First report of successful Naegleria detection from environmental resources of some selected areas of Rawlakot, Azad Jammu and Kashmir, Pakistan

Naegleria belongs to the free-living amoeba family and is well-known as a human pathogen. It is recognized as etiological agent of primary amoebic meningoencephalitis involving central nervous system which always leads to death. To date, there is not a single report demonstrating Naegleria isolation and identification from environmental sources of Rawlakot, Azad Jammu and Kashmir Pakistan, and thus the aim of this study. Naegleria was isolated on non-nutrient agar plates seeded with heat killed E. coli and confirmed by morphological properties of the both stages of cyst or trophozoites. Furthermore, PCR was conducted along with direct sequencing of the PCR product for molecular identification. PCR and sequencing data verified the amplification of Naegleria sp. (07) and Vahlkampfia sp. (01) from both water and soil samples. Interestingly two species were successfully isolated and cultured on both 30 and 45°C. To the best of our knowledge this is the first report demonstrating the Naegleria isolation and molecular characterization from environmental sources of Rawlakot, Azad Jammu and Kashmir, Pakistan. The author is anxious for further evaluation of the pathogenic potential of the identified species and explores drinking water across Pakistan to investigate its quality and frequency of FLA, which might be a possible human hazard in future

Challenges and opportunities in the establishment of a hereditary breast cancer clinic at an academic medical center in a low-middle income country

It is now standard of care to offer genetic testing to patients at risk of hereditary breast cancer and make management decisions based on these results. Although great strides have been made in ensuring access to genetic testing and genetic counseling by establishing hereditary breast cancer clinics in well-resourced countries, these are essentially non-existent in low-middle income countries like Pakistan. We established a hereditary breast cancer clinic involving a multidisciplinary team, including a medical geneticist and a genetic counselor. Our efforts were based on consensus guidelines and included educating medical providers about the importance of genetic testing in breast cancer care and the mandatory presence of a genetics team member at the weekly Breast Tumor Board meeting. This resulted in an increase in the number of referrals of breast cancer patients for genetic testing. In this report, we describe the challenges we faced in setting up such a system in Pakistan and the measures to overcome them. There is a need to establish such hereditary breast cancer clinics, which can also be replicated at other centers in low-resource settings, to improve standardized assessment and management of the patients with hereditary breast cancer according to consensus guideline

eP039: Does Lynch syndrome cause predisposition to breast cancer? Experience from a hereditary breast cancer clinic in Pakistan

Background: Lynch Syndrome (LS), also known as hereditary non-polyposis colorectal cancer syndrome, is an autosomal dominant disorder caused by the presence of germline pathogenic (P) or likely pathogenic (LP) variants in DNA mismatch repair (MMR) genes, which include MLH1, MSH2, MSH6, PMS2, and EPCAM. Presence of a disease-causing variant in any of these MMR genes increases an individual’s lifetime risk of developing colorectal cancer (CRC) (61%), endometrial (57%), ovarian (38%), renal pelvis or ureter (28%), prostate (24%), breast (19%), small bowel (11%), gastric (9%), brain (8%), hepatobiliary cancer (4%) and pancreatic cancer (2%). It is still unclear whether LS causes a predisposition to breast cancer, with current data suggesting a risk \u3c 15%, which is close to the general population risk of 13%. Guidelines for high-risk surveillance and management of individuals with LS are thus not well-established for breast cancer, which at present is to be managed based on family history.Identification of germline disease-causing variants in MMR genes is thus pivotal for optimizing the treatment and surveillance of patients with LS and for the identification of at-risk family members, to reduce cancer-related morbidity and mortality. This is best done using Next Generation Sequencing (NGS) multi-gene panel testing, which increases the diagnostic yield, but also increases the chances of finding variants of uncertain significance (VUS). As the Pakistani population remains under-represented, the likelihood of finding a VUS is high, making it difficult to use these results for clinical decision-making.Objectives:• To study the presence of disease-causing variants as well as VUSs in MMR genes causing LS in a series of patients diagnosed with breast cancer who underwent genetic testing using a multi-gene NGS panel• To study the clinical characteristics of patients with LS who presented with breast cancer• To use immunohistochemistry (IHC) on breast tumour tissue samples to clarify whether VUSs in MMR genes in breast cancer patients are associated with loss of staining.Methods: Retrospective chart review of patients who visited the hereditary cancer (HBC) clinic and proceeded with Hereditary Breast and Gynecological Cancer multi-gene NGS panel testing (at Prevention Genetic and Invitae Genetics, USA) from May 2017 to Oct 2021, at an Academic Medical Centre, Aga Khan University Hospital, Karachi, Pakistan. IHC was performed using standard antibodies against the protein products of MLH1, MSH2, MSH6 and PMS2.Results and Discussion: A total of 460 breast cancer patients qualified and proceeded with testing, considering their personal and/or family history of disease, based on NCCN criteria. 94 patients (20.4%) had a positive genetic test result, which included Pathogenic (P) and Likely Pathogenic (LP) variants. Of the remaining patients, 167 (36.3%) had one or more VUS[s], and 199 (43.3%) had a negative genetic test result.Five out the total 460 patients (1.1%) or five of the 94 patients with positive results (5.3%), tested positive for LS, with an average age at diagnosis of 40 years. One patient had a personal history of colon cancer at age 38 and had then presented with breast cancer at age 43; and had a family history of colon cancer. Another patient who presented with breast cancer only, harbored a pathogenic variant in MSH6 as well as BRCA1, having a positive family history of breast and uterine cancer. In the remaining three patients, personal or family history was not indicative of any possible established link with LS, and their diagnoses would have been missed if MMR genes were not included in the multi-gene panel. Thus, multi-gene testing including MMR genes increased the diagnostic yield by 4.3% (4/94), even after excluding the patient with a personal history of colon cancer. Details of these patients are mentioned in Table 1.Out of the total 460 patients who underwent testing, 28 (6.1%) harbored a VUS in MMR genes, namely PMS2 (n=12), MSH2 (n=7), MSH6 (n=8) and one patient had a VUS in both PMS2 and MSH2. No VUS in MLH1 was identified in breast cancer patients.IHC was done on 18/28 (64.3%) and no loss of staining was observed on any tissue sample, possibly indicating that the variants are not disease-causing. We also observed six recurrent VUSs in unrelated patients, which included, (variant.1) v.1: MSH2 NM_000251.2 (p.L135V) (Rs193096019 MAF in South Asians=0.05%), v.2: MSH6 NM_000179.2 (p.L1356Dfs*4), (rs775836476, MAF in South Asians=0.04%), v.3: MSH6, (p.R911Q), (Rs761622304,MAF in South Asians= 0.006%), v.4: PMS2 NM_000535.5 (p.R294W) (rs563433235, MAF in South Asians= 0.03%), v.5: PMS2 (p.L454S) (absent from population database), v.6: PMS2 (p.D784N) (unreliable region coverage).It is worth noting that, v. 2, 4 and 6 have been reported in diseased individuals, while v.5 is a novel variant, absent from the gnomAD, highlighting the need for further functional studies to understanding the role of these variants in tumorigenesis.Conclusions: In our experience with genetic testing in the HBC, a small proportion of patients were diagnosed with Lynch Syndrome, some presenting with breast cancer alone, without a personal or family history of LS associated tumors. This may justify the need to include MMR genes in multi-gene panels being offered to breast cancer patients. VUSs in these genes remain challenging, especially in underrepresented populations, and IHC may be a way to at least partially clarify their significance. More ethno-specific genomic studies, as well as better functional studies are required provide better clinical care

Association between Ki-67 proliferative index and oncotype-Dx recurrence Score in hormone receptor-positive, HER2-negative early breast cancers. A systematic review of the literature

Background: Oncotype-Dx (ODx) is a 21-gene assay used as a prognostic and predictive tool for hormone receptor (HR)-positive and human epidermal growth factor receptor 2 (HER2)-negative, node-negative, or 1 to 3 lymph node-positive early breast cancers (EBCs). The cost of the test, which is not available in low-middle income countries (LMICs), is not within the means of most individuals. The Ki-67 index is a marker of tumor proliferation that is cost-effective and easily performed and has been substituted in many cases to obtain prognostic information.Objective: We aimed to identify the correlation between the ODx recurrence score (RS) and the Ki-67 index in HR-positive EBCs and to determine whether Ki-67, like the ODx, can help facilitate clinical decision-making.Design: Systematic review correlating Ki-67 index and ODx in HR-positive and HER2-negative EBCs as per Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.Data sources and methods: We searched different databases between January 2010 and May 2023 and included retrospective/prospective cohorts, clinical trials, case-control, and cross-sectional studies involving HR-positive and HER2-negative EBCs correlating the Ki-67 index and ODx RS categories.Results: Of the 18 studies included, 16 indicated a positive or weakly positive correlation between ODx and the Ki-67 index. The combined P value of the included studies is P = .000), which shows a statistical significance between the 2. Our review also discusses the potential of machine learning and artificial intelligence (AI) in Ki-67 assessment, offering a cost-effective and reproducible alternative.Conclusion: Even although there are limitations, studies indicate a favorable association between ODx and the Ki-67 index in specific situations. This implies that Ki-67 can offer important predictive details, especially regarding the likelihood of relapse in HR-positive EBC. This is particularly significant in LMICs where financial constraints often hinder the availability of costly diagnostic tests

eP077 - Germline pathogenic variants in Pakistani patients evaluated at a hereditary breast cancer clinic

Background: Breast cancer is the most common malignