Comparison of Clinicopathological Parameters, and Treatment Responses in Younger and Older Chronic Myeloid Leukaemia Patients Treated with Imatinib

@#Introduction: Differences in baseline characteristics and response to treatment in different age groups of patients with chronic myeloid leukaemia (CML) in resource-limited countries have not been extensively studied. We aimed to determine the differences in clinicopathological parameters at diagnosis and response to imatinib in adult CML patients with younger (under 60 years; YCML) and older (60 years and older; OCML) age treated at our institution from March 2001 to March 2021. Methods: A retrospective analysis of consecutive adult CML patients receiving imatinib was performed. Clinicopathological parameters and treatment response were reviewed and analysed using hospital medical records and electronic data reports. Results: The median age at diagnosis was 50 years. OCML patients (n=17) had significantly more comorbidities. The YCML group (n=50) generally had a palpable spleen >5cm from the costal margin, mild anaemia, hyperleukocytosis and thrombocytosis. A starting dose of 400 mg/day was observed in 84% of YCML and in 65% of OCML. Cumulative complete cytogenetic response was 50% in YCML versus 70.6% in OCML, p=0.158. OCML tended to have a higher percentage of major molecular response (MMR) (52.9% versus 32%) and a shorter time to MMR, 22 months (range 5-70) versus 35 months (range 8-53). OCML experienced more haematological and non-haematological treatment-related adverse events after imatinib therapy. Conclusion: Although OCML patients had more comorbidities and treatment intolerances, overall long-term treatment response was comparable to YCML. In OCML, a more personalised approach to initial and subsequent dosing of imatinib may be considered.

Calreticulin Mutations in Myeloproliferative Neoplasms Patients Diagnosed in UKM Medical Centre

@#Introduction: Calreticulin (CALR) mutations are one of the molecular markers that has been incorporated for the diagnosis of myeloproliferative neoplasms (MPN) in the revised 2017 WHO Classification of Haematopoietic and Lymphoid Tumors. This study was performed to determine the prevalence of CALR mutations in patients with MPN diagnosed in UKMMC and to compare their demographics plus laboratory features with other MPN patients. Methods: A total of 59 MPN patients who tested negative for JAK2V617Fmutation were selected and 21 MPN patients positive for JAK2V617F were included as controls. Screening for CALR exon 9 was done by multiplex polymerase chain reaction (PCR) followed by Sanger sequencing. Results: A total of six JAK2 V617F negative MPN samples were found to be positive for CALR mutations. Out of these six, three patients with CALR mutations were of type I mutation, two were type II while one was a mutation in the stretch III region. None of the twenty one JAK2 V617F positive MPN samples were positive for CALR mutation. Clinical phenotypes for those positive for CALR were restricted to Essential Thrombocythemia (ET), Primary Myelofibrosis (PMF) and one case of atypical Chronic Myeloid Leukaemia (CML). Conclusion: CALR mutations constituted 10.16% from the MPN patients who were negative for JAK2V617F mutation with no significant differences in platelet counts, hemoglobin (Hb), hematocrit and white cell counts as compared to MPN patients with JAK2 V617F mutations. Testing for CALR mutations among those who are negative for JAK2V617F within Malaysian population maybe worthwhile and require larger scale studies.

Detection of α-thalassaemia in neonates on cord blood and dried blood spot samples by capillary electrophoresis

Haemoglobin Bart’s (Hb Bart’s) level is associated with α-thalassaemia traits in neonates, enabling early diagnosis of α-thalassaemia. The study aimed to detect and quantify the Hb Bart’s using Cord Blood (CB) and CE Neonat Fast Hb (NF) progammes on fresh and dried blood spot (DBS) specimen respectively by capillary electrophoresis (CE). Methods: Capillarys Hemoglobin (E) Kit (for CB) and Capillarys Neonat Hb Kit (for NF) were used to detect and quantify Hb Bart’s by CE in fresh cord blood and dried blood spot (DBS) specimens respectively. High performance liquid chromatography (HPLC) using the β-Thal Short Programme was also performed concurrently with CE analysis. Confirmation was obtained by multiplex ARMS Gap PCR. Results: This study was performed on 600 neonates. 32/600 (5.3%) samples showed presence of Hb Bart’s peak using the NF programme while 33/600 (5.5%) were positive with CB programme and HPLC methods. The range of Hb Bart’s using NF programme and CB programme were (0.5–4.1%) and (0.5-7.1%), respectively. Molecular analysis confirmed all positive samples possessed α-thalassaemia genetic mutations, with 23/33 cases being αα/--SEA, four -α3.7/-α3.7, two αα/-α3.7 and three αα/ααCS. Fifty Hb Bart’s negative samples were randomly tested for α-genotypes, three were also found to be positive for α-globin gene mutations. Thus, resulting in sensitivity of 91.7% and 88.9% and specificity of 100% for the Capillarys Cord Blood programme and Capillarys Neonat Fast programme respectively. Conclusion: Both CE programmes using fresh or dried cord blood were useful as a screening tool for α-thalassaemia in newborns. All methods show the same specificity (100%) with variable, but acceptable sensitivities in the detection of Hb Bart.

