Clinico-hematological Features Research Articles

Frequency of clinico-hematological features and JAK 2 status of young adults presenting with polycythemia in RMI hospital, Peshawar.

Objective: To determine the frequency of clinico-hematological features and JAK 2 status of young adults presenting with Polycythemia Vera at the department of hematology. Study Design: Cross-sectional study. Setting: Department of Hematology, RMI Hospital, Peshawar. Period: 25th November 2023 to 25th May 2024. Methods: A total of 190 patients aging between 18-45 years and presented as suffering from Polycythemia Vera as per diagnosis of consultant hematopathologists, were included in this study. Samples with clear evidence of hemolysis, coagulation, or clots were excluded. Patient’s demographics, details of physical appearance, clinical findings and history of adverse events were recorded. Hematological and pathological investigations were done and analysis of JAK2 V617F mutation was conducted using polymerase chain reaction from the diagnostic lab of the institute. The primary outcome of the study was to determine the frequency of clinico-hematological features and JAK 2 status. Results: The Mean±SD of age in our study was 36.81±6.11 years with an age range of 21-45 years. Out of total patients, 49 (25.79%) patients were asymptomatic. The major symptoms affecting the daily life activities were headache 65 (34.21), abdominal discomfort 41 (21.58%), blurred vision 27 (14.21%) and fatigue 27 (14.21%). These patients had plethoric face 61 (32.11%), splenomegaly 64 (33.68%) and budd-chiari syndrome 8 (4.21%). The results of hematopathological findings showed abnormal hemoglobin (17±3.92 gm/dL), hematocrit (52.94±4.8 g %), platelet (579.63±227.68 103/μL) and leukocyte (10.32±2.6, 103/μL) levels while 177 (93.16 %) patients had mutated JAK2 levels. Conclusion: Plethoric face and splenomegaly are the commonly found clinical findings in patients with Polycythemia Vera while these patients also have abnormal levels of hemoglobin, hematocrit, platelet and leukocyte. JAK 2 status serves conclusive role in the diagnosis of Polycythemia Vera.

The Frequency, Clinico-Hematological Features, and Outcome of Acute Promyelocytic Leukemia with Variant RARA Rearrangements: A Single Center Experience from India.

The Frequency, Clinico-Hematological Features, and Outcome of Acute Promyelocytic Leukemia with Variant RARA Rearrangements: A Single Center Experience from India.

Plasma Cell Leukemia-Clinicopathological Profile from a Tertiary Care Center in Western India.

Poornima ManimaranIntroduction Plasma cell leukemia (PCL) is very uncommon and aggressive neoplasm constituting 2 to 4% of all plasma cell dyscrasias. By definition, clonal plasma cells should make up 20% of peripheral blood or have an absolute plasma cell count of 2 × 10 9 cells/cu.mm. PCL can be primary or secondary. In this study, the clinicohematological features of PCL, and correlation of immunophenotypic profile and conventional therapies with overall survival was analyzed. Materials and Methods This retrospective study involved PCL patients who were diagnosed across a 12-year period, from 2010 to 2021, at a tertiary care center in western India. Clinical, biochemical, peripheral smear, bone marrow aspirate, immunophenotyping, and molecular analysis were performed. Results Total 39 PCL patients were included in the study among which 36 were primary PCL patients. Splenomegaly (10/27), hepatomegaly (6/26), and lymphadenopathy (5/23) were noted. At presentation, all patients had anemia (<11g/dL), thrombocytopenia (33/39), hypercalcemia (>11mg/dl) 10/33 (30.3%) and lytic lesions was noted in 18/26 (69.2%). Immunophenotype of these patients showed CD 38 positivity, CD 138 positivity, CD56 positivity, and CD 117 negativity were 100, 62, 41.6, and 89%, respectively. Overall survival of our patients was 4.1 months and overall survival of patients treated with VTD (bortezomib, thalidomide, dexamethasone) and VCD (bortezomib, cyclophosphamide, dexamethasone) regimen was 3.4 and 4.1 months, respectively, which was not statically significant ( p -value 0.816). CD117 and CD56 markers were also not having any prognostic significance ( p -value 1.000 and 0.873, respectively). Conclusion Because of rarity of the disease, prospective studies are very limited and hence management and outcome of the disease are difficult to analyze. The current treatment protocols have no survival advantage and hence newer therapeutic approach is mandatory to attain better outcome.

SEVERITY AND CLINICO-HAEMATOLOGICAL FEATURES OF APLASTIC ANEMIA IN ADULTS PRESENTING TO PUBLIC SECTOR TERTIARY CARE HOSPITAL OF LAHORE: A DESCRIPTIVE CROSS SECTIONAL STUDY

Background &amp; Objective: Aplastic anemia is a rare and heterogeneous disorder. Literature shows inconsis- tencies in frequency and its clinico-hematological findings. Objective of this study was to assess frequency of different severity grades and clinico-hematological features in newly diagnosed cases of aplastic anemia in adults.&#x0D; Methods: In this descriptive, cross-sectional study, conducted in Allama Iqbal Medical College Lahore from October 2021 through April 2022, a total of 100 diagnosed cases of acquired aplastic anemia were included. Modified Camitta's criteria were applied to assess the severity of aplastic anemia. Clinical features such as pallor, fever and bleeding manifestations were determined by history and physical examination. About 3ml whole blood was collected in EDTA vial and run for complete blood count on automated hematology analyzer for hematological Parameters (Hb, Platelets, total leukocyte count and absolute Neutrophil count). Data were entered and analyzed using SPSS version 20. Quantitative variables like age, Hb, TLC and platelet count were expressed as mean ±Standard deviation. Qualitative variables such as gender, severity of aplastic anemia and clinical features were expressed as percentages.&#x0D; Results: In this study, 55% participants were male and 45% were female. All patients had pallor, 61% had fever and 66% had bleeding on presentation. Regarding severity of aplastic anemia, 56% were categorized as severe, 24% as very severe and 20% were as non severe aplastic anemia.&#x0D; Conclusion: Severe aplastic anemia is frequent among male population and at younger age. This information has prognostic implications. Therefore, all patients with aplastic anemia should be assessed for severity for further clinical management.

