580 km (range: 6–3200 km). 109 (29%) opted for standard of care and were admitted for induction chemotherapy. Of the 271 that did not take treatment the major reason was lack of financial resources in 219 (81%). There were 27 (24·7%) inductions deaths and of these, 12 (44·5%) were due to multidrug‐resistant gram‐negative bacilli and 12 (44·5%) showed evidence of a fungal infection. The overall survival at 1 year was 70·4% ± 10·7%, 55·6% ± 6·8% and 42·4% ± 15·6% in patients aged ≤15 years, 15 ‐ 60 years and ≥60 years, respectively. In conclusion, the biggest constraint is the cost of treatment and the absence of a health security net to treat all patients with this diagnosis.

The t(6;9)(p22;q34) in myeloid neoplasms: a retrospective study of 16 cases

Among patients with acute myeloid leukemia (AML), the t(6;9) (p22;q34) is a rare but defined subset with a poor prognosis. We report 16 patients with the t(6;9), of whom 13 had AML, 2 had myelodysplastic syndrome (MDS), and 1 had chronic myeloid leukemia in myeloid blast crisis (CML-BC). All except for one were evaluated at diagnosis. The median age was 34.5 (range: 7-62 years), with 12 adults and 12 males. Trilineage dysplasia was present in 13 (81%). Marrow basophilia was seen in only two patients, one of whom had CML-BC. HLA-DR was positive in all 12 patients assessed, CD33 in 11, CD13 in 10, and CD34 in seven. Four patients had one other abnormality apart from the t(6;9). These were the t(9;22) in the patient with CML and deletion 9q, addition 13q, and an isochromosome 8q in the other three patients. There were no complex karyotypes. Fms-related tyrosine kinase 3--internal tandem duplication (FLT3-ITD) mutations were seen in seven of 13 patients. Follow-up details were available for six patients. Three received palliative care, and follow-up details were not available for the other seven. The response to chemotherapy was poor in the remaining patients. The only patients who survived were three out of the four who had allogeneic hematopoietic stem cell transplantation (HSCT).

Infections in haematological malignancies: an autopsy study of 72 cases

Autopsy material from 72 patients with haematological malignancies treated in India was reviewed. Thirty-seven patients (51%) had documented infections; 20 (27%) had bacterial infections, 14 of which were Gram-negative organisms (Pseudomonas species in 10); tuberculosis was present in 2 patients (2.7%). Twenty-one patients (29%) had systemic fungal infections; invasive pulmonary aspergillosis and gastrointestinal candidiasis were present in 10 patients each. Only 3 patients (4%) had viral infection, all of which were due to cytomegalovirus. Eleven patients (15%) had polymicrobial infections. No patient had any parasitic infection. Systemic fungal infections due to Aspergillus and Candida predominated, while Gram-negative bacterial infections were also common.

RNA expression of genes involved in cytarabine metabolism and transport predicts cytarabine response in acute myeloid leukemia

BACKGROUND Variation in terms of outcome and toxic side effects of treatment exists among acute myeloid leukemia (AML) patients on chemotherapy with cytarabine (Ara-C) and daunorubicin (Dnr). Candidate Ara-C metabolizing gene expression in primary AML cells is proposed to account for this variation. METHODS Ex vivo Ara-C sensitivity was determined in primary AML samples using MTT assay. mRNA expression of candidate Ara-C metabolizing genes were evaluated by RQPCR analysis. Global gene expression profiling was carried out for identifying differentially expressed genes between exvivo Ara-C sensitive and resistant samples. RESULTS Wide interindividual variations in ex vivo Ara-C cytotoxicity were observed among samples from patients with AML and were stratified into sensitive, intermediately sensitive and resistant, based on IC50 values obtained by MTT assay. RNA expression of deoxycytidine kinase (DCK), human equilibrative nucleoside transporter-1 (ENT1) and ribonucleotide reductase M1 (RRM1) were significantly higher and cytidine deaminase (CDA) was significantly lower in ex vivo Ara-C sensitive samples. Higher DCK and RRM1 expression in AML patients blast correlated with better DFS. Ara-C resistance index (RI), a mathematically derived quotient was proposed based on candidate gene expression pattern. Ara-C ex vivo sensitive samples were found to have significantly lower RI compared with resistant as well as samples from patients presenting with relapse. Patients with low RI supposedly highly sensitive to Ara-C were found to have higher incidence of induction death (p = 0.002; RR: 4.35 [95% CI: 1.69-11.22]). Global gene expression profiling undertaken to find out additional contributors of Ara-C resistance identified many apoptosis as well as metabolic pathway genes to be differentially expressed between Ara-C resistant and sensitive samples. CONCLUSION This study highlights the importance of evaluating expression of candidate Ara-C metabolizing genes in predicting ex vivo drug response as well as treatment outcome. RI could be a predictor of ex vivo Ara-C response irrespective of cytogenetic and molecular risk groups and a potential biomarker for AML treatment outcome and toxicity. Original submitted 22 December 2014; Revision submitted 9 April 2015.