Hb lepore/β0-thalassaemia with α+-thalassaemia interactions, a potential diagnostic pitfall

Haemoglobin (Hb) Lepore is a variant Hb consisting of two α-globin and two δβ-globin chains. In a heterozygote, it is associated with clinical findings of thalassaemia minor, but interactions with other haemoglobinopathies can lead to various clinical phenotypes and pose diagnostic challenges. We reported a pair of siblings from a Malay family, who presented with pallor and hepatosplenomegaly at the ages of 21 months and 14 months old. The red cell indices and peripheral blood smears of both patients showed features of thalassaemia intermedia. Other laboratory investigations of the patients showed conflicting results. However, laboratory investigation results of the parents had led to a presumptive diagnosis of compound heterozygote Hb Lepore/β-thalassaemia and co-inheritance α+-thalassaemia (-α3.7). Hb Lepore has rarely been detected in Southeast Asian countries, particularly in Malaysia. These two cases highlight the importance of family studies for accurate diagnosis, hence appropriate clinical management and genetic counseling.

Hb Lepore/β0-Thalassaemia With α+-Thalassaemia Interactions, A Potential Diagnostic Pitfall

Haemoglobin (Hb) Lepore is a variant Hb consisting of two α-globin and two δβ-globin chains. In a heterozygote, it is associated with clinical findings of thalassaemia minor, but interactions with other haemoglobinopathies can lead to various clinical phenotypes and pose diagnostic challenges. We reported a pair of siblings from a Malay family, who presented with pallor and hepatosplenomegaly at the ages of 21 months and 14 months old. The red cell indices and peripheral blood smears of both patients showed features of thalassaemia intermedia. Other laboratory investigations of the patients showed conflicting results. However, laboratory investigation results of the parents had led to a presumptive diagnosis of compound heterozygote Hb Lepore/β-thalassaemia and co-inheritance α+-thalassaemia (-α3.7). Hb Lepore has rarely been detected in Southeast Asian countries, particularly in Malaysia. These two cases highlight the importance of family studies for accurate diagnosis, hence appropriate clinical management and genetic counseling.

Double Philadelphia chromosome-positive B acute lymphoblastic leukaemia in an elderly patient

A rare case of double Philadelphia chromosome-positive B Acute lymphoblastic Leukaemia (B-ALL) is reported here. A 60-year-old lady presented with one month history of fever, submandibular lymphadenopathy, loss of appetite and weight loss. Physical examination revealed multiple palpable cervical lymph nodes. Blood film showed leucocytosis with 72% blasts. Bone marrow assessment confirmed a diagnosis of B-ALL with presence of double Philadelphia (Ph) chromosomes. As she was very ill, she was initially treated with an attenuated regimen of induction chemotherapy consisting of rituximab, cyclophosphamide, vincristine and prednisolone (R-CVP) along with intrathecal chemotherapy comprising methotrexate, cytarabine and hydrocortisone. Bone marrow examination post-induction chemotherapy showed >5% blasts. She was subsequently re-induced with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP) along with intrathecal chemotherapy, following which she went into complete remission. Consolidation chemotherapy consisting of methotrexate, methylprednisolone, cytarabine, intrathecal chemotherapy and imatinib was subsequently administered followed by maintenance chemotherapy consisting of vincristine, prednisolone and imatinib (IDEAMOP). She developed spontaneous bruises and relapsed four months into her maintenance chemotherapy with 90% blasts in the bone marrow which was treated with fludarabine, cytarabine and granulocyte colony stimulating factor (FLAG). Unfortunately she developed neutropenic sepsis which was complicated by invasive lung aspergillosis. Bone marrow examination post-FLAG showed 80% blasts. Despite aggressive antifungal therapy, her lung infection worsened and she finally succumbed to her illness 13 months after the initial diagnosis. We highlight a rare case of elderly B-ALL with double Ph chromosomes which carries a poor prognosis despite aggressive treatment for the disease and its complications.

Molecular characteristic of alpha thalassaemia among patients diagnosed in UKM Medical Centre

