Application of HRM Analysis in Detection of PDGFRA Exon 10 Polymorphism in CML Patients with Imatinib Resistance

@#Introduction: Imatinib mesylate has been widely used as a standard treatment for chronic myeloid leukemia (CML). It acts as a selective competitive inhibitor of the BCR-ABL tyrosine kinase. Despite the excellent efficacy on CML treatment, some patients developed resistance to the treatment. Mutation in the PDGFRA may be one of the factors involved in the mechanism of resistance that affects the response to imatinib. The mutational status of PDGFRA is highly relevant for prognosis and treatment prediction in CML patients. Thus, this study is intended to establish and validate a High Resolution Melting (HRM) analysis for PDGFRA exon 10 c.1432 T>C polymorphism in CML patients. Methods: High resolution melting (HRM) analysis was used to identify the c.1432 T > C polymorphism in PDGFRA exon 10 (n =86; response = 43; resistance = 43). The results from HRM analysis were compared and validated with Sanger sequencing. The association between the polymorphism and treatment response was assessed by statistical analysis using binomial logistic regression analysis. Results: HRM analyses showed two different melt curves. One curve followed the shape of the reference, homozygous wild type (TT) and the other curve showed a different melting profile than the reference with the TC genotype (heterozygous variant). The results revealed that heterozygous variant (TC) genotype showed a high risk of acquiring resistance with an OR of 3.795; 95% CI: 1.502-9.591, with a statistically significant association, p = 0.005. HRM analysis also showed 100% sensitivity and specificity in the detection of PDGFRA exon 10. Conclusion: The HRM analysis of PDGFRA exon 10 c.1432 T>C was successfully established. The exon 10 c.1432 T>C polymorphism shows a higher risk for the development of resistance toward imatinib treatment.

Linezolid-resistant Enterococcus casseliflavus and Enterococcus gallinarum isolated from poultry farms in Kelantan, Malaysia

Aims@#Linezolid has become a decisive therapy in treating infections with vancomycin-resistant Enterococcus (VRE). Currently, the emergence of linezolid-resistant Enterococcus further complicates the therapeutic options and leads to global health threat not only in hospital setting but in the community. The study aimed at antimicrobial pattern of Enterococcus isolated from 6 poultry farms in Kelantan, Malaysia.@*Methodology and results@#Between February and December 2019, 300 broiler cloacal swab sample (Gallus gallus domesticus) were collected and screened for linezolid-resistant enterococci (LRE) using a standard biochemical and antimicrobial susceptibility tests. Among all the samples, 32.3% (n=97/300) grew Enterococcus, 71.1% (n=69/97) of it were identified Enterococcus casseliflavus by molecular identification, whilst remaining isolates 28.9% (n=28/97) were further identified as Enterococcus gallinarum by 16S rRNA sequencing. None of the isolates were found to exhibit high-level resistance to vancomycin. However, 3/97 (3.1%) were exhibit resistance to high-level gentamicin based on Kirby-Bauer disk diffusion test. Whereas 48/97 (49.5%) of isolates were observed to be resistant to ampicillin, 28/97 (28.9%) were resistant to penicillin. Surprisingly, among the two strains isolated, 18.6% (n=18/97) of it were resistant to linezolid. Isolates showed resistance to linezolid by disk diffusion test were verified by VITEK-2 automated system (bioMérieux, USA) with MIC ≥8 µg/mL. All antimicrobial susceptibility test and minimal inhibitory concentration (MIC) results were interpreted according to Clinical and Laboratory Standard Institute (CLSI). @*Conclusion, significance and impact of study@#In conclusion, this study has reported the prevalence of linezolid resistant Enterococcus (LRE) in highly intrinsic antibiotic resistant of E. casseliflavus and E. gallinarum in Malaysia poultry farms, alongside with the truancy of vanA strains. The emergence of LRE strains is an alarming problem to the animal husbandry and healthcare setting worldwide. This could lead to potentially untreatable and life-threatening enterococcal infections. Even more worrying is the spread of LRE to geographical regions where these strains were previously unreported, which may pose a global health threat. Antimicrobial surveillance in poultry husbandry is thus, dimly necessary to prevent wide spread of multidrug-resistant bacteria.

