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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. A surrogate approach for the rapid baseline detection of Philadelphia chromosome (Ph)–like acute lymphoblastic leukemia cases, followed by its kinase gene alterations. Early detection of Ph–like acute lymphoblastic leukemia cases is extremely important to start personalized treatments to improve the overall survival/outcomes of these cases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: The published literature on hematological, clinical, flowcytometric‐immunophenotyping, and minimal residual disease outcomes of the prognostically important genetic subtypes of acute lymphoblastic leukemia (ALL) is scarce from low‐income countries. For newer classifications such as BCR::ABL1‐like ALLs, the scarcity of patient‐level data is even more pronounced. Methods: The authors performed comprehensive detection of recurrent gene fusions and BCR::ABL1‐like ALL cases followed by immunophenotypic profiling and obtained clinical outcome parameters for a large cohort (n = 1021) of patients from India. This cohort included a significant number of patients with BCR::ABL1‐like ALL subtype and other genetic subtypes of ALL. Results: Patients with BCR::ABL1‐positive and BCR::ABL1‐like ALL were significantly older, had male preponderance, and expressed a higher white blood cell count than BCR::ABL1‐negative cases (p <.05). Logistic regression modeling of B‐lineage‐ALL (B‐ALL) subtypes revealed that cluster of differentiation (CD)36 is a strong statistically significant predictive marker of BCR::ABL1‐like ALL (p <.05). Furthermore, patients with BCR::ABL1‐like ALLs show a significantly higher frequency of CD36 expression compared to BCR::ABL1‐negative ALLs (p <.05). In terms of clinical symptoms, lymphadenopathy is a strong statistically significant predictive marker in BCR::ABL1‐like ALLs compared to BCR::ABL1‐negative ALL cases (p <.05). In terms of treatment outcomes, minimal residual disease (MRD) positivity in BCR::ABL1‐positive ALL cases were statistically significant (p <.05), and BCR::ABL1‐like ALL cases had high MRD‐positivity as compared to BCR::ABL1‐negative ALL cases but did not show statistical significance. Conclusions: The findings evince the use of novel therapies and personalized treatment regimens to improve the overall survival of the newer incorporated entities in B‐ALLs. This is the first report characterizing the hematological, clinical, flowcytometric‐immunophenotyping, and minimal residual disease outcomes of the prognostically significant subtypes of ALLs in patients from India. Plain Language Summary: Characterizing the hematological, clinical, flowcytometric‐immunophenotyping, and minimal residual disease outcomes of the prognostically significant subtypes (n = 1021) of acute lymphoblastic leukemia (ALLs) in patients from India.We have made two independent logistic regression models of cluster of differentiation (CD) markers and clinical symptoms to differentiate prognostically significant subtypes of ALLs.Logistic regression analysis of CD markers revealed CD36 as a strong predictor in BCR::ABL1‐like ALL cases compared to BCR::ABL1‐negative ALL cases.Logistic regression analysis of clinical symptoms revealed lymphadenopathy significantly predicts BCR::ABL1‐like ALLs (p <.05).In terms of treatment outcomes, BCR::ABL1‐positive ALL had statistically significant minimal residual disease (MRD) (p <.05), and BCR::ABL1‐like ALL cases had high MRD‐positivity but did not show statistical significance as compared to BCR::ABL1‐negative ALLs. This study characterizes prognostically significant genetic subtypes of B‐acute lymphoblastic leukemia (B‐ALL). Logistic regression modeling of cluster of differentiation markers and clinical symptoms is used to predict various genetic subtypes of B‐ALL. [ABSTRACT FROM AUTHOR]

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. The identification of Philadelphia chromosome (Ph)–like acute lymphoblastic leukemia (ALL) cases with NanoString nCounter digital‐barcoded gene expression profiling was examined in Indian patients. A higher incidence of Ph‐like ALL cases (32.05%) was identified. Furthermore, genomic and proteomic characterization of Ph‐like ALL cases was performed in Indian patients. This is the first report on the incidence and characterization of Ph‐like ALL cases from developing nations. [ABSTRACT FROM AUTHOR]

For the detection of BCR-ABL1-like ALL cases, two methodologies, specifically Gene expression profiling (GEP) or Next-generation targeted sequencing (NGS) and TaqMan based low-density (TLDA) card, are being used. NGS is very costly and TLDA is not widely commercially available. In this study, we quantified the expression of 8 selected overexpressed genes in 536 B-ALL cases. We identified 26.67% (143/536) BCR-ABL1-like ALLs using hierarchical clustering and principal component analysis. BCR-ABL1-like ALL cases were significantly older at presentation (p = 0.036) and had male preponderance (p = 0.047) compared to BCR-ABL1-negative ALL cases. MRD-positivity and induction failure were more commonest in BCR-ABL1-like ALL cases (30.55 vs.19.35% in BCR-ABL1-negative ALL cases). Lastly, we built a PHi-RACE classifier (sensitivity = 95.2%, specificity= 83.7%, AUC= 0.927) using logistic regression to detect BCR-ABL1-like ALL cases promptly at diagnosis. This classifier is beneficial for hematologists in quick decision making at baseline to start tailored treatment regimes. [ABSTRACT FROM AUTHOR]

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Background: Aberrant phenotypes in acute leukemia have been reported with varying frequencies in independent studies and their association with prognostic factors is still a matter of debate.Aim: This study aims to identify the frequency of aberrant immunophenotypes in de novo acute myeloid leukemia (AML) and to evaluate their association with initial clinical and hematological features.Materials and Methods: A total of 181 patients of de novo AML were included during the time (July 2010-June 2012). The immunophenotype of all cases of AML was studied by using flow cytometry.Results: Aberrant lymphoid antigen expression was seen in 43.1% cases. Most frequent aberrant lymphoid antigen was CD7, seen in 26.5% cases. All French-American-British (FAB) subtypes except AML-M3 expressed aberrant lymphoid antigens. The expression was most common in AML-M4 in the current study. CD34 expression in AMLs was significantly associated with the expression of aberrant lymphoid antigens. Lymphoid antigen expression in adult AML was significantly associated with higher white blood cell (WBC) count (>50,000/mm3) and higher number of peripheral blasts (>70%).Conclusion: In summary, CD7 is the most common aberrant lymphoid antigen expressed in AML. FAB subtype AML-M3 is usually not associated with aberrant lymphoid antigen expression. AML cases with CD34 positivity are more likely to express aberrant lymphoid markers. The current study also supports that aberrant lymphoid antigen expression in adult AML is associated with adverse presenting hematological features (WBC count >50,000/mm3, peripheral blasts >70%). Pediatric Ly + AML cases are not associated with adverse presenting clinical and biological features. [ABSTRACT FROM AUTHOR]

Background: Identification of aberrant antigen expression is important in characterizing neoplastic population among non.neoplastic bone marrow counterparts and further in the detection of minimal residual disease. (MRD). Flow cytometry (FCM) is an important tool in identifying aberrant phenotypes. Incidence of aberrant phenotypes varies considerably in independent studies and its association with prognostic factors is still debatable.Aim: To identify the prevalence of aberrant phenotypes on immunophenotyping in a large series of de novo acute lymphoblastic leukemia (ALL) and to evaluate any association with initial clinical and hematological features.Materials and Methods: In the current study, 303 patients of de novo ALL were included from the Department of Hematology, PGIMER, Chandigarh during the time period (July 2010 to June 2012). The immunophenotype of all cases of ALL was studied using FCM.Results: Aberrant myeloid antigen expression was seen in 42.5% cases. Most frequent aberrant myeloid antigen was CD13 (32.2% cases), followed by CD33 (27.2% cases) and CD117 (18.5% cases). The expression of CD117 was relatively frequent in comparison to earlier reports which describe its rare expression. Adult T- ALL showed higher expression of CD33 and CD117 than pediatric T-ALL (P = 0.032 and 0.043, respectively). Myeloid antigen expression in ALL was associated with lower WBC count (P < 0.05) and lower number of peripheral blasts (P < 0.05). Expression of CD34 was higher in My + ALL group (P < 0.05) than My- ALL group.Conclusion: In summary, CD117 is a relatively frequently expressed myeloid marker contrary to earlier reports which describes its rare expression. Pediatric and adult ALL cases with low blast count and CD34 positivity are more likely to express aberrant myeloid markers. Current study also supports that myeloid antigen expression in both adult and pediatric ALL is not associated with adverse presenting clinical and biological features. [ABSTRACT FROM AUTHOR]

Cure rates for adult acute lymphoblastic leukemia (ALL) in developing countries are significantly lower because of problems unique to these countries. We assessed some of the problems in adult ALL patients (>12 years of age) in a tertiary care hospital of northwest India with modified BFM regimen. The diagnosis of ALL was made according to FAB criteria. The protocol consisted of Phase I & II induction, consolidation, reinduction and maintenance phases. CNS prophylaxis was administered with 24 Gy radiation and intrathecal methotrexate. One hundred and eighteen patients (72.9% males), aged 12 - 68 years (median 23 years) were treated from January 1997 till December 2003. Follow-up of patients was done till December 2005. Complete remission (CR) was achieved in 85.6% patients after induction therapy and 40% patient relapsed. Most patients (23.7%) relapsed during the maintenance phase or after completion of chemotherapy. At least 15% of patients (15/101) after successful induction abandoned the treatment because of financial constraints, prolonged travel time to treatment facility and switching over to alternative medicines. Fatal infectious complications occurred in 19.5% of patients. The 3-year and 5-year event free survival rates were 29.8% and 21.6% respectively. In conclusion, modified BFM regimen resulted in high induction rates but relatively poor 5-year event free survival. Infections related death and post induction abandonment of treatment were the main reasons for poor overall results. [ABSTRACT FROM AUTHOR]

Testicular involvement in a case of acute lymphoblastic leukemia (ALL) is well reported, but occurrence of “isolated” malignant hydrocele is extremely uncommon. We herein report a case of a 22-year-old man who presented to our hematology clinic with fever and easy fatiguability of 2 weeks’ duration. Examination revealed pallor, cervical lymphadenopathy, and bilateral scrotal swellings. He was diagnosed as a case of Philadelphia-positive ALL (B-cell type) based on peripheral smear, bone marrow examination, and flow cytometry of the marrow aspirate. Ultrasonography of scrotum revealed bilateral hydrocele with normal testes. Cytopathological analysis of the hydrocele fluid showed the presence of lymphoblasts. The patient was treated with modified BFM-90 protocol along with imatinib mesylate (600 mg/day). He achieved complete remission with a minimal residual disease of ,0.001% at the end of induction therapy. However, the hydrocele persisted and a repeat cytological examination of the aspirate did not reveal any lymphoblasts. The patient was treated with consolidation (high-dose methotrexate), bilateral testicular irradiation, and re-induction following which the hydrocele disappeared. The patient is currently on maintenance phase of BFM-90 protocol and is alive at one year of follow-up. Contiguous spread from the subclinical testicular involvement is hypothesized as the mechanism for development of hydrocele in the current case. The role of cytopathology in the early diagnosis of testicular involvement in ALL is emphasized here. [ABSTRACT FROM AUTHOR]