Alpha (α) thalassaemia is the most common inherited disorder in Malaysia. The clinical severity is dependant on the number of α genes involved. Full blood count (FBC) and haemoglobin (Hb) analysis using either gel electrophoresis, high performance liquid chromatography (HPLC) or capillary zone electrophoresis (CE) are unable to detect definitively alpha thalassaemia carriers. Definitive diagnosis of α-thalassaemias requires molecular analysis and methods of detecting both common deletional and non-deletional molecular abnormailities are easily performed in any laboratory involved in molecular diagnostics. We carried out a retrospective analysis of 1623 cases referred to our laboratory in Universiti Kebangsaan Malaysia Medical Centre (UKMMC) for the diagnosis of α-thalassaemia during the period October 2001 to December 2012. We examined the frequency of different types of alpha gene abnormalities and their haematologic features. Molecular diagnosis was made using a combination of multiplex polymerase reaction (PCR) and real time PCR to detect deletional and non-deletional alpha genes relevant to southeast Asian population. Genetic analysis confirmed the diagnosis of α-thalassaemias in 736 cases. Majority of the cases were Chinese (53.1%) followed by Malays (44.2%), and Indians (2.7%). The most common gene abnormality was αα/--SEA (64.0%) followed by αα/-α3.7 (19.8%), -α3.7 /--SEA (6.9%), αα/ααCS (3.0%), --SEA/--SEA (1.2%), -α3.7/-α3.7 (1.1%), αα/-α4.2 (0.7%), -α4.2/--SEA (0.7%), -α3.7/-α4.2 (0.5%), ααCS/-- SEA (0.4%), ααCS/ααCd59 (0.4%), ααCS/ααCS (0.4%), -α3.7/ααCd59 (0.3%), αα/ααCd59 (0.1%), αα Cd59/ ααIVS I-1 (0.1%), -α3.7/ααCS (0.1%) and --SEA /ααCd59 (0.1%). This data indicates that the molecular abnormalities of α-thalassaemia in the Malaysian population is heterogenous. Although α-gene deletion is the most common cause, non-deletional α-gene abnormalities are not uncommon and at least 3 different mutations exist. Establishment of rapid and easy molecular techniques is important for definitive diagnosis of alpha thalassaemia, an important prerequisite for genetic counselling to prevent its deleterious complications.

HbA2 levels in normal, B-thalassaemia and haemoglobin E carriers by capillary electrophoresis

Objective: The capillary electrophoresis (CE) is a new system that utilizes the principle of electrokinetic separation of molecules in eight electrolyte buffer-fi lled silica capillaries. In this study, we established the normal ranges of haemoglobin A2 (HbA2) and haemoglobin F (HbF) levels for normal individuals using this system and also the HbA2 level in β thalassaemia and haemoglobin E (HbE) individuals. Materials and Methods: 154 samples from normal individuals, 218 samples from β thalassaemia heterozygotes and 91 samples from HbE heterozygotes were subjected to high performance liquid chromatography (HPLC) and CE analysis. Results: The normal ranges for HbA2 and HbF by CE were 2.75% (SD 0.26%) and 0.03% (SD 0.24%) respectively, which were signifi cantly lower than that of HPLC 2.88% (SD 0.25%) and 0.58% (SD 0.61%) (p <0.001). The HbA2 level for HbE heterozygotes was 3.58% (SD 0.44%), which was signifi cantly higher than normal (p <0.001) but lower than that of β-thalassaemia heterozygotes (p<0.001) and the true HbE level was 24.28% (SD 3.38%). Conclusion: The CE system provided a fully automated and high throughput system for haemoglobin analysis. We established the normal ranges for HbA2 and HbF levels by CE. We also determined the ranges for HbA2 in beta thalassaemia and HbE heterozygotes using this system.

Co-inheritance of compound heterozygous Hb Constant Spring and a single –α3.7 gene deletion with heterozygous δβ thalassaemia: A diagnostic challenge

Haemoglobin Constant Spring (Hb CS) mutation and single gene deletions are common underlying genetic abnormalities for alpha thalassaemias. Co-inheritance of deletional and non-deletional alpha (α) thalassaemias may result in various thalassaemia syndromes. Concomitant co-inheritance with beta (β) and delta (δ) gene abnormalities would result in improved clinical phenotype. We report here a 33-year-old male patient who was admitted with dengue haemorrhagic fever, with a background history of Grave’s disease, incidentally noted to have mild hypochromic microcytic red cell indices. Physical examination revealed no thalassaemic features or hepatosplenomegaly. His full blood picture showed hypochromic microcytic red cells with normal haemoglobin (Hb) level. Quantitation of Hb using high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) revealed raised Hb F, normal Hb A2 and Hb A levels. There was also small peak of Hb CS noted in CE. H inclusions was negative. Kleihauer test was positive with heterocellular distribution of Hb F among the red cells. DNA analysis for α globin gene mutations showed a single -α-3.7 deletion and Hb CS mutation. These fi ndings were suggestive of compound heterozygosity of Hb CS and a single -α-3.7 deletion with a concomitant heterozygous δβ thalassaemia. Co-inheritance of Hb CS and a single -α-3.7 deletion is expected to result at the very least in a clinical phenotype similar to that of two alpha genes deletion. However we demonstrate here a phenotypic modifi cation of α thalassemia presumptively as a result of co-inheritance with δβ chain abnormality as suggested by the high Hb F level.

Extensive myelofibrosis responsive to treatment for acute erythroblastic leukaemia

Intense myelofibrosis is rarely associated with de novo acute myeloid leukaemia (AML) except in acute megakaryoblastic leukaemia (AML-M7) where there is diffuse marrow fibrosis as a consequence of proliferation of neoplastic myeloid cells. AML associated with significant myelofibrosis developing both de novo or secondary to primary (idiopathic) myelofibrosis is characterised by a fulminant course and extremely poor prognosis, primarily due to treatment-resistant disease. The prognostic value of degree of marrow fibrosis in de novo AML has been poorly investigated. We describe a case of extensive myelofibrosis associated with acute erythroblastic leukaemia (AML-M6) that responded to induction therapy of the leukaemia.