In this study, clinico-hematological, genetic and outcome profile of children with BMF was evaluated to delineate the underlying genotype and phenotype.
Cases were evaluated as two groups: Group 1 ...(n = 56; DBA-23, FA-18, DC-2, UBMFS-13) included children with suspected IBMFS based on clinical phenotype and accessible lab investigations and Group 2 (n = 53) included children with IAA treated with IST. Targeted NGS was carried out in a subset of these children (n = 42) and supplemented with WES wherever required.
We identified causative mutation in overall 15 of 27 tested children (55.5%) in group 1 and 2 of 15 tested children (13.3%) in group 2. In DBA, a mutation was noted in 50% cases with involvement of RPS 19 (75%) and RPL5 (25%) genes. Phenotypic abnormalities were present in 69.5% and response to steroids in 68.4% of cases at a median follow up of 33 months. In children with IAA, overall response (complete + partial) was present in 51% at a median follow up of 23 months. The 3-year OS and FFS for the cohort of IAA were 68% and 48% respectively. Targeted sequencing could also pick up germline mutations in 50% of UBMFS cases and nearly 19% of IAA cases.
•Targeted NGS panel could detect pathogenic mutations in 50% of DBA cases and is not inferior to whole exome sequencing•85% of our DBA cases with RPS19 mutations were steroid responsive on follow-up•Up to 50% of unclassified inherited bone marrow failure syndromes could also be classified on targeted sequencing•Fanconi Anemia cases can have atypical phenotype with findings of ringed sideroblasts and or coeliac disease•Targeted NGS testing ina subset of IAArevealed a pathogenic/VUSvariation in 19% cases
Gene expression profiling is the criterion standard for recognizing Ph-like ALL signatures among B-ALLs. The prerequisite of GEP is the accurate normalization of target genes with stable expression ...of housekeeping genes in a quantitative PCR. HKGs exhibit differential expression in the different experimental conditions and affect the target genes' expression, leading to imprecise qPCR results. The selection of stable HKGs is crucial in GEP experiments, especially in identifying high-risk Ph-like ALL cases. We have evaluated the expression stability of nine HKGs (
GAPDH, ACTB, GUSB, RNA18S, EEF2, PGK1, B2M, TBP
and
ABL1
) in identified Ph-like ALLs and Ph-negative (
n
= 23 each) using six algorithms, 4 traditional softwares; geNorm, BestKeeper, NormFinder, Delta Cq value method, and two algorithms, RefFinderTM and ComprFinder. Further, we have validated the expression of 8 overexpressed normalized genes in Ph-like ALL cases (
JCHAIN, CA6, MUC4, SPATS2L, BMPR1B, CRLF2, ADGRF1 and NRXN3
). GeNorm, BestKeeper, NormFinder, Delta Cq value method, RefFinderTM and ComprFinder algorithm analysis revealed that
EEF2, GAPDH
, and
PGK1
form the best representative HKGs in Ph-like ALL cases, while RNA18s, ß-actin, and
ABL1
in Ph-negative ALLs. Lastly, we performed a correlation analysis and found that the combination of
EEF2, GAPDH
, and
PGK1
represents the best combination with a very high correlation in Ph-like ALL cases. This is the first report that shows
EEF2, GAPDH,
and
PGK1
are the best HKG genes and can be used in the diagnostic panel of Ph-like ALL cases using qPCR at baseline diagnosis.
Epstein-Barr virus (EBV) is involved in the development of a wide range of B cell lympho-proliferative disorders. Its association with plasma cell disorders (PCD) however is not clear, especially in ...immunocompetent patients. To explore any relationship, 39 patients of suspected PCD with positive M-band on electrophoresis and 50 healthy controls were enrolled. EBV DNA in peripheral blood was quantified using quantitative Real Time Polymerase Chain Reaction (qPCR). Of 39 patients, 15 (38.5%) had EBV DNA compared to 8/50 (16%) controls (
p = 0.0008
). The mean viral copy number was found to be significantly high in patients compared to controls (1.8 × 10
5
; range = 2.6 × 10
3
–7.6 × 10
5
copies/ml and 1.7 × 10
4
; range = 7.0 × 10
2
–6.1 × 10
4
copies/ml respectively;
p = 0.003
). This is the first study, which characterizes the frequency of EBV in circulation in patients of PCD. The significance of increased prevalence of circulating EBV and a higher viral load in our immunocompetent patients however, needs further evaluation.