Homozygous HbS in A Malay Patient: A Rare Variant of Sickle Cell Disease in Malaysia

@#Sickle cell disease in Malay ethnicity is uncommon, with few cases been reported only in Malaysian Indians. Detecting sickle haemoglobin in patients with osteoarticular manifestation is not as simple as those with haemolysis crisis, due to its extremely low incidence in this country. We hereby report a case of a 19-year-old Malay female who presented with a long-standing history of disabling movement of both hip joints, intermittent painful swollen right elbow, and chronic back pain. Imaging investigations revealed features of chronic osteomyelitis and avascular necrosis while blood investigations demonstrated features of mild normochromic normocytic anaemia and extravascular haemolysis. Further blood smear and haemoglobin analysis eventually confirmed the presence of homozygous sickle haemoglobin manifesting as sickle cell anaemia. Our case has highlighted the importance of prompt identification and thorough evaluation of the cause of anaemia in a patient with disabling chronic osteoarticular problem.

Classifying DLBCL according cell of origin using Hans algorithm and its association with clinicopathological parameters: A single centre experience

@#Introduction: In recent years, "double hit" and "double protein" involving gene rearrangement and protein expression of c-MYC and BCL2 and/or BCL6 are the most used terms to describe poor prognostic factors in diffuse large B-cell lymphoma (DLBCL). This study was to determine the frequency of double or triple protein expression by using immunohistochemistry (IHC) and comparing the result with clinicopathological features and cell of origin (COO) classification. Methods: We conducted a cross-sectional study by using 29 archived formalin-fixed paraffin embedded tissue blocks of DLBCL. All the samples were evaluated for the subgrouping of COO DLBCL was determined by expression of CD10, BCL6 and MUM1 based on Hans classification. In addition, expressions of c-MYC, BCL2 and BCL6 were detected by IHC. Results: Among the 29 cases, MYC, BCL2 and BCL6 proteins were detected in 72.4%, 62.1% and 62.1% of patients, respectively. Concurrent expression (c-MYC positive/BCL2 positive and/or BCL6 positive) was present in 58.6% of patients. 34.5% were categorised as germinal centre like (GCB) subgroup and 65.5% were categorised as nongerminal centre like (non-GCB) subgroup. Among the clinicopathological features, the double/triple protein expression lymphoma was significantly associated with elevated LDH level (p=0.018), IPI score (p=0.003), Ann Arbor stage (p=0.011) and complete response rate (p=0.011). Conclusion: Double/triple protein lymphoma was strongly associated more adverse clinical risk factors. Thus, analyses of MYC, BCL2 and BCL6 expression by IHC represents a rapid and inexpensive approach to risk-stratify patients with DLBCL at diagnosis.

Acute Abdomen: Unmasked the Bleeding Site in Severe Haemophilia A

@#Haemophilia A is an inherited bleeding disorder, commonly involve soft tissues and joints. Gastrointestinal tract bleeding, are not uncommon but seldom highlighted. A 23-year-old male with underlying severe haemophilia A was presented with a generalised abdominal pain for 2 days, abdominal distension, diarrhoea and vomiting. He did not have any trauma to the abdomen. Abdominal examination revealed generalized tenderness with sign of guarding on palpation. Laboratory investigations revealed isolated, prolonged activated partial thromboplastin time (APTT) with normal total white blood cell count and haemoglobin level. In view of acute abdomen, which was not resolved by conservative treatment, an emergency laparotomy was done with FVIII concentrate and recombinant factor VII (rFVII) coverage. Intraoperative findings noted patchy gangrenous spots of about 30 cm in length in the small bowel. Histopathology examination revealed an evidence of haemorrhage within the submucosal and intramuscularis layer from the resected specimen. This case highlighted the possibility of gastrointestinal bleeding without prior trauma, which can be presented as acute abdomen in severe haemophilia patient.

Autologous peripheral blood stem cell transplantation among lymphoproliferative disease patients: factors influencing engraftment

Objectives: Autologous peripheral blood stem cells transplantation [APBSCT] is a therapeutic option which can be used in various hematological, neoplastic disorders including lymphoproliferative disease [LPD]. Differences in patient populations and treatment modalities in different transplant centers mean it is important to improve the knowledge of the different factors affecting engraftment after APBSCT for the success of this procedure. We sought to determine the factors influencing neutrophil and platelet engraftment after APBSCT in patients with LPD