The Clinical Profile of Children in India with Pigmentary Anomalies along the Lines of Blaschko and Central Nervous System Manifestations

Abstract:  The aim of this study was to describe the clinical profile of children with congenital pigmentary anomalies along Blaschko lines and the associated manifestations in the central nervous system. Twenty‐six children aged 12 years or less (14 boys and 12 girls), with hypopigmentation and hyperpigmentation along Blaschko lines and central nervous system manifestations were included during the period June 2001 to December 2003. Detailed physical, cutaneous, and systemic examinations were carried out. Relevant investigations included histopathology, karyotyping, electroencephalogram, computerized tomography scans, and magnetic resonance imaging of the brain whenever possible. Twenty children had hyperpigmentation along the lines of Blaschko, four had hypopigmentation, and two had a combination of the two. Eight children had diffuse involvement. Of these, two each had hypomelanosis of Ito, incontinentia pigmenti and linear and whorled nevoid hypermelanosis. The remaining 20 patients could not be categorized as any of the described entities. The majority (92.3%) of patients manifested skin and central nervous system disorders before the age of 2 years. Hyperpigmentation along Blaschko lines was significantly higher in patients with central nervous system manifestations (p = 0.01). Developmental delay was the most frequent central nervous system presentation. Multiple systems were affected, including the eyes. Histology was useful to distinguish incontinentia pigmenti from other types of nevoid hyperpigmentation.

Spectrum of BCR-ABL kinase domain mutations in patients with chronic myeloid leukemia from India with suspected resistance to imatinib-mutations are rare and have different distributions

The introduction of tyrosine kinase inhibitors hasrevolutionized the treatment of chronic myeloidleukemia (CML). Imatinib mesylate, which compe-titively targets the adenosine 5-triphosphate (ATP)-binding site of the kinase domain of BCR-ABL [1] isnow the first choice of therapy for CML. Theincidence of CML in India varies from 0.8 to 2.2and 0.6 to 1.6 per 100 000 population in males andfemales, respectively. The overall incidence of CMLis 42.1% of all the adult leukemias as seen in ouroutpatient department from the year 1997 to 2008(1226 out of 2975 adult leukemias). Despite theimpressive rate of complete hematological responseand complete cytogenetic remissions, some casesshow primary or secondary resistance to imatinib.Several mechanisms have been attributed as thecause for clinical resistance to imatinib [2,3]. Amongthese, point mutations in the BCR-ABL kinasedomain appear to be the most common mechanismoccurring in 30–90% of patients who developresistance [4,5]. A point mutation in BCR-ABLkinase domain can cause an amino acid change andimpair imatinib binding by interrupting the criticalcontact point or by altering the conformation of theprotein [6]. To date, more than 50 different kinasedomain mutations have been identified and theyconfer different degrees of in vitro resistance toimatinib. There have been very few reports on thespectrum of BCR-ABL kinase domain mutations inAsian patients with CML [7–9]. In a recent studyfrom the Korean population, CML patients withimatinib resistance showed high rates (63%) ofmutations in the BCR-ABL kinase domain [7]. Sincethe response rate to imatinib is suboptimal in theIndian population [10,11], it is possible that this canbe attributed to a different mutation spectrum in thispopulation. The aim of the present study wastherefore to analyze the frequency and the spectrumof BCR-ABL kinase domain mutations in Indianpatients with CML with clinical resistance toimatinib.Between January 2004 and January 2009, periph-eral blood samples were collected from 76 patientswith CML with suspected clinical resistance toimatinib diagnosed in different phases of the disease(54 in chronic phase, 14 in accelerated phase, and 8in blast crisis). Eligible patients visiting the outpatientclinic at the department of Haematology, ChristianMedical College, Vellore were enrolled in the Glivec(imatinib) International Patient Assistance Program(GIPAP). Patients with CML in the chronic phase,accelerated phase, and blast crisis stage receivedstandard doses of 400, 600, and 800 mg/day ofimatinib, respectively.Primary resistance to imatinib was defined asfailure to achieve hematological remission within 3–6 months or failure to achieve any level of cytogeneticresponse at 6 months, major cytogenetic response at12 months, or complete cytogenetic response at 18months. Secondary resistance was defined as relapse

Cytogenetic analysis of acute myeloid leukemia with t(8;21) from a tertiary care center in India with correlation between clinicopathologic characteristics and molecular analysis

Abstract The t(8;21)(q22;q22) is the most common translocation in acute myeloid leukemia (AML). We describe the clinicopathologic and cytogenetic profile of 117 patients with t(8;21) AML. There were 76 males and 88 adults. The median age was 26 years. Most patients (80%) had AML M2. Dysplasia was present in 68% of patients and eosinophilia in 18%. Eight patients had fewer than 20% blasts. Additional chromosomal aberrations were seen in 103 patients (88%) with loss of a sex chromosome (LSC) in 78 patients (66%) and deletion 9q in 21 (18%). The other recurrent abnormalities were trisomies 4, 8 and 15, monosomy 17 and deletion 7q (less than 5% each). Three- or four-way variant t(8;21) were seen in 6% of patients and 3% had tetraploidy. Aberrant expression of CD19 was seen in 54% of patients. FLT3 mutations were seen in 7.5% of patients (3/40) and c-KIT mutations in 6.6% (2/30). None had NPM1 or JAK2 V617F mutations. One patient had a granulocytic sarcoma. Complete remission was achieved in 96% of the 26 newly diagnosed patients after first induction. The median follow-up was 25 months (range 4–68). The overall survival was 69% at 31 months.