Next Generation Sequencing-Based Germline Panel Testing for Breast and Ovarian Cancers in Pakistan.

BACKGROUND: Pathogenic germline mutations in BRCA1/2 constitute the majority of hereditary breast and/or ovarian cancers worldwide. Incidence and mortality rate of breast and ovarian cancers in Pakistani women is high. Thus, to establish the diagnosis for targeted therapy in Pakistan, we conducted Next-generation sequencing-based germline testing for the detection of BRCA1/2 oncogenic variants associated with breast and ovarian cancer subtype. METHODS: Peripheral blood of 24 women, diagnosed with breast and epithelial ovarian cancers, was taken from the recruited cases with the consent of performing germline genetic testing. DNA was isolated from the peripheral blood and subjected to indexed BRCA Panel libraries. Targeted NGS was performed for all coding regions and splicing sites of BRCA1 and BRCA2 genes using AmpliSeq for Illumina BRCA Panel and Illumina MiSeq sequencer (placed at AFIP). Analysis of the sequencing results has been done by using Illumina bioinformatics tools. RESULTS: We detected 421 variants having a quality score of 100 in all cases under study. The list of identified variants in BRCA1 and BRCA2 genes was narrowed down after filtering out those which did not pass q30 and those with a minor allele frequency (MAF) > 0.05 based on gnomAD browser. To classify these variants, clinical significance was predicted using external curated databases. As a result, we interpreted (n = 4) 16.7% pathogenic variants in BRCA1 and (n = 6) 25% variants of uncertain significance (VUS) in both genes. Descriptive statistics depicted that the age and BMI of BRCA positive cases are less than BRCA negative cases. CONCLUSION: Our findings exhibit an initial report for the NGS based cancer genetic testing in Pakistan.  This will enable us to pursue screening and diagnosis of hereditary BRCA mutation utilizing the latest state-of-the-art technique locally available in Pakistan ultimately resulting in targeted cancer therapy.

Additional chromosomal abnormalities in Philadelphia positive chronic myeloid leukemia.

OBJECTIVE: To determine the frequency of additional chromosomal abnormalities in Philadelphia chromosome positive Chronic Myeloid Leukemia (CML) by conventional cytogenetic analysis. METHODS: This descriptive cross sectional study was conducted at Armed Forces Institute of Pathology (AFIP), Rawalpindi, from January 2012 to December 2016. A total number of 528 newly diagnosed CML patients were included in the study. The subjects were tested for the presence of Philadelphia (Ph) chromosome and other additional cytogenetic abnormalities by conventional cytogenetic analysis interpreted according to International System of Human Cytogenetic Nomenclature (ISCN) criteria. Molecular analysis for BCR-ABL was also performed for each patient. The additional cytogenetic abnormalities were then classified into major route abnormalities and minor route abnormalities. RESULTS: Out of the 528 newly diagnosed CML patients, 378 (71.6%) were males and 150 (28.4%) were females. The age of patients ranged between 18 to 74 years. Four hundred and ninety-eight (94.3%) patients showed Philadelphia chromosome on karyotyping while 30 (5.7%) were negative for the Philadelphia chromosome. On analysis of these 498 Philadelphia positive patients, additional cytogenetic aberrations were detected in 26 (4.9%) patients. Of these, 7 (1.3%) had major route abnormalities while 19 (3.6%) had minor route abnormalities. CONCLUSION: The frequency of additional chromosomal abnormalities in our study were not in accordance with previous local and international studies.

Pattern of Blood Stream Infections and their antibiotic susceptibility profile in a Neonatal intensive care unit of a tertiary care hospital; a current perspective.

OBJECTIVE: To determine the pattern of blood stream infections and their antibiotic susceptibility profile with infectivity predictors in a neonatal setting. METHODS: The descriptive cross-sectional study was conducted at the Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from December 1, 2016,to April 30, 2018, and comprised blood culture samples received in Bactec/BactAlert paediatric bottles from neonates aged 0-30 days admitted in the neonatal intensive care unit. The samples were processed as per the standard guidelines. Antibiotic susceptibility was checked as per guidelines of the Clinical and Laboratory Institute. VITEK 2 system was used for rapid identification and minimum inhibitory concentrations of the drugs. SPSS 24 was used for data analysis. RESULTS: Out of 640 samples, 172(27%) were culture-positive. Among them, 98(57%) were gramnegative rods, 50(29%) gram-positive cocci and 24(14%) were fungi. Of the 172 pathogens identified, Klebsiella pneumoniae was 39(22.7%) followed by Candida species 24(14%) and methicillin-resistant Coagulase-negative staphylococci 20(11.6%). Of Klebsiella pneumoniae isolates, 26(58%) were extended spectrum -lactamase producers. Among Acinetobacterbaumanii, 11(58%) were extensively drug resistant and Carbapenem-resistant strains were 20(91%). Also, 4(8%) isolates of Enterococcus faecium were vancomycin-resistant. CONCLUSIONS: Majority of the isolates causing blood stream infections in neonatal intensive care unit were multi drug resistant, posing a therapeutic challenge for the neo natologists .

Native valve dual pathogen endocarditis caused by Burkholderia cepacia and Aspergillus flavus - a case report.

INTRODUCTION: Infective endocarditis (IE) is an important clinical condition with significant morbidity and mortality among the affected population. A single etiological agent is identifiable in more than 90 % of the cases, however, polymicrobial endocarditis (PE) is a rare find, with a poor clinical outcome. Here we report a case of native valve dual pathogen endocarditis caused by Burkholderia cepacia and Aspergillus flavus in an immunocompetent individual. It is among unique occurrences of simultaneous bacterial and fungal etiology in IE. CASE PRESENTATION: A 30-year-old male was admitted to a cardiology institute with complaints of low grade intermittent fever and progressive shortness of breath for last two months. He was a known case of rheumatic heart disease and had suffered an episode of IE three years ago. On the basis of clinical presentation and the results of radiological investigations, a diagnosis of infective endocarditis was made. Paired blood samples for culture and sensitivity, sampled before the commencement of antimicrobial therapy, yielded growth of Burkholderia cepacia which was highly drug resistant. Sensitivity results-directed therapy consisting of tablet Trimethoprim-Sulfamethoxazole, two double-strength tablets 12 hourly, and Meropenem, 1 g IV every 8 h, was commenced. Despite mild relief of fever intensity, overall clinical condition did not improve and double valve replacement therapy was carried out. Excised valves were sent for microbiological analysis. Burkholderia cepacia was grown on tissue culture with a similar antibiogram to that previously reported from the blood culture of this patient. Direct microscopy of section of valvular tissue with 10 % KOH revealed abundant fungal hyphae. Patient serum galactomannan antigen assay was also positive. Histopathological examination of vegetations also revealed hyphae typical of species of the genus Aspergillus. The patient was successfully treated with meropenem, trimethoprim-sulfamethoxazole and voriconazole. CONCLUSION: The hallmark of successful treatment in this case was exact identification of pathogens, antibiogram-directed therapy and good liaison between laboratory experts and treating clinicians.

Spectrum of liposarcomas-a study of 106 cases.

BACKGROUND: Liposarcoma is a malignant tumour that arises in fat cells in deep soft tissue. This study was conducted to access the spectrum of liposarcomas METHODS: This descriptive study was conducted at Armed Forces Institute of Pathology (AFIP), Rawalpindi from 1st January 2008 to 31st December, 2012 and included all the cases diagnosed as liposarcomas. Records of the malignant tumors of soft tissue that presented during this period were analysed and out of this spectrum of liposarcomas were studied. RESULTS: A total of 19367 malignant tumours were diagnosed during study period. Out of these, 615 were malignant soft tissue tumours. Out of these 106 cases were liposarcoma with an overall frequency of 0.54% of the malignant neoplasm and 17.24% of soft tissue sarcomas .The age ranged from 26-85 years. Out of these 106 cases 77 were male and 29 were female with a male to female ratio of 2.6:1. The most common tumour seen was pleomorphic liposarcoma (42.5%) followed by myxoid liposarcoma (22.6%), dedifferentiated liposarcoma (19.8%) and well differentiated liposarcoma (15.1%). Fifty five (51.9%) of liposarcomas originated in lower extremity, the second most commonly involved site was upper extremity (22.6%) CONCLUSION: Pleomorphic Liposarcoma is the commonest liposarcoma of extremities and their frequency is much high in our population.

Malignant lymphomas in Pakistan according to the WHO classification of lymphoid neoplasms.

OBJECTIVE: To determine the spectrum of malignant lymphomas in our set up, according to the WHO classification. METHODS: All the cases diagnosed as malignant lymphoma, during the year 2005, were retrieved from the institution based tumour registry record and classified according to WHO criteria depending on the immunohistochemical results of a panel of lymphoma markers. RESULTS: The male to female ratio was 2.5:1 for almost all types of malignant lymphomas. The age range was 3 to 80 years. The frequency of Hodgkin's lymphoma, Burkitt's lymphoma and lymphoblastic lymphoma were higher amongst the children, whereas follicular lymphomas, mantle cell lymphoma and CLL/SLL were more frequently reported in 5th, 6th and 7th decades. Of the total cases 62% were nodal and 38% extranodal (majority in the GI tract). Non Hodgkin's lymphoma was more (73%) frequent than Hodgkin's disease. Mixed cellularity and nodular sclerosis were the main histological variants of Hodgkin's disease. CONCLUSIONS: Immunohistochemistry is not very frequently used in our set up and also at very few other centres. Therefore, its application should be encouraged to raise the quality of data on lymphoid neoplasms and contribute to their control.

The pathological spectrum of malignant testicular tumours in northern Pakistan.

OBJECTIVE: To determine the pattern of testicular malignant tumours in northern Pakistan. METHODS: The study included all malignant testicular tumours, diagnosed from January 2001 to June 2006. All histologically diagnosed malignant tumours of testis were retrieved from the case files. Basic epidemiological data regarding each case was collected from the request forms. The data was then analysed for the site of involvement, age distribution and histological types of tumours. RESULTS: During the study period a total of 107 patients had testicular malignancies, constituting 0.74% of all malignant tumours and 1.24% of all male malignancies diagnosed during this period. Peak incidence was in 30-40 years age group and a second peak was observed in the above 60 age group. Germ cell tumours (67%) were the most frequent histological type followed by non Hodgkin lymphoma; the latter particularly was responsible for the peak in the older age groups. Statistical analysis showed that there was no significant increase registration of testicular tumours as compared to our previous analysis. CONCLUSION: The study points out that the incidence of testicular cancer is low in our set up as in other Asian countries. The histological pattern is predictable except that the testicular non Hodgkin lymphoma is more contrary, to reported elsewhere and probably responsible for the second peak observed in the present series.