Methods: We conducted a retrospective review of 70 patients with LPD [35 with lymphoma and 35 with multiple myeloma] who had undergone APBSCT between January 2008 and December 2016. Data obtained included disease type, treatment, and stem cell characteristics. Kaplan-Meier analysis was performed for probabilities of neutrophil and platelet engraftment occurred and was compared by the log-rank test. The multivariate Cox proportional hazards regression model was used for the analysis of potential independent factors influencing engraftment. A p-value < 0.050 was considered statistically significant

Results: Most patients were ethnic Malay, the median age at transplantation was 49.5 years. Neutrophil and platelet engraftment occurred in a median time of 18 [range 4–65] and 17 [range 6–66] days, respectively. The majority of patients showed engraftment with 65 [92.9%] and 63 [90.0%] showing neutrophil and platelet engraftment, respectively. We observed significant differences between neutrophil engraftment and patient's weight [< 60/>/= 60 kg], stage of disease at diagnosis, number of previous chemotherapy cycles [< 8/>/= 8], and pre-transplant radiotherapy. While for platelet engraftment, we found significant differences with gender, patient's weight [< 60/>/= 60 kg], pre-transplant radiotherapy, and CD34+ dosage [< 5.0/>/= 5.0 × 106/kg and < 7.0/>/= 7.0 × 106/kg]. The stage of disease at diagnosis [p = 0.012] and pre-transplant radiotherapy [p = 0.025] were found to be independent factors for neutrophil engraftment whereas patient's weight [< 60/>/= 60 kg, p = 0.017], age at transplantation [< 50/>/= 50 years, p = 0.038], and CD34+ dosage [< 7.0/>/= 7.0 × 106/kg, p = 0.002] were found to be independent factors for platelet engraftment

Conclusions: Patients with LPD who presented at an early stage and with no history of radiotherapy had faster neutrophil engraftment after APBSCT, while a younger age at transplantation with a higher dose of CD34+ cells may predict faster platelet engraftment. However, additional studies are necessary for better understanding of engraftment kinetics to improve the success of APBSCT

Placebo Controlled Trials: Interests of Subjects versus Interests of Drug Regulators

The use of placebo-controlled trials in situations where established therapies are available is considered ethically problematic since the patients randomised to the placebo group are deprived of the beneficial treatment. The pharmaceutical industry and drug regulators seem to argue that placebo-controlled trials with extensive precautions and control measures in place should still be allowed since they provide necessary scientific evidence for the efficacy and safety of new drugs. On the other hand, the scientific value and usefulness for clinical decision-making may be much higher if the new drug is compared directly to existing therapies. As such, it may still be unethical to impose the burden and risk of placebo-controlled trials on patients even if extensive precautions are taken. A few exceptions do exist. The use of placebo-controlled trials in situations where an established, effective and safe therapy exists remains largely controversial.

Primary Mediastinal Large B-Cell Lymphoma and Its Mimickers: A Rare Case Report With Literature Review

Primary mediastinal large B-cell lymphoma (PMLBL) is an uncommon non-Hodgkin lymphoma with a distinct clinicopathological entity in the WHO classification of lymphoid malignancies. It is known to originate from B-cells of the thymus. It mimics thymic neoplasms and other lymphomas clinically and histopathologically. We reported a 33-year-old obese man who presented with shortness of breath off and on for 4 years. Radiologically, there was a huge anterior mediastinal mass. Tru-cut biopsy was initially diagnosed as type-A thymoma. Histopathological examination of the excised specimen revealed PMLBL with stromal fibrosis and sclerosis which created a diagnostic difficulty. The neoplastic cells varied from medium-sized to large pleomorphic cells, including mononuclear cells with centroblastic and immunoblastic features as well as bi-lobed Reed Sternberg (RS)-like cells and horse-shoe like hallmark cells. Some interlacing spindle cells and epithelioid cells were also present. Immunohistochemically, tumour cells expressed diffuse positivity for LCA, CD20, CD79a, CD23, Bcl2, MUM-1 and heterogenous positivity for CD30 and EMA, and were negative for CD10, CD15 and ALK. Ki67 scoring was very high. Tumour cells infiltrated into peri-thymic fat and pericardium. No malignant cells were detected in the pleural fluid and there was no bone marrow infiltration. The patient showed partial response to 6 cycles of RICE chemotherapy, and was planned for second line chemotherapy using hyper-CVAD regimen followed by autologous stem cell transplantation. This case illustrates the importance of thorough sampling and immunohistochemistry in differentiating PMLBL from its differential diagnoses.