A surrogate molecular approach for the detection of Philadelphia chromosome-like B-acute lymphoblastic leukemia.

BACKGROUND: Philadelphia chromosome (Ph)-like B-acute lymphoblastic leukemia (B-ALL) is a clinically significant, high-risk genetic subtype of B-ALL cases. There are few data on the incidence, characterization, and treatment outcomes of Ph-like ALL cases from low- and middle-income countries. There is a pressing need to establish a well-organized/cost-effective approach for identifying Ph-like ALL instances. METHODS: Multiplex reverse transcriptase polymerase chain reaction, nCounter NanoString, and fluorescence in situ hybridization were used to detect and characterize Ph-like ALL cases among recurrent genetic abnormalities (RGA)neg B-ALL cases. At the end of induction therapy, flow cytometry-minimal residual disease (MRD) assay was used to quantify MRD positivity in Ph-like ALL cases. RESULTS: Of 130 newly diagnosed B-ALL cases, 25% (BCR::ABL1), 4% (ETV6::RUNX1), 5% (TCF3::PBX1), 2% (KM2TA::AFF1), and 65% RGAneg B-ALL cases were revealed by multiplex reverse transcriptase polymerase chain reaction. Among RGAneg B-ALL cases, 24% Ph-like ALL cases using nCounter NanoString were identified, with 48% CRLF2high cases with 45% CRLF2::P2RY8 and 18% CRLF2::IGH rearrangements(∼r) revealed by fluorescence in situ hybridization. In 52% of CRLF2low cases, 17% ABL1 and JAK2∼r 8% EPOR::IGH & PDGRFB∼r were identified. Ph-like ALL cases had higher total leukocyte count (p < .05), male preponderance (p < .05), and high MRD-positivity/induction failure compared with RGAneg B-ALL cases. Furthermore, in Ph-like ALL cases, 11 significant genes using quantitative polymerase chain reaction were identified and validated. CRLF2, IGJ, CEACAM6, MUC4, SPATS2L and NRXN3 genes were overexpressed and show statistical significance (p < .05) in Ph-like ALL cases. CONCLUSIONS: This study showed the high incidence of Ph-like ALL cases with kinase activating alterations and treatment outcomes from low- and middle-income region. Furthermore, a surrogate cost-effective multiplex panel of 11 overexpressed genes for the prompt detection of Ph-like ALL cases is proposed. PLAIN LANGUAGE SUMMARY: Identification of recurrent gene abnormalities (RGA)neg B-acute lymphoblastic leukemia (B-ALL) cases using multiplex-reverse transcriptase polymerase chain reaction. Identification and characterization of Philadelphia (Ph)-like ALL cases using nCounter NanoString gene expression profiling and fluorescence in situ hybridization. Furthermore, Ph-like ALL cases were characterized according to CRLF2 expression and kinase-activating genomic alterations. Minimal residual disease of Ph-like ALL cases were quantified using flow cytometry-minimal residual disease assay. A surrogate molecular approach was established to detect Ph-like ALL cases from low- and middle-income countries.

Novel lncRNAs LINC01221, RP11-472G21.2 and CRNDE are markers of differential expression in pediatric patients with T cell acute lymphoblastic leukemia.

INTRODUCTION: Pediatric T-cell acute lymphoblastic leukemia (T-ALL) poses significant challenges due to its aggressive nature and resistance to standard treatments. Long non-coding RNAs (lncRNAs) have emerged as potential biomarkers and therapeutic targets in leukemia. This study aims to characterize the lncRNA landscape in pediatric T-ALL, identify specific lncRNAs signatures, and assess their clinical relevance. METHODS: RNA sequencing was performed on T-ALL patient and control samples. Differential expression analysis identified dysregulated lncRNAs and mRNAs. Functional enrichment analysis revealed potential roles of these lncRNAs in cancer pathogenesis. Validation of candidate lncRNAs was conducted using real-time PCR. Clinical correlations were assessed, including associations with patients' clinical characteristics and survival outcomes. RESULTS: Analysis identified 674 dysregulated lncRNAs in pediatric T-ALL, with LINC01221 and CRNDE showing the most interactions in cancer progression pathways. Functional enrichment indicated involvement in apoptosis, survival, proliferation, and metastasis. Top 10 lncRNAs based on adjusted p value < 0.05 and Fold Change > 2 were selected for validation. Seven lncRNAs LINC01221, PCAT18, LINC00977, RP11-620J15.3, RP11-472G21.2, CTD-2291D10.4, and CRNDE showed correlation with RNA sequencing data. RP11-472G21.2 and CTD-2291D10.4 were highly expressed in T-ALL patients, with RP11-620J15.3 correlating significantly with better overall survival (p = 0.0007) at a median follow up of 32 months. The identified lncRNAs were further analysed in B-ALL patients. Distinct lncRNAs signatures were noted, distinguishing T-ALL from B-ALL and healthy controls, with lineage-specific overexpression of LINC01221 (p < 0.0001), RP11-472G21.2 (p < 0.001) and CRNDE (p = 0.04) in T-ALL. CONCLUSION: This study provides insights into the lncRNA landscape of pediatric T-ALL, offering potential diagnostic and prognostic markers. RP11-620J15.3 emerges as a promising prognostic marker, and distinct lncRNAs signatures may aid in the differentiation of T-ALL subtypes. Further research with larger cohorts is warranted to validate these findings and advance personalized treatment strategies for pediatric T-ALL patients.

Integrated analysis of transcriptome and genome variations in pediatric T cell acute lymphoblastic leukemia: data from north Indian tertiary care center.

INTRODUCTION: T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease with poor prognosis and inferior outcome. Although multiple studies have been perform on genomics of T-ALL, data from Indian sub-continent is scarce. METHODS: In the current study we aimed to identify the genetic variability of T-ALL in an Indian cohort of pediatric (age ≤ 12 years) T-ALL patients (n = 25) by whole transcriptome sequencing along with whole exome sequencing and correlated the findings with clinical characteristics and disease outcome. RESULTS: The median age was 7 years (range 3 -12 years). RNA sequencing revealed a definitive fusion event in 14 cases (56%) (including a novel fusions) with STIL::TAL1 in 4 (16%), followed by NUP21::ABL1, TCF7::SPI1, ETV6::HDAC8, LMO1::RIC3, DIAPH1::JAK2, SETD2::CCDC12 and RCBTB2::LPAR6 in 1 (4%) case each. Significant aberrant expression was noted in RAG1 (64%), RAG2 (80%), MYCN (52%), NKX3-1 (52%), NKX3-2 (32%), TLX3 (28%), LMO1 (20%) and MYB (16%) genes. WES data showed frequent mutations in NOTCH1 (35%) followed by WT1 (23%), FBXW7 (12%), KRAS (12%), PHF6 (12%) and JAK3 (12%). Nearly 88.2% of cases showed a deletion of CDKN2A/CDKN2B/MTAP genes. Clinically significant association of a better EFS and OS (p=0.01) was noted with RAG2 over-expression at a median follow up of 22 months, while a poor EFS (p=0.041) and high relapse rate (p=0.045) was observed with MYB over-expression. CONCLUSION: Overall, the present study demonstrates the frequencies of transcriptomic and genetic alterations from Indian cohort of pediatric T-ALL and is a salient addition to current genomics data sets available in T-ALL.

X-Linked Lymphoproliferative Syndrome: A Spectrum of Clinical and Immunological Profile and Novel Pathogenic Variants from Chandigarh, India.

INTRODUCTION: X-linked lymphoproliferative syndrome (XLP) is a rare primary immune deficiency. Two types of XLP have been described: XLP-1 and XLP-2. METHODS: We found 7 patients with XLP (3 had XLP-1 and 4 had XLP-2) after reviewing the data from Pediatric Immunodeficiency Clinic from 1997 to 2021. RESULTS: Mean age at diagnosis was 3.8 years, and mean delay in diagnosis was 2.6 years. Five patients had recurrent episodes of infections. Four patients developed at least one episode of hemophagocytic lymphohistiocytosis (HLH) (2 with XLP-1 and 2 with XLP-2). Of these, 2 had recurrent HLH (both with XLP-2). Epstein-Barr virus (EBV) infection was detected in 2 (1 with XLP-1 and 1 with XLP-2). Both these patients had HLH. One child with XLP-2 had inflammatory bowel disease. Hypogammaglobulinemia was seen in 3 (2 with XLP-1 and 1 with XLP-2). Genetic analysis showed previously reported variants in 5, while 2 had novel variants (one in exon 7 of XIAP gene [c.1370dup p.Asn457Lysfs Ter16] and other had splice site variant in intron 1 of SH2D1A gene [c.138-2_138-1insG]). Episodes of HLH were managed with intravenous immunoglobulin (IVIg), methylprednisolone, oral prednisolone, cyclosporine, and rituximab. Inflammatory bowel disease was managed using oral prednisolone and azathioprine. One patient underwent haploidentical hematopoietic stem cell transplantation. One child with XLP-2 and WAS died because of fulminant pneumonia. DISCUSSION/CONCLUSIONS: XLP should be considered as a strong possibility in any patient with features of HLH, repeated infections with hypogammaglobulinemia, persistent EBV infection, and early-onset IBD.

Idiopathic hypereosinophilic syndrome presenting with stroke.

Hypereosinophilic syndrome (HES) is characterized by eosinophilia associated with organ damage. The disorder has substantial clinical heterogeneity and a highly variable prognosis. This report describes an interesting autopsy case of a 62-year-old lady presenting with itching and stroke-like symptoms. She was diagnosed with an "idiopathic" variant of HES after a thorough exclusion of all known causes. Despite adequate measures, she deteriorated rapidly. At autopsy, acute cerebral infarcts were identified in multiple vascular territories including infarcts in watershed areas. Additionally, her heart showed classic pathological features of eosinophilic myocarditis spanning all three stages.

Genomic and proteomic characterization of Philadelphia-like B-lineage acute lymphoblastic leukemia: A report of Indian patients.

BACKGROUND: The gold standard for the identification of Philadelphia (Ph)-like acute lymphoblastic leukemia (ALL) patients is gene expression profiling. Because of its diverse nature, its identification is extremely difficult and expensive. On the genomic and proteomic landscape of Ph-like ALL patients, there is a paucity of published literature from developing countries. METHODS: The authors used digital barcoded nCounter NanoString gene expression profiling for its detection, followed by molecular and proteomic characterization using fluorescence in situ hybridization and liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: The authors found 32.05% Ph-like ALL patients and their median age at presentation was considerably higher than Ph-negative ALL cases (p = .0306). Furthermore, we identified 20% CRLF2 overexpressed cases having 8.33% CRLF2-IGH translocation with concomitant R683S mutation and 8.33% CRLF2-P2RY8 translocation. In 80% of CRLF2 downregulated cases, we identified 10% as having JAK2 rearrangement. Minimal residual disease-positivity was more common in Ph-like ALL cases (55.55% vs. 25% in Ph-negative ALL cases). Immunoglobulin J chain (Jchain), small nuclear ribonucleoprotein SmD1 (SNRPD1), immunoglobulin κ constant (IGKC), NADH dehydrogenase (ubiquinone) 1 α subcomplex subunit 2 (NDUFA2), histone H2AX (H2AFX), charged multivesicular body protein 4b (CHMP4B), and carbonyl reductase (NADPH) (CBR1) proteins were identified to be substantially expressed in Ph-like ALL patients, using LC-MS/MS. Gene enrichment analysis indicated that involvement of spliceosomal mediated messenger RNA splicing pathway and four microRNAs was statistically significant in Ph-like ALL patients. CONCLUSIONS: For the first time, we have described incidence, molecular, and proteomic characterization of Ph-like ALL, in developing nations. PLAIN LANGUAGE SUMMARY: In developing countries, detecting Philadelphia (Ph)-like B-lineage acute lymphoblastic leukemia is complicated and challenging due to its diverse genetic landscape. There is no well-defined and cost-effective methodology for its detection. The incidence of this high-risk subtype is very high in adult cases, and there is an urgent need for its accurate detection. We have developed an online PHi-RACE classifier for its rapid detection, followed by delineating the genomic and proteomic landscape of Ph-like acute lymphoblastic leukemias for the first time in Indian patients.

'Evaluation of adverse prognostic gene alterations & MRD positivity in BCR::ABL1-like B-lineage acute lymphoblastic leukaemia patients, in a resource-constrained setting.

BACKGROUND: Early detection of BCR::ABL1-like ALL could impact treatment management and improve the overall survival/outcome. BCR::ABL1-like ALL cases are characterised by diverse genetic alterations activating cytokine receptors and kinase signalling. Its detection is still an unmet need in low-middle-income countries due to the unavailability of a patented TLDA assay. METHODS: This study's rationale is to identify BCR::ABL1-like ALLs using the PHi-RACE classifier, followed by the characterisation of underlying adverse genetic alterations in recurrent gene abnormalities negative (RGAneg) B-ALLs (n = 108). RESULTS: We identified 34.25% (37/108) BCR::ABL1-like ALLs using PHi-RACE classifier, characterised by TSLPR/CRLF2 expression (11.58%), IKZF1 (Δ4-7) deletion (18.9%) and chimeric gene fusions (34.61%). In overexpressed TSLPR/CRLF2 BCR::ABL1-like ALLs, we identified 33.33% (1/3) CRLF2::IGH and 33.33% (1/3) EPOR::IGH rearrangements with concomitant JAK2 mutation R683S (50%). We identified 18.91% CD13 (P = 0.02) and 27.02% CD33 (P = 0.05) aberrant myeloid markers positivity, which was significantly higher in BCR::ABL1-like ALLs compared to non-BCR::ABL1-like ALLs. MRD positivity was considerably higher (40% in BCR::ABL1-like vs. 19.29% in non-BCR::ABL1-like ALLs). CONCLUSIONS: With this practical approach, we reported a high incidence of BCR::ABL1-like ALLs, and a lower frequency of CRLF2 alteration & associated CGFs. Recognising this entity, early at diagnosis is crucial to optimise personalised treatment strategies.

Characteristics and outcome of infectious complications after autologous hematopoietic cell transplantation in multiple myeloma patients.

BACKGROUND: Infections are a significant cause of morbidity and mortality after autologous hematopoietic cell transplantation (AHCT) in multiple myeloma (MM) patients. There has been a rapid advancement and evolution in MM treatment landscape in the last decade. There is limited information on post-AHCT infectious complications among MM patients with or without levofloxacin prophylaxis from developing countries. MATERIALS AND METHODS: We performed a retrospective study to explore the incidence, pattern, and clinical outcome of infections following AHCT in MM patients from 2010 to 2019 at our center. Patient-specific, disease-specific, and transplant-specific details were retrieved from the case files. The characteristics of infectious complications (site, intensity, organism, treatment, and outcomes) were analyzed. All patients who underwent transplantation from 2010 to 2016 received levofloxacin antibiotic prophylaxis. Common terminology criteria for adverse events (CTCAE) criteria (v5.0) were used for the grading of infections and regimen-related toxicity. International Myeloma Working Group updated criteria were used for the assessment of disease response before transplant and at day +100. RESULTS: Ninety-five consecutive patients with newly diagnosed multiple myeloma (NDMM) (n = 85), RRMM (n = 7), plasma cell leukemia (n = 2), and Polyneuropathy, Orgaomegaly, Endocrinopathy, Monoclonal gammopathy, skin abnormalities (POEMS) syndrome (n = 1) underwent AHCT during the study period. Their median age was 55 years (range 33-68); 55.8% were males. Immunoglobulin IgG kappa was the most common monoclonal protein (32.6%), International Staging System stage III disease was present in 45.3%, and 84.2% of patients achieved more than very good partial response before AHCT. The median time from diagnosis to AHCT was 10 months (range 4-144). Eighty-nine patients (93.7%) developed fever after AHCT. Fever of unknown focus, microbiologically confirmed infections, and clinically suspected infections were found in 50.5%, 37.9%, and 5.3% of patients, respectively. Clostridiodes difficile-associated diarrhea was observed in eight patients (8.4%). Neutrophil and platelet engraftment occurred after a median of 11 days (range 9-14) and 12 days (range 9-23), respectively. The median duration of hospital stay was 16 days (range 9-29). Only two patients (2.1%) required readmission for infections within 100 days of AHCT. Transplant-related mortality (TRM) in the study population was 4.2% (n = 4). The levofloxacin prophylaxis group (n = 32, 33.7%) had earlier neutrophil engraftment (day +10 vs. day +11) and platelet engraftment (day +11 vs. day +12), but time to fever onset, duration of fever, hospital stay, TRM, and day +100 readmission rates were not significantly different from those of patients without levofloxacin prophylaxis. There was no significant difference in the spectrum of infections between patients with and without levofloxacin prophylaxis. The overall survival and progression-free survival of the study population at 5 years were 72.7% and 64.8%, respectively. CONCLUSION: This study shows that the incidence of infections and TRM are higher in MM patients from lower-middle income countries after AHCT than in those from developed countries. The majority of such patients lack clinical localization and microbiological proof of infection. There was no significant difference in the spectrum of infections and their outcomes in patients with and without levofloxacin prophylaxis.

Hematological characteristics, cytogenetic features, and post-induction measurable residual disease in thymic stromal lymphopoietin receptor (TSLPR) overexpressed B-cell acute lymphoblastic leukemia in an Indian cohort.

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.

An Evaluation of a Fluorescence In Situ Hybridization Strategy Using Air-dried Blood and Bone-marrow Smears in the Risk Stratification of Pediatric B-Lineage Acute Lymphoblastic Leukemia in Resource-limited Settings.

Cytogenetic abnormalities (CAs), one of the strongest influencers of therapeutic outcome in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), can be identified by different techniques. Despite several technological advances, many centers with resource-limited settings continue to use either reverse-transcriptase polymerase chain reaction (RT-PCR) and/or fluorescence in situ hybridization (FISH) to identify prognostically relevant CAs. We evaluated a simple and cost-effective triple-probe FISH strategy on air-dried blood and bone-marrow smears and compared its performance with a multiplex RT-PCR-based approach in the prognostication of pediatric BCP-ALL patients. Three hundred twenty BCP-ALL patients were tested prospectively and in parallel by FISH on air-dried blood or bone-marrow smears and RT-PCR. The FISH strategy correctly diagnosed all genetic abnormalities identified by RT-PCR. Prognostically relevant genetic abnormalities were missed by RT-PCR in 24 (8.1%) patients. In another 20 children (6%), with samples inadequate for RT-PCR testing (dry taps or due to poor sample quality), a successful FISH testing could be performed on bone-marrow aspirate or trephine-imprint smears. In addition, FISH detected ploidy changes, which could be confirmed by FxCycle Violet-based flow-cytometry. FISH testing on air-dried smears identified more prognostically relevant CAs, provided information on the ploidy status, and could be successfully performed in children with difficulty in bone-marrow sampling.

Genetic basis of unexplained erythrocytosis in Indian patients.

OBJECTIVE: To evaluate the spectrum of genetic defects in Indian patients with unexplained erythrocytosis. METHODS: Fifteen families (18 patients) with unexplained erythrocytosis were enrolled after excluding polycythemia vera and secondary erythrocytosis. Focused Sanger sequencing from genomic DNA was performed for EPOR (exon 8), VHL (exons 2-3), EGLN1 (exons 2-5), EPAS1 (exon 12), and all exons of HBB, HBA1, and HBA2 genes. RESULTS: Eleven of the 18 patients (including two pairs of brothers) had Chuvash polycythemia, that is, homozygosity for VHL:c.598C > T (p.Arg200Trp). Three patients (two of whom were brothers) had HBB mutations associated with increased oxygen-affinity hemoglobin-one had a heterozygous Hb McKees Rocks HBB:c.438T > A (p.Tyr146*), and two brothers showed heterozygous Hb Rainier HBB:c.437A > G (p.Tyr146Cys). No pathogenic variants were found in the remaining four cases. CONCLUSION: A gene-by-gene Sanger sequencing approach could determine a genetic basis for erythrocytosis in 11 of the 15 (73%) Indian families, with homozygous VHL:c.598C > T (p.Arg200Trp) being the commonest pathogenic variant. This first study from the Indian subcontinent provides a rationale for analyzing this variant in patients with suspected congenital erythrocytosis from this region. Rare first occurrences of Hb McKees Rocks and Hb Rainier in Indians are also being reported.

Childhood Blastic Plasmacytoid Dendritic Cell Neoplasm Mimicking Acute Rheumatic Fever: Report of an Unusual Clinical Presentation and Review of Literature.

BACKGROUND: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is very rarely diagnosed in children with less than 50 cases in the literature. OBSERVATION: We report a case of childhood BPDCN who mimicked acute rheumatic fever at presentation. Majority of the reported childhood BPDCN received acute lymphoblastic leukemia-like chemotherapy with/without stem cell therapy, whereas those who received acute myeloid leukemia-like therapy predominantly succumbed to disease or sepsis. Overall 68% of the patients were alive and achieved complete remission with an overall prognosis slightly better in children compared with adults. CONCLUSIONS: The case is reported due to its unique unusual clinical presentation and its rarity in pediatric population.

A Young Child with Eosinophilia, Rash, and Multisystem Illness: Drug Rash, Eosinophilia, and Systemic Symptoms Syndrome After Receipt of Fluoxetine.

Drug rash, eosinophilia, and systemic symptoms (DRESS) syndrome is a severe systemic hypersensitivity reaction that usually occurs within 6 weeks of exposure to the offending drug. Diagnosis is usually straightforward in patients with pyrexia, skin rash, hepatitis, and eosinophilia with a preceding history of exposure to agents often associated with DRESS syndrome, such as aromatic anticonvulsants and sulfa drugs, but diagnosis of DRESS may still be a challenge. We report a 4-year-old child with probable DRESS syndrome complicated by multiple hematologic complications that developed 1 month after exposure to fluoxetine, a drug not known to be associated with such severe reactions.

Frequency of Paroxysmal Nocturnal Hemoglobinuria Clones by Multiparametric Flow Cytometry in Pediatric Aplastic Anemia Patients of Indian Ethnic Origin.

BACKGROUND: The literature on paroxysmal nocturnal hemoglobinuria (PNH) in aplastic anemia (AA) is largely focused on adults with few studies in children. Moreover, large studies are conspicuously absent from developing countries. Knowledge of the prevalence and utility of their detection is required before widespread use of PNH screening in pediatric AA in resource-limited settings. METHODS: We performed a retrospective audit over a period of 9 years to study the prevalence of PNH clones by flow cytometry (FCM) in children ≤12 years of age presenting with AA, and analyzed their response to immunosuppressant therapy. RESULTS: Nine (12.9%) out of 70 patients had PNH clones comprising >1% of the target cell population, including five patients (7.14%) with PNH clone size >10%. The clone size in monocytes ranged from 3.7% to 95.2% (median 21.1%) and in neutrophils from 1.6% to 87.6% (median 19.5%). Fluorescent aerolysin (FLAER)-based FCM screening significantly improved the detection of PNH clones compared to non-FLAER based screening techniques (18.4% vs. 6.25%). One child showed chronic intravascular hemolysis and another developed arterial stroke during the course of illness. None of our PNH-positive AA patients tested for chromosome breakage studies (n = 8) showed increased clastrogen-induced breakage. CONCLUSIONS: A lower frequency but moderate/large-sized PNH clones were seen in our pediatric AA population, compared to western data. FLAER-based FCM screening significantly improved the detection of PNH clones. We recommend routine FLAER-based screening of PNH in pediatric AA patients.

ß-Thalassemia Intermedia Caused by Compound Heterozygosity for Hb Lepore-Hollandia and ß-Thalassemia is Rare in the Indian Population.

Compound heterozygosity for one of the Hb Lepore mutations and ß-thalassemia (ß-thal) is a rare cause of non transfusion-dependent thalassemia. We report a 4-year-old boy who presented clinically as homozygous/compound heterozygous ß-thal intermedia (ß-TI), an impression that was corroborated by the initial hemoglobin (Hb) high performance liquid chromatography (HPLC). However, the correct diagnosis of a rare compound heterozygous Hb Lepore-Hollandia/ß-thal was revealed after parental studies and molecular analyses including ß-globin gene sequencing. Our patient highlights the importance of a logical stepwise multi modality approach and the vital importance of parental screening and molecular studies in accurate characterization of complex hemoglobinopathies. Correct diagnosis is especially crucial if pre natal detection is anticipated for future pregnancies. Molecular analyses alone may not compensate for the unavailability of parental testing. This is because the molecular results may be misinterpreted, especially if limited tests are conducted. The infrequent prior reports of this combination from distant parts of the Indian subcontinent suggests that the origin of Hb Lepore-Hollandia from sporadic mutations occurs in isolated families.

An analysis of transplant glomerulopathy and thrombotic microangiopathy in kidney transplant biopsies.

Glomerular diseases of the transplanted kidney are the most important cause of poor long- term outcome. The estimation of the magnitude of this problem and an elucidation of pathogenic mechanism is essential for improvement of graft survival. This study from the Indian subcontinent aims (i) to determine the incidence of transplant glomerulopathy (TG) and thrombotic microangiopathy (TMA) in a large cohort of indicated renal transplant biopsies, (ii) to evaluate the histological and ultrastructural features of TG and TMA, and (iii) to assess the relationship between the two glomerular lesions. Of a total of 1792 indication renal transplant biopsies received over 5 years (2006-2010), 266 biopsies (of 249 patients) had significant glomerular pathology and were further analyzed along with immunofluorescence, electron microscopy (EM), and C4d immunohistochemistry. TG is the most common glomerular lesion followed by TMA seen in 5.97% and 5.08% of allograft biopsies, respectively, which constitutes 40.23% and 34.2% of biopsies with significant glomerular lesions. Pathologic antibody-mediated rejection (AMR) is associated with both TG and TMA in 71% and 46.5%, respectively. A coexistent TG was found in 18.4% of biopsies with TMA. Endothelial swelling with subendothelial widening, a feature of TMA, is also seen in early TG by EM. Our findings support the concept that TG evolves from a smoldering TMA of various causes.

Primitive neuroectodermal tumor of the uterine cervix diagnosed during pregnancy: a rare case with review of literature.

Primitive neuroectodermal tumors of the cervix are very rare. A 28-year-old pregnant woman presented with a cervical mass. The tumor was staged as IB2. The biopsy from tumor was suggestive of malignant small round cell tumor. She then underwent termination of pregnancy followed by radical hysterectomy. Based on morphologic and immunohistochemical profile, a diagnosis of peripheral primitive neuroectodermal tumor of the cervix was made. The patient received adjuvant chemotherapy and radiotherapy. The patient is alive and disease-free 33 months post-surgery. The present case highlights the importance of keeping primitive neuroectodermal tumors in the differential diagnosis of small cell neoplasms of the uterine cervix. Pregnancy should not be a barrier to early detection and treatment of this potentially aggressive tumor. The optimal treatment methods have not yet been established because of the rarity of the tumor.

Detection of circulating normal and tumor plasma cells in newly diagnosed patients of multiple myeloma and their associations with clinical and laboratory parameters.

INTRODUCTION: Circulating plasma cells (CPCs) are frequently noted in variable frequencies in the entire spectrum of plasma cells neoplasms. With advent of high sensitivity multi-parametric flow cytometry, it is not only possible to detect CPCs present in very low numbers, but also to categorise them into circulating tumor plasma cells (CTPCs) and circulating normal plasma cells (CNPCs), based on their marker-profile. This study used multi-colour flow cytometry to evaluate the load of both CTPCs & CNPCs at the time of diagnosis and at six months' time-point of therapy, and evaluated associations of both with clinical and laboratory parameters. METHODS: Twenty one newly diagnosed MM patients were enrolled. Six to nine millilitres of EDTA-anticoagulated peripheral blood sample was used for flow cytometry. A ten colour antibody panel was used for analysis of CPCs, which were categorised further into CTPCs and CNPCs. Approximately 4.8 million events were acquired for the analysis. The percentage &absolute numbers of CTPCs and CNPCs were noted and the proportion of CTPCs out of all CPCs (CTPCs + CNPCs) were also calculated for evaluating their statistical associations. RESULTS: All 21 patients of newly diagnosed MM showed presence of CPCs (CTPCs and/or CNPCs) at the time of diagnosis. The CTPCs were detected in 76 % of the study population. The median percentage and absolute counts of CTPCs were 0.52 % and 54.9 cells /µL, respectively. CNPCs were found in 95 % and the median percentage and absolute counts of CNPCs were 0.025 % and 2.66 cells/µL. After six months of therapy, CPCs (CTPCs and/or CNPCs) were found in all nine patients evaluated for this assay. CTPCs were found 33 %, with a median of 0.075 % and CNPCs were found in 89 % with a median of 0.01 %. Our study showed that the load of CTPCs was found to be higher in patients with presence of lytic bone lesions, plasmacytoma, presence of PCs on peripheral blood film by light microscopy, presence of Chr 1p32 deletion, expression of CD56 and CD81 on CTPCs, and in patients with absence of very good partial response (VGPR). Conversely, the load of CTPCs was significantly lower in patients with concomitant amyloidosis. Also, percentage of bone marrow plasma cells exhibited a significant positive correlation with the absolute count of CTPCs. We observed that the mean percentage of CNPCs was significantly higher in female patients. The load of CNPCs was lower in patients with thrombocytopenia and with hypoalbuminemia. CONCLUSION: Increased burden of CTPCs was associated with presence of lytic lesions, plasmacytomas, Chr 1p32 deletion, expression of CD56 and CD81 on tumor cells and with failure to achieve very good partial response. The CNPCs were lower in patients with thrombocytopenia and with hypoalbuminemia. To best ot our knowledge, this is the first study from India on the relevance of circulating tumor plasma cells and the first study in the world to analyse the associations of circulating normal plasma cells in newly diagnosed patients of multiple myeloma. The study also highlights the utility of multi-parametric flow cytometry in identification and enumeration of circulating plasma cells. MICRO ABSTRACT: Circulating plasma cells indicates poorer outcomes in patients of multiple myeloma. Twenty one newly diagnosed multiple myeloma patients were evaluated by flow cytometry to enumerate and characterise circulating tumor plasma cells (CTPCs) and circulating normal plasma cells (CNPCs). Higher load of CTPCs correlated with known poor prognostic markers and poor response to therapy.

The frequency and clinical outcome of mono-hit and multi-hit TP53 aberrations in newly diagnosed multiple myeloma.

We investigated the frequency and outcome of mono-hit and multi-hit TP53 aberrations [biallelic or ≥1 TP53 mutations (TP53mut) or TP53mut with variant allele frequency (VAF) ≥55%] in an Indian cohort of newly diagnosed multiple myeloma (NDMM) patients. We employed fluorescence insitu hybridisation (FISH; n=457) and targeted next-generation sequencing (NGS; n=244) on plasma cell-enriched samples. We also studied the impact of TP53mut in cases with and without TP53 deletions (TP53del). In our cohort with a median age of 60 years, TP53del and TP53mut were seen in 12.9% (n=59/457; 14-95% cells) and 10.2% (n=25/244; 30 variants; VAF 3.4-98.2%; median 38.2%) respectively. Mono-hit and multi-hit-TP53 aberrations were observed in 10.2% and 7.8%, respectively. Compared to TP53-wild-type (TP53wt), mono-hit and multi-hit TP53 aberrations were associated with significantly poorer progression-free survival (PFS) (22.6 vs 12.1 vs 9.5 months; p=0.004) and overall survival (OS) [not reached (NR) vs 13.1 vs 15.6 months respectively; p=0.024]. However, multi-hit TP53 did not significantly differ in OS/PFS compared to mono-hit cases. Compared to TP53wt, PFS and OS were significantly poorer in patients with TP53mut only (9.5 vs 22.6 months and 12.1 months vs NR, respectively; p=0.020/0.004). TP53mut retained its significance even in the presence of any Revised International Staging System (HR 2.1; 95% CI 1.1-3.8; p=0.015) for OS. The detection of additional cases with TP53 aberrations, as well as poor survival associated with the presence of mutation alone, supports TP53mut testing in NDMM at least in patients without TP53del and other high-risk cytogenetic abnormalities.