Based on the immunophenotype, acute lymphoblastic leukemia (ALL) can be categorized into B-cell or T-cell lineages. B-cell precursor ALL (BCP-ALL) cases show various genetic/molecular abnormalities, ...and varying frequencies of chimeric fusion transcripts in BCP-ALL cases are reported from different parts of the world. We studied the immunophenotypic aberrancy profiles of a large number of BCP-ALL cases with respect to various common chimeric fusion transcripts.
Flow cytometric immunophenotyping and multiplex reverse-transcription polymerase chain reaction assays were performed for 986 BCP-ALL cases.
Among 986 BCP-ALL cases, the incidence of various fusion transcripts was 38.36% in adult cases and 20.68% in pediatric cases. Adult BCP-ALL patients with t(9;22)(
) fusion transcripts and expression of aberrant myeloid markers were significantly older at presentation (p=0.0218) with male preponderance (p=0.0246) compared to those without aberrant myeloid expression. In pediatric patients with the t(12;21)(
) chimeric fusion transcript, aberrant expression of CD13 was observed in 39.13%, CD33 in 36.95%, and CD117 in 8.69% of patients, respectively. Pediatric BCP-ALL patients with the
fusion transcript and expression of aberrant myeloid markers were not significantly different compared to those without with respect to demographic and clinical/hematological characteristics (p=0.5955). Aberrant myeloid markers were rarely or never expressed in pediatric and adult BCP-ALL patients with the t(4;11)(
) and t(1;19)(
) fusion transcripts.
Aberrant myeloid markers were frequently expressed among BCP-ALL patients with the t(9;22)(
) and t(12;21) (
) fusion transcripts. However, BCP-ALL patients with the t(4;11)(
) and t(1;19)(
) fusion transcripts rarely or never expressed aberrant myeloid markers. Aberrant myeloid CD markers can be used in predicting chimeric fusion transcripts at baseline so as to plan appropriate tyrosine kinase inhibitor therapy in cases of BCP-ALL with specific chimeric fusion transcripts. This study has delineated the relationship of chimeric fusion transcripts with the aberrant expression of myeloid markers in a large cohort of BCP-ALL cases.
BRAF
V600E is a disease defining mutation for hairy cell leukemia (HCL), which helps in its diagnosis and differentiation from morphologically similar splenic marginal zone lymphoma (SMZL) and ...HCL-variant (HCL-v). Forty eight cases:HCL(n = 34), SMZL(n = 11) and HCL-v(n = 3) were included. Of these, 32 were retrospective and 16 were prospective. DNA was extracted, in retrospective cases from cells obtained by smears from bone marrow aspirate/trephine imprint (BMA/BMTx) slides, and in prospective cases from peripheral blood (PB)/BMA samples.
BRAF
V600E mutation testing was done using ARMS-PCR.
BRAF
V600E mutation was positive in all HCL and negative in all SMZL and HCL-v cases. DNA extracted from BMA/BMTx slides gave results comparable to DNA extracted from PB/BMA samples. Median age of presentation for HCL (53 years) and SMZL (56 years) were quite similar, however, HCL-v (71 years) cases presented at an older age. Statistically significant differences between the three groups were seen for total leucocyte, platelet, absolute lymphocyte and monocyte counts, bone marrow-infiltration pattern, reticulin fibrosis, and an expression of CD11c, CD25, CD103, CD123, and CD200. The use of BMA/BMTx smears for DNA extraction was found to be a useful alternative to DNA extraction from formalin-fixed paraffin-embedded biopsy sections. ARMS-PCR is an efficient and specific technique to detect
BRAF
V600E mutation in HCL patients.
Sustained release nanoformulations of second line antitubercular drugs levofloxacin and ethionamide had shown promise in pharmacokinetics and acute and sub-acute toxicity studies. The present study ...evaluated the clastogenicity potential of the nanoformulations of these antitubercular agents. Clastogenicity was evaluated by (a) in vitro micronucleus assay (b) in vivo micronucleus assay in Swiss albino mice and (c) sister chromatid exchange (SCE) in CHO cell lines. Ethionamide and levofloxacin loaded nanoparticles were 312 ± 64 nm and 245 ± 24 nm in size respectively and drug encapsulation was 35.2 ± 3.1% w/w and 45.6 ± 9.4% w/w, respectively. The frequency of MN-NCE/1000 NCE and MN-PCE/1000 PCE were significantly reduced in mice treated with ethionamide nanoparticle (3.5 ± 0.9, 13.8 ± 16.68) and levofloxacin nanoparticles (5.6 ± 2.7, 16.7 ± 12.7) compared to the mice treated with free ethionamide (11.5 ± 4.1, p = 0.23 and 45.19 ± 19.21, p = 0.38) and free levofloxacin (14.7 ± 1.88, p < 0.0001 and 54.6 ± 18.1, p = 0.0017), respectively. For in vitro, micronucleus assay frequencies of micronuclei per thousand bi-nucleated cells (MN-BN/1000 BN) was 188.3 ± 20.20 and 148 ± 20.42 for ethionamide and levofloxacin nanoparticles as compared to 232.6 ± 16.04 (p = 0.52) and 175 ± 5.56 (p = 0.45) for free ethionamide and levofloxacin, respectively. The average number of SCE per cell for nanoformulation of ethionamide were not different from that of free drug (4.9 ± 0.51 vs 4.1 ± 0.55, p = 0.86). The SCE per cells were not significant difference for nanoformulation of levofloxacin (2.33 ± 1.36 vs 5.46 ± 0.25, p = 0.88). In vitro and in vivo assays have shown relatively less clastogenic potential of equivalent dose of ethionamide nanoparticles as compared to the conventional formulation.
Background
The standard dose (SD) of horse anti-thymocyte globulin (hATG) ATGAM (Pfizer, USA) or its biosimilar thymogam (Bharat Serum, India) for the treatment of Aplastic Anemia (AA) is ...40 mg/kg/day for 4 days in combination with cyclosporine. Data on the impact of hATG dose on long-term outcomes are limited. Here, we describe our comparative experience using 25 mg/kg/day (low-dose LD) hATG for 4 days with SD for the treatment of AA.
Methods
We retrospectively studied patients with AA (age > 12 years) who received two doses of hATG combined with cyclosporine. Among 93 AA patients who received hATG, 62 (66.7%) and 31 (33.3%) patients received LD and SD hATG with cyclosporine, respectively. Among these,seventeen(18.2%) patients also received eltrombopag with hATG and cyclosporine. Overall response rates complete response (CR) and partial response (PR) of LD and SD hATG groups at 3 months (50% vs. 48.4%;
p
= 0.88), 6 months (63.8% vs. 71.4%;
p
= 0.67), and 12 months (69.6% vs. 79.2%;
p
= 0.167) were comparable. The mean (Standard Deviation) 5-year Kaplan–Meier estimate of overall survival and event-free survival was 82.1 (4.6)% and 70.9 (5.5)% for the study population. The mean (standard deviation) 5-year Kaplan–Meier estimate of overall survival and event-free survival of those who received LD hATG versus SD hATG dose was 82.9 (5·3)% versus 74.8 (10·3)% (
P
= 0·439), and 75.2 (6.2)% versus 61.4(11.2)% (
P
= 0·441).
Conclusion
Our study revealed that the response rates of patients with AA and LD were similar to those of patients with SD to hATG combined with cyclosporine in a real-world setting.
The overexpression of cytokine receptor-like factor-2 (CRLF2) identified by anti-thymic stromal lymphopoietin receptor/TSLPR flow cytometry (FCM) has been reported as a screening tool for the ...identification of
BCR-ABL1-
like B-cell acute lymphoblastic leukemia/B-ALL with
CRLF2
re-arrangement. TSLPR expression was studied prospectively in consecutive 478 B-ALLs (≤ 12 years (
n
= 244); 13–25 years (
n
= 129); > 25 years (
n
= 105)) and correlated with various hematological parameters and end-of-induction measurable residual disease (day 29; MRD ≥ 0.01% by 10-color FCM). TSLPR positivity in ≥ 10% leukemic cells was detected in 14.6% (
n
= 70) of B-ALLs.
CRLF2
re-arrangement was detected in eight cases (11.4%) including
P2RY8-CRLF2
(
n
= 6), and
IgH
-
CRLF2
(
n
= 2) with a median TSLPR positivity of 48.8% and 99% leukemic cells, respectively. Recurrent gene fusions/RGF (
BCR-ABL1
(17.1%);
ETV6-RUNX1
(4.2%),
TCF3-PBX1
(1.4%)), other
BCR-ABL1-
like chimeric gene fusions/CGFs (
PDGFRB
-rearrangement (2.9%),
IgH-EPOR
(1.4%)),
CRLF2
extra-copies/hyperdiploidy (17.1%), and
IgH
translocation without a known partner (10%) were also detected in TSLPR-positive patients. CD20 positivity (52.9% vs 38.5%;
p
= 0.02) as well as
iAMP21
(4.3% vs 0.5%;
p
= 0.004) was significantly more frequent in TSLPR-positive cases. TSLPR-positive patients did not show a significantly higher MRD, compared to TSLPR-negative cases (37% vs 33%). Increasing the threshold cut-off (from ≥ 10 to > 50% or > 74%) increased the specificity to 88% and 100% respectively in identifying
CRLF2
translocation. TSLPR expression is not exclusive for
CRLF2
translocations and can be seen with various other RGFs, necessitating their testing before its application in diagnostic algorithms. In patients with high TSLPR positivity (> 50%), the testing may be restricted to
CRLF2
aberrancies, while patients with 10–50% TSLPR positivity need to be tested for both
CRLF2-
and non-
CRLF2 BCR-ABL1
-like CGFs.
Genetic work-up of unexplained erythrocytosis that is suspected to be inherited in nature currently requires either laborious exon-by-exon gene panel testing by Sanger sequencing or expensive ...next-generation sequencing. A high prevalence of Chuvash polycythemia (61%) has been previously reported among north Indian erythrocytosis patients. We assessed PCR-RFLP for
VHL
c.598C > T mutation as a first-line test in 99 persons with
JAK2
V617F-negative, unexplained erythrocytosis. We enrolled two groups: Group A (n = 38) had erythrocytosis patients (n = 33) or their first-degree relatives (n = 5), and, Group B with 61 healthy blood donation volunteers who were deferred after the discovery of unexplained high hemoglobin levels. Detailed history and clinical examination, hemogram, erythropoietin levels and PCR–RFLP for the
VHL
:c.598C > T;p.R200W mutation were done. In Group A, three (8%) persons aged 9, 13 and 30-years were homozygous for
VHL
:c.598C > T. Two were heterozygous (parents of a known case of Chuvash polycythemia). None of the Group B subjects had the Chuvash mutation. Erythropoietin levels in group A were low in 5/26 cases (19%) and normal in 18/26 (69%). In Group B, seven (11%) donors had normal values while the remaining 54 (89%) had high erythropoietin levels. Despite a lower frequency (8%) compared to literature, our results suggest that the relatively simpler PCR-RFLP for
VHL
:c.598C > T mutation may be considered for the initial genetic screening of unexplained, suspected congenital erythrocytosis in regions where Chuvash polycythemia comprises a large proportion of inherited erythrocytosis, after polycythemia vera and common acquired secondary causes are excluded.
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