Demographic features and hematological profile in patients of chronic lymphocytic leukemia in southern Punjab Pakistan.

Objective: To generate data regarding clinicohematological findings of CLL in patients of Southern Punjab Pakistan. Study Design: Descriptive Cross-sectional study. Setting: Hematology Section Pathology Department, Nishtar Medical University, Multan. Period: January 2016 to September 2019. Material &amp; Methods: A total of 44 patients of all age groups diagnosed as CLL were enrolled. Diagnosis was made on morphology and immunohistochemistry of bone marrow trephine biopsies and cases without immunohistochemical panel were excluded from the study. Clinical features and baseline tests were evaluated in all cases. The data was analyzed using SPSS Version 23.0. Results: Out of 44 patients, majority were males (70.5%). Mean age was 51±12.6 years. B symptoms were present in 71% patients. Lymph nodes were enlarged in 44%. Splenic and hepatic enlargement was seen in 32% and 15.9% respectively. Anemia was seen in 45.5% and thrombocytopenia in 34.1%. Bone marrow was infiltrated in 100% patients and the most common pattern of infiltration was diffuse (65%). CD20 and CD5 were positive in all of the cases (100%) and CD23 in 96%. Seropositivity for both anti HCV and HBsAg was present in 2.3%. Conclusion: The clinicohematological features of CLL in our patients show some differences to the cited literature with a greater majority presenting at younger age with diffuse bone marrow involvement having lower positivity for HBsAg, anti HCV and less hepatosplenomegaly.

Clinico-hematological features and outcome of patients affected by Congo–Crimean hemorrhagic fever: An experience from a single center

BACKGROUND: Congo–Crimean hemorrhagic fever is a tick-borne zoonotic viral disease caused by Crimean–Congo hemorrhagic fever virus (CCHFV). The outbreak in Pakistan is increased during Eid-ul-Adha. We describe a cluster of cases that presented to our hospital. AIMS: The aim of this study was to determine the outcome of Crimean–Congo hemorrhagic fever-positive cases from January 2011 to August 2019. MATERIALS AND METHODS: Retrospective data were retrieved using the International Classification of Diseases version 9. We received 2101 samples for testing. Polymerase chain reaction (PCR)-positive cases were included in the study. History of bleeding and animal contact was recorded. Variables analyzed were age, gender, profession, and coinfection with other viral illnesses. RESULTS: A total of 70 PCR-positive cases were included in the study (frequency 3.3%). Sixty-one were males and nine were females. Fever was present in all cases. Epistaxis was noted in 54% of patients. Thrombocytopenia was present in all patients. Eighteen patients were butchers, six were shopkeepers, nine students, and few laborers. All females were housewives. Twenty-two patients had a history of contact with animals. Seven patients were coinfected with viral hepatitis. There were 23 (33%) deaths. CONCLUSION: Overall mortality was 33%. Twenty-two patients had a history of contact with animals. There is a strong need for public education, especially during the month of Eid-ul-Adha.

A Retrospective Observational Study of Dicentric (9;12): A Unique, Nonrandom Translocation Defining a Cytogenetic Subgroup with Favorable Outcome in Acute Lymphoblastic Leukemia

Abstract Introduction Cytogenetic abnormalities are integral to the risk stratification of acute lymphoblastic leukemia (ALL). Objectives The present study aimed to highlight a rare, yet nonrandom cytogenetic abnormality notably dicentric (9;12), which was observed in ALL patients who presented to our institute. The study analyzed the frequency, clinicohematological features, and treatment response of these patients. Materials and Methods A single-group observational study was conducted from April 2014 to April 2020. Cytogenetic analysis was done on bone marrow aspirate samples of the patients referred to the cytogenetics laboratory with clinical diagnosis of acute leukemia. Cytogenetic, clinical, and hematological data were collected from respective departmental records, case files, and patients. Results Dic(9;12) was identified in 1.2% of ALL (19 out of 1,544 patients). They showed striking preponderance in teen and young adult males with characteristic precursor B cell immunophenotype. Majority of these patients displayed favorable risk profiles such as low total count, mild lymphadenopathy and splenomegaly, mild-to-moderate elevation of lactate dehydrogenase, and good response to first induction chemotherapy. Rare coexistence of dic(9;12) with well-established cytogenetic markers such as t(9;22) and t(1;19) was observed. Conclusion Dic(9;12) is one of the most specific cytogenetic markers of precursor B cell (pre-B) ALL. It defines a subgroup with favorable clinical and biological profile. We suggest inclusion of dic(9;12) in cytogenetic risk stratification of precursor B cell ALL. Long-term follow-up studies are recommended to establish the prognostic significance of this cytogenetic subgroup, which may benefit from less intensive chemotherapy.

Clinico-Hematological and cytogenetic spectrum of adult myelodysplastic syndrome: The first retrospective cross-sectional study in Iranian patients

BackgroundMyelodysplastic syndrome (MDS), a heterogeneous group of hematopoietic malignancy, has been shown to present different cytogenetic abnormalities, risk factors, and clinico-hematological features in different populations and geographic areas. Herein, we determined the cytogenetic spectrum and clinico-hematological features of Iranian MDS patients for the first time.MethodsThis prospective cross-sectional study was conducted on 103 patients with MDS in Ahvaz, southwest of Iran, from 2014 to 2018. Clinical presentations, complete blood counts (CBC), and bone marrow (BM) biopsy samples were assessed. Perls' staining was used to evaluate BM iron storage. The cytogenetic evaluation was performed using the conventional G banding method on the BM.ResultsPatients’ median age was 62.3 (ranged from 50–76), and the majority were male (72.8%). The most common clinical symptom at the time of admission was fatigue (n = 33) followed by pallor (n = 27). The most common subgroup was MDS-Multi Lineage Dysplasia (MDS-MLD) (n = 38, 36.8%), followed by MDS-Single Lineage Dysplasia (MDS-SLD) (n = 28, 18.4%). A normal karyotype was observed in 59 patients (57.3%), while 44 patients (42.7%) had cytogenetic abnormalities. Trisomy 8 (+ 8) was the most common cytogenetic abnormality (n = 14) followed by del 17p (n = 9) and monosomy 7 (− 7) (n = 7). Twelve patients (11.65%) were transformed to AML.ConclusionOur data betokened that among our MDS patients, Trisomy 8 is the predominant cytogenetic abnormality, and MDS-MLD and MDS-SLD are the most common of subtypes. Noteworthy, the male: female ratio was slightly higher in Iran than in previous reports from other parts of the world. Our study is the first report of the clinical, hematological, and cytogenetic spectrum of MDS patients in Iran

Next-generation sequencing reveals gene mutations landscape and clonal evolution in patients with acute myeloid leukemia

ABSTRACT Objectives The study aims to understand geneome diversification and complexity that developed in Acute myeloid leukemia (AML). Methods Next-generation sequencing (NGS) was used to identify the genetic profiles of 22 genes relevant to hematological malignancy in 204 patients with de novo non-M3 AML. Results At time of initial diagnosis, at least one mutation was identified in 80.9% of patients (165/204). The most commonly mutated gene was NPM1 (22.1%), followed by ASXL1 (18.1%), TET2 (18.1%), IDH2 (15.7%), CEBPA (14.7%), FLT3-ITD (13.2%) and DNMT3A (11.8%). Mutations landscape analysis indicated several patterns of co-occurring and mutual exclusive gene mutations. Some correlation was observed between gene mutations and clinicohematological features. Multivariate analysis showed that age >60 years, karyotypes, IDH2 and KIT mutations were the independent unfavorable prognostic factors for OS; NPM1-mut/ FLT3-ITD-wt was independently correlated with prolonged OS; whereas the independent poor risk factors for RFS were karyotypes, high WBC and RUNX1 mutation. According to different genotype demonstrated by multivariate analysis, 163 patients with intermediate-risk cytogenetics were classified into three subgroups: patients with NPM1-mut/ FLT3-ITD-wt or biallelic CEBPA mutation as favorable risk, patients with KIT, IDH2, TP53 or NRAS mutations as unfavorable risk, and the remaining was the intermediate risk. We also obtain information of clonal evolution during leukemia progression by observing five patients who underwent repeat NGS at relapse in our cohort. Conclusion NGS techniques is a useful tool for discovering related gene mutations and clonal evolution in AML genomes, leading to novel targeted therapeutic approaches that could improve patients outcomes.

Epidemiology of aplastic anemia: a study of 1324 cases

ABSTRACT Objective: Prevalence of aplastic anemia (AA) is high in the Asian population. This study was done to explore the etiology and association of AA with various socio-economic and environmental factors. Study design and setting: Study included 1324 consecutive AA cases registered at Armed Forces Bone Marrow Transplant Centre Rawalpindi, Pakistan, from March 2001 to August 2016. The study questionnaire was completed through an interview. It included patients’ socio-demographic details, personal and family medical history, environmental attributes and clinico-hematological features. Results: The median age of patients was 20 years, 997 were male and 327 female. Distribution of non-severe, severe and very severe AA was 230 (17.4%); 598 (45.2%) and 496 (37.4%), respectively. The majority of patients were from low (n = 761, 57.5%) or middle socioeconomic class (n = 543, 41%). Consanguinity among patients (n = 806, 61%) was slightly higher than the national statistics. History of chemical exposures included fertilizers (n = 116, 8.7%), pesticides (n = 56, 4.2%) and industrial chemicals (n = 37, 2.8%). PNH clone was found in 63 of AA patients. After excluding 298 patients undergoing HSCT and 660 deaths/lost to follow-up, disease evolution was observed in 38(10.4%) patients out of 366 evaluable patients. These included PNH = 18, MDS = 11 and AML = 9. Discussion: Due to lack of funding and adequate human resource at the center, age and sex-matched controls could not be included. Other limitations were a lack of molecular testing to exclude the possibility of inherited bone marrow failure syndromes on a genetic basis. Conclusion: Younger age, male predominance and higher consanguinity point toward genetic factors in AA etiology among the South Asian population.

Profile of Pediatric Chronic Myeloid Leukemia in the Era of Imatinib: A Study from South India

Abstract Introduction: Chronic myeloid leukemia (CML), a chronic hematologic malignancy, is rare in pediatric patients. Studies of the tyrosine kinase inhibitor imatinib are required so that uniform guidelines may focus on disease therapy and follow-up for children. We analyzed the clinicohematologic features of the disease, treatment response to imatinib, follow-up measures, and the impact of the disease on the patients and their family. Materials and Methods: All pediatric patients diagnosed with CML and treated and followed-up were studied regarding demographics, clinical features at presentation, and diagnostic profile, including laboratory parameters, peripheral blood smear test, fluorescent in situ hybridization and karyotyping, and reverse-transcriptase polymerase chain reaction for the BCR-ABL fusion gene. Treatment modalities, adverse reactions, remedial measures, assessment at every follow-up visit, patient’s education, parents’ socioeconomic status, and economic and psychological stresses were also evaluated. Results: Six patients were administered upfront therapy with a standard dose of imatinib. Hematological and biochemical parameters were monitored after the drug administration. We assessed the treatment response using molecular detection of the BCR-ABL transcripts. All patients who complied with drug therapy showed a complete molecular response and minimal toxic symptoms. However, parents found it difficult to cope socially and economically. Conclusion: Imatinib mesylate is effective and has a good molecular response, minimal toxicity, and good patient compliance. However, due to its cost, families reacquire financial debt, and the disease creates uncertainty about the child’s future, thereby necessitating psychosocioeconomic support for parents. Changes in the policies of cancer support groups are urgently needed to provide lifelong, lifesaving drugs free of cost.

Invariant phenotype and molecular association of biallelic TET2 mutant myeloid neoplasia

AbstractSomatic TET2 mutations (TET2MT) are frequent in myeloid neoplasia (MN), particularly chronic myelomonocytic leukemia (CMML). TET2MT includes mostly loss-of-function/hypomorphic hits. Impaired TET2 activity skews differentiation of hematopoietic stem cells toward proliferating myeloid precursors. This study was prompted by the observation of frequent biallelic TET2 gene inactivations (biTET2i) in CMML. We speculated that biTET2i might be associated with distinct clinicohematological features. We analyzed TET2MT in 1045 patients with MN. Of 82 biTET2i cases, 66 were biTET2MT, 13 were hemizygous TET2MT, and 3 were homozygous TET2MT (uniparental disomy); the remaining patients (denoted biTET2− hereafter) were either monoallelic TET2MT (n = 96) or wild-type TET2 (n = 823). Truncation mutations were found in 83% of biTET2i vs 65% of biTET2− cases (P = .02). TET2 hits were founder lesions in 72% of biTET2i vs 38% of biTET2− cases (P < .0001). In biTET2i, significantly concurrent hits included SRSF2MT (33%; P < .0001) and KRAS/NRASMT (16%; P = .03) as compared with biTET2−. When the first TET2 hit was ancestral in biTET2i, the most common subsequent hits affected a second TET2MT, followed by SRSF2MT, ASXL1MT, RASMT, and DNMT3AMT. BiTET2i patients without any monocytosis showed an absence of SRSF2MT. BiTET2i patients were older and had monocytosis, CMML, normal karyotypes, and lower-risk disease compared with biTET2− patients. Hence, while a second TET2 hit occurred frequently, biTET2i did not portend faster progression but rather determined monocytic differentiation, consistent with its prevalence in CMML. Additionally, biTET2i showed lower odds of cytopenias and marrow blasts (≥5%) and higher odds of myeloid dysplasia and marrow hypercellularity. Thus, biTET2i might represent an auxiliary assessment tool in MN.

Biallelic TET2 Inactivation in Myeloid Neoplasia: From Clonal Hierarchy to Clinical Phenotypes

Genomic data has led to the identification of bio-markers of morphological features and disease sub-entities in myeloid neoplasia (MN). Somatic TET2 mutations (TET2MT) are frequently found in MN, particularly in chronic myelomonocytic leukemia (CMML). TET2MT are mostly loss-of-function and hypomorphic hits leading to inactivation of TET2 protein. In fact, impaired TET2 activity skews the differentiation of hematopoietic stem cells toward proliferating myeloid precursors favoring myeloid tumorigenesis. However, the contribution of TET2MT to clinico-hematological features in MN has been controversial, possibly due to studies containing too few patients relative to the combinatorial diversity of co-occurring lesions.We recently reported on the clonal architecture of TET2MT in patients with MN. Of these, 40% of the patients harbored biallelic TET2MT (biTET2MT). Further analysis showed a frequent occurrence of biallelic TET2 inactivation (biTET2i). To date, only a few studies have investigated the clinical consequences of biTET2i in MN. We hypothesized that the presence of biTET2i identifies a group of patho-morphological features that independently define a distinct MN subtype. To test our hypothesis, we studied correlations between mutational configuration, clinico-hematological/morphological features and survival outcomes in cases that were biTET2ivs. not (biTET2-), combining whole exome and targeted deep sequencing, SNP-arrays and conventional cytogenetics.Among 1,001 clinically annotated MN patients, 82 were biTET2i (66 biTET2MT, 13 hemizygous TET2MT and 3 homozygous TET2MT, i.e. UPD) and 919 were biTET2- (96 monoallelicTET2MT and 823 wild type). TET2 hits were ancestral lesions in 72% of biTET2ivs. 38% in biTET2- cases (P<.0001). When the 1stTET2 hit was ancestral in biTET2i, the most common subsequent hit was a 2ndTET2MT, followed by SRSF2MT, ASXL1MT, KRASMT/NRASMT and DNMT3AMT. Truncation mutations (frameshift or nonsense variants) were found in 83% of biTET2ivs. 65% of biTET2- cases (P=.02). A second TET2 hit in biTET2MT cases significantly increases the accrual of additional truncating changes. Furthermore, biTET2i were significantly enriched for additional hits in SRSF2MT (33%; P<.0001) and KRASMT/NRASMT (16%; P=.03) while biTET2- for TP53MT (11%; P=.03). SRSF2MT was also found to be significantly associated with biTET2i when compared to monoallelicTET2MT (P=.02). In contrast, biTET2i cases showed absence of SRSF2MT in the absence of monocytosis. We then assessed associations of biTET2i with specific genotype/phenotype. Clinical analyses revealed that cases with biTET2i compared to cases with biTET2- were older (91% ≥60 years vs. 74%, P=.0004) and more commonly had normal karyotype (65% vs. 45%; P=.0007). BiTET2i were enriched in patients with CMML1/2 (44% vs. 9%; P<.0001), and predominantly in lower-risk cases (62% vs. 47% in biTET2-; P=.003). While a second TET2 hit occurred frequently, biTET2i did not portend faster progression but rather associated with monocytic differentiation, consistent with its prevalence in CMML. In addition, among biTET2i with SRSF2MT or KRASMT/NRASMT, CMML was diagnosed in 70% (P=.001) and 77% (P=.01) of the cases, respectively, significantly higher than what was seen in the biTET2i population (44%). In biTET2- cases, leukopenia (81%; P<.0001), neutropenia (52%; P=.008), pancytopenia (27%; P=.008) and increased marrow blast percentages (≥5% in 33%; P=.01) were more prevalent than in biTET2i cases, which in return co-segregated with monocytosis (84%; P<.0001), marrow hypercellularity (cellularity >70% in 67%; P<.0001) and marked myeloid dysplasia (68%; P=.0003). Given our observation of a highly significant (P<.0001) relationship between biTET2i, CMML diagnosis and/or monocytosis, we also evaluated patients without frank diagnosis of CMML (CMML-) and compared biTET2ivs.biTET2- for associations with monocytosis and myeloid dysplasia, two hallmarks of CMML. Increased monocyte counts among CMML-cases were significantly overrepresented in biTET2i cases (72%; P=.03) vs.biTET2- (55%) as was myeloid dysplasia (72% vs. 46%; P=.0001). Lastly, biTET2i as a sole hit or in combination with other hits did not influence survival outcomes.In sum, biTET2i invariantly associates with distinct morphological and clinical phenotype. It may thus represent an early diagnostic marker of morphologic MN sub-entities. DisclosuresNazha:MEI: Consultancy. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Maciejewski:Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy.

Comparative Study of Clinico-hematological Features, Molecular Spectrum and Response to Imatinib in Chronic Myelogenous Leukemia Patients: Pediatric and Adolescent Versus Adults.

Chronic myelogenous leukemia (CML) is a rare disease in children, accounting for approximately 3% of leukemias in children and adolescents, with an annual incidence of 1 case per million children in western countries. This study was conducted, at PGIMER, Chandigarh. Ninety eight patients, 48 in children and adolescents group, and 50 in adult group were included in the study. Their hematological profiles along with the bone marrow findings were analyzed. The diagnosis of CML was confirmed by cytogenetics and/or molecular analysis. The complete hematological response (CHR) was analyzed at 3months and cytogentic response (CgR) at 12months after starting imatinib therapy. Compared to adults, pediatric and adolescent patients were more symptomatic at presentation (93.5 vs. 75%). Among symptomatic patients, massive splenomegaly (>10cm), higher total leucocyte and platelet counts were seen more frequently in pediatric patients. The most common transcript in both groups was e14a2. The distribution of pediatric and adolescent cases in Sokal, Hasford and EUTOS score, showed only statistically significant difference for low risk Sokal group, which had more patients in pediatric group. Compared to adults, pediatric and adolescent patients had similar CHR rate (91.3 vs. 92%), but showed lesser major CgR rate (90.9 vs. 95.5%) however, this was not statistically significant.

Profile of Pediatric Chronic Myeloid Leukemia in the Era of Imatinib - a Study from a Developing Country

INTRODUCTIONChronic myeloid leukemia (CML) is very rare in children constituting 3% of pediatric leukemias and 10% of all CML cases. There is a lack of uniform guidelines, though the leukemic biology and clinicohematological features are the same as adults. Since the advent of imatinib, treatment modalities have evolved. However the effect of long term treatment on the child and family are areas to be studied, especially in developing countries.AIMTo study the clinicohematologic profile, diagnostic and therapeutic methods, follow up measures, impact of the disease on the patient's education and the psychosocioeconomic compromise of the parents.MATERIALS AND METHODSInstitutional ethics committee approval and informed consent from parents was obtained. All cases of pediatric CML were included. The patients and parents were interviewed and their medical records reviewed. The demography, clinical and hematological features at presentation, treatment, compliance, toxicity and follow up details were collected. The parents were provided with a validated questionnaire to assess the psychosocioeconomic impact.RESULTSIn 2011 - 2014, 7 patients below 18 years of age were diagnosed to have CML. Incidence: 5.6 % of pediatric leukemias and 9 % of CML in our centre. 1 patient opted out of treatment; 6 are undergoing treatment and follow up. 3 (50%) were above 14 years of age, 2 (33%) between 10 and 14 years, 1 (17%) below 10 years. Male to female ratio is 2:1. The predominant presenting symptoms were abdominal pain, fever, weight loss and weakness. 4 patients (67%) complained of bone pain, pallor and throat pain. 4 patients (67%) had splenomegaly.All patients presented at the chronic phase of CML. 50% had WBC count less than 20,000/µl and 1 patient (17%) had hyperleukocytosis. FISH and karyotyping detected t(9;22) in all patients. RT-PCR for bcr-abl was done. All patients were started on upfront therapy with standard dose of T.Imatinib. None needed escalated therapy. Toxic symptoms noted were gastrointestinal intolerance, skin rashes, hyperpigmentation, bone pain, myalgia and cramps which were tolerable in 5 (83%) patients. 1 (17%) had to be hospitalized. The dosage was reduced and then gradually increased. 5 patients (83%) were compliant.RESPONSE TO TREATMENT AND FOLLOWUP3 (50%) patients obtained full treatment response attaining complete molecular response (CMR) at 12-18 months, maintaining CMR at about 4 years of follow up.2 (33%) patients are also responding well with major molecular response at 1 year (follow up 14 months) and complete cytogenetic response at 6 months (follow up 9 months). 1 patient showed treatment failure due to default follow up and loss of compliance.PSYCHOSOCIOECONOMIC EFFECT:4 (66%) patients absented from school for more than 6 months and lost an academic year.2 patients who were above 14 years of age absented for a few months which did not affect their studies.5 (83%) patients belonged to upper middle socioeconomic class; they travelled to the hospital by bus and met medical expenses by insurance and support from funding agencies and friends. 1 (17%) belonged to upper class who met expenses by himself.Of the parents of patients evaluated, 33% responded to feeling guilty. 16% felt guilt related to self, 33% felt guilt with regards to interpersonal relationships. 50% were depressed with 33% admitting to feeling gloomy, 33% feeling easily upset and 33% worried about the future. 50% showed difficulty in coping, 50% felt burdened, 50% felt their activities were affected. However, nobody turned to drugs or alcohol. 33% had problems in their interpersonal relationships; 17% had issues with family, 33% did not want to mingle with others. 17% felt stigmatized; 17% felt neglected, 17% felt their social life was disturbed. 33% felt economical constraints; 33% worried about making ends meet, 17% were required to sell/ mortgage assets.DISCUSSION AND CONCLUSION:In correlation with literature, pediatric CML is rare in our centre. Patients responded well with minimum toxicity to Imatinib when compliance was good. Schooling was affected by the disease. Parents showed signs of depression and difficulty in coping.The government should initiate strategies to provide psychosocioeconomic support to CML patients and family members so they may cope with the disease. DisclosuresNo relevant conflicts of interest to declare.

Co-Existing Gene Mutations at Diagnosis and at Relapse in De Novo Acute Myeloid Leukemia with MLL Translocations

Background and purpose: The Mixed-lineage leukemia (MLL) gene located at 11q23 can be fused to a variety of partner genes through chromosomal translocations (MLL-T) in acute leukemia. The co-existence of commonly known mutated genes has not been comprehensively studied in MLL-T AML. We aimed to determine (1) the prevalence and clinical relevance of gene mutations at diagnosis, and (2) the genetic evolution profile at relapse in de novo AML with MLL-T.Materials and methods: Bone marrow samples from 80 de novo AML with MLL-T patients were analyzed on 20 gene mutations involving activating signaling pathway (class I), transcription pathway (class II), epigenetic regulators (class III), and tumor suppressors, TP53 and WT1 (class IV). MLL-T was all confirmed by FISH analysis. The common MLL fusion transcripts were detected by RT-PCR, including 26 MLLT3, 13 MLLT10, 12 MLLT4, 11 ELL, 3 MLLT1, 3 AFF1, and 2 EPS1. Another 6 infrequent or rare partner genes (one each of MLLT6,CBL, ARHGEF12, TET1, SEPT6, and SEPT9) were identified by cDNA panhandle PCR. In the remaining 4 cases, the partner genes were not identified. Mutational analyses were performed with PCR-based assays followed by direct sequencing. Twenty-two of 26 patients who relapsed had relapse samples for comparative analysis.Results: The frequencies of gene mutations of class I, II, III, and IV, in 80 de novo MLL-T AML patients were 49.4%, 3.8%, 10.3% and 1.3%, respectively. Together, 53.8% of patients with MLL-T had at least one mutated gene. KRAS (17.7%), FLT3-TKD (11.5%), and NRAS (11.4%) mutations occurred most frequently. Among the epigenetic regulators, 7.9% of patients had DNMT3A, 2.7% TET2, 1.4% ASXL1, and none had IDH1/2 or EZH2 mutations. Three patients had 3 co-existing mutations and 9 patients had two mutations, with 6 of the 12 carrying FLT3-TKD mutations. Gene mutations of transcription pathway and tumor suppressors rarely occurred, only involving NPM1 in two cases and one each for RUNX1 and WT1 mutation. The patients with DNMT3A mutations were significantly associated with older age (P=0.005), FAB AML-M4 (P<0.0001), and higher circulating monocytes (P=0.022). Patients with NRAS mutations had lower circulating monocytes (P<0.0001). KRAS-mutated patients had a higher percentage of marrow leukemic cells (P=0.002). No differences were observed between clinico-hematological features and mutation status of other genetic subtypes. Of the 65 MLL-T AML patients who received standard chemotherapy, 53 achieved complete remission. There were no differences in the outcomes between AML patients with MLLT3-MLL and MLL-MLLT10; patients with both subtypes had a superior event-free survival compared to those with other MLL-fusion transcripts (P=0.036) and a trend towards favorable overall survival (P=0.066). We failed to find significant differences between outcomes and the mutation status of each functional class. All the 3 patients carrying FLT3-ITD had an overall survival less than 6 months. Of the 22 paired diagnosis and relapse samples, 3 patients retained the same NRAS mutations at relapse whereas another one changed from G12D to G12C at relapse. Another 3 relapsed with identical KRAS mutations; while one acquired KRAS mutation at relapse. Two patients had stable DNMT3A mutations at both phases. All the 4 patients who harbored FLT3-TKD mutations at diagnosis lost the mutations at relapse. PTPN11 mutation was found in only one case at diagnosis, while none at relapse. One each had identical mutated gene of CBL and WT1 at both diagnosis and relapse. One each acquired TP53 and WT1 at relapse.Conclusions: Our results showed that co-existing mutations of RAS and FLT3-TKD were most frequently detected at diagnosis in de novo MLL-T AML. Gene mutations, except FLT3-ITD, had no impact on outcomes in MLL-T AML. FLT3-TKD mutations were absent in all relapse samples while clonal evolution of WT1, TP53 or KRAS mutations might contribute to the relapse of leukemia in a subset of MLL-T AML patients.Grant support: NHRI-EX93-9011SL, NHRI-EX96-9434SI, NSC95-2314-B-195-001, NSC96-2314-B-195-006-MY3, NSC97-2314-B-182-011-MY3, MMH-E-101-09 and OMRPG3C0021. DisclosuresNo relevant conflicts of interest to declare.

Frequency of Helicobactor pylori infection among adult patients with chronic immune thrombocytopenia: A Pakistani Perspective

Objective: Many studies have reported an association between chronic immune thrombocytopenia and Helicobactor pylori infection. Significant improvement in platelet count is seen after eradication therapy. We looked for the frequency of H. pylori infection in immune thrombocytopenia and clinico-hematological features. Material and Method: A prospective case-control study was conducted from January 2009 to December 2011. One hundred and ten chronic immune thrombocytopenic patients and 40 control subjects were enrolled. H. pylori infection was documented by H. pylori stool antigen enzyme immunoassay method. Results: H. pylori infection in immune thrombocytopenic patients was seen in 45 cases (40.9%) and in 24 (60%) controls; not significant statistically. No significant differences were seen in H. pylori positive and negative patients with respect to maternal characteristics and clinical features except for mean platelet count, which was lower in H. pylori negative group (P = 0.035). Conclusion: Unlike many related studies, we found no significant difference between the frequencies of cases versus controls. Degree of thrombocytopenia appears more marked in H pylori negative patients.

Relationship between the 46/1 haplotype of the JAK2 gene and the JAK2 mutational status and allele burden, the initial findings, and the survival of patients with myelofibrosis

An association has been reported between a specific haplotype of the JAK2 gene, the homozygous 46/1 haplotype, and a predisposition to the development of chromosome Philadelphia-negative myeloproliferative neoplasms. Concerning myelofibrosis (MF), controversy remains on the relationship between the above JAK2 haplotype and the patients' clinicohematological features and survival. Among 132 patients with MF (60% primary MF, 20% postpolycythemia vera MF, 20% post-essential thrombocythemia MF; 59% JAK2V617F positive) who were analyzed for the JAK2 46/1 haplotype, 29 were found to be homozygous and 53 heterozygous. The homozygous 46/1 haplotype was more often observed in JAK2V617F-positive patients (29.5 versus 11%, p = 0.012). Moreover, among JAK2V617F-positive patients, those who were homozygous for the 46/1 haplotype had a higher allele burden than the remainder (92 versus 48%, p = 0.0017). Overall, patients with homozygous 46/1 haplotype showed significantly higher hemoglobin values and higher leukocyte counts, but no association was seen with other clinicohematological features. Finally, no relationship was observed between the JAK2 46/1 haplotype and either the patients' prognostic score or survival.

Clonal Leukemic Evolution In Myelodysplastic Syndromes With ASXL1 and EZH2 mutations: A Comparative Analysis Of 58 Paired Samples

Background and purposeASXL1 and EZH2 are histone modifiers. Mutations of ASXL1 and EZH2 genes have been described in both myelodyspolastic syndromes (MDS) and acute myeloid leukemia (AML). The role of ASXL1 or EZH2 mutations in the progression from MDS to secondary AML (sAML) is unclear. We aimed to determine the clinical relevance of ASXL1 and EZH2 mutations in patients with MDS and to investigate the role of ASXL1 or EZH2 mutation with its cooperating mutated genes in sAML progression. MethodsASXL1 and EZH2 mutations were analyzed on bone marrow samples from 126 patients with de novo MDS (45 RAEB1, 53 RAEB2, 24 RCMD and 4 RARS). Paired matched MDS/sAML samples were available for ASXL1 mutational analysis in 58 patients and for EZH2 in 54 patients. Mutational analysis of ASXL1 exon 12 was performed by PCR assays followed by direct sequencing, EZH2 mutations were first screened with denaturing high-performance liquid chromatography on amplified PCR fragments covering the whole coding sequencing from exons 2 to 20 of EZH2 gene followed by sequencing of the abnormal profile. Additional 20 known gene mutations in myeloid neoplasms were also examined in patients carrying ASXL1 or EZH2 mutations. Relative allele frequency was determined by pyrosequencing. ResultsAmong the 126 patients, ASXL1 mutations were detected in 18 (14.3%) patients and EZH2 in 11 patients (8.7%), 3 of them had both mutations. Taken together, 20.6% of patients carried mutations of ASXL1 and/or EZH2. ASXL1-mutated patients had male predominance (17 out of 18 patients, P=0.012) and fewer circulating blasts (P=0.007). ASXL1-mutated and -unmutated patients had no difference in hemoglobin levels, white blood cell counts, platelet counts, cytogenetics, WHO subtypes, bone marrow blasts, IPSS-R or risk to sAML. ASXL1 mutations had no impact on overall survival (P=0.765) or time to sAML transformation (P=0.605). No significant difference was observed between EZH2 mutation status and clinicohematologic features or outcomes. Of the 58 paired samples, ASXL1 mutations were detected in 8 cases at diagnosis of MDS, all were also present at the time of sAML progression with no difference in the mutant allele burden (P=0.614), 2 patients acquired ASXL1 mutations. Five had EZH2 mutations at both phases of disease which exhibited a similar allele frequency (P= 0.434), none lost and one acquired EZH2 mutation at sAML phase. Progression to sAML was accompanied by additional gene mutations including RUNX1 (n=4), TET2 (n=3), CEBPα (n=2), PTPN11 (n=2), and one each for FLT3-ITD, CBL, MLL-PTD, DNMT3A, IDH2 and EZH2 in ASXL1-mutated patients; TET2 (n=4), RUNX1 (n=3), N-RAS (n=2) and single cases for DNMT3A, IDH1, and ASXL1 in EZH2-mutated patients. Cooperating mutations were either detected in both MDS and sAML samples or newly appeared in sAML samples except one who lost JAK2V617F mutation while acquired EZH2 and N-RAS mutations during sAML progression. ConclusionsOur study on a large cohort of paired MDS and sAML samples demonstrated that ASXL1 or EZH2 mutations remained stable at both phases of disease in most patients. Clonal evolution can occur and cooperation of additional gene mutations is frequently detected in patients harboring ASXL1 or EZH2 mutations in the progression of MDS to sAML. Grant supportThis work was supported by NHRI-EX102-10003NI and DOH102-TD-C-111-006, Taiwan. Disclosures:No relevant conflicts of interest to declare.

Masked Polycythemia Vera (mPV): Results Of An International Study

IntroductionPatients with masked polycythemia vera (mPV) present with hemoglobin (HB) values borderline inferior to those required by the World Health Organization (WHO) diagnostic criteria (HB >16.5g% and >18.5 g% in female and male respectively) but are suspected to have PV for presenting clinico-hematological features. The diagnostic HB values required by the WHO guidelines have been considered surrogate of increased red cell mass (RCM). However, in both males and females HB values are not always equal to an absolute erythrocytosis and conversely a HB concentration below the required cut off level could be associated with an increased red cell mass. We believe that bone marrow (BM) morphology may play an important role in differentiating PV from other MPN entities and reactive changes. In this study we investigated JAK2V617/Exon12-positive patients showing BM features characteristic for PV and classified as mPV or overt PV according to their hemoglobin levels and evaluated their respective outcomes including thrombosis, progression to myelofibrosis (MF), acute leukemia (AL) and overall survival with the purpose to support the diagnosis of PV also in patients presenting with lower HB levels than required by WHO criteria. Materials and MethodsA clinicopathological database of patients who were diagnosed and treated for PV was created by clinicians and hematopathologists from seven international centers of excellence for MPNs. Eligibility criteria for entry included the presence of JAK2V617F (or Exon12) mutation, EPO measurement, no evidence of subnormal ferritin levels and the availability of representative, treatment-naive BM biopsies (hematoxylin-eosin staining and silver impregnation after Gomori or Gordon-Sweet). All BM biopsies were centrally re-reviewed by one of the authors (J.T.) who was completely blinded to outcome data. In both mPV and overt PV patients, phlebotomy and cytoreductive therapy were used with the same frequency (p=0.101). Results and DiscussionAmong 397 mutated patients, with PV BM morphology as defined by WHO, 257 (65%) met the full WHO-2008 criteria. The remaining 140 patients (35%) were classified as mPV for levels of HB at diagnosis, ranging from 16.5 to 18.5 g/dL in men and from 15.0 to 16.5 g/dL in women, and frequent presence of subnormal EPO levels (65%).At baseline, mPV patients were more males, had more previous arterial thrombosis and presented more frequently with platelet counts exceeding the required level of WHO-defined ET (450 x 109/L), often mimicking clinically this entity at onset.After a median follow-up of 3.8 (0-29.8) and 4.5 (0-21.1) years in mPV and overt PV, respectively, time to the first thrombotic event was superimposable in the two groups while time to progression to MF and AL was significantly different .These combined events were recorded in 10% of mPV and 5.8% of overt PV, corresponding to an annual rate of 1.60 % pts/year in mPV and 0.97 % pts/year in overt PV, respectively (p=0.010). In addition, an inferior overall survival was documented in mPV patients in comparison with overt PV (p=0.011). The annual rate of death in mPV was almost twice that of overt PV, mainly due to an excess of hematological transformation (overt MF and AL). Multivariable analysis identified age older than 65 years (hazard ratio (HR) 6.63, P<.0001), WBC >10 x109/L (HR 2.99, P=.005) and diagnosis of mPV (HR 2.38, P=.036) as independent risk factors for survival in these patients.These results show that in JAK2 mutated patients with borderline HB levels, BM features may be a valid support to confirm the suspicion of PV diagnosis. Of interest, recent data demonstrated that all patients with increased RCM also had BM morphology typical of PV. It is tempting to speculate that the clinico-hematologic phenotype of mPV may represent an early stage of a JAK2 mutated MPN, presenting as a variant of PV, with a more rapid progression to MF and AL. The recognition of these early PV patients has an important clinical impact since, in spite of not meeting the full WHO criteria, mPV share the same vascular risk as overt PV.In conclusion, these data suggest that mPV is not necessarily only an early form of classical PV but a distinct entity with disease outcomes akin to primary myelofibrosis. Disclosures:No relevant conflicts of interest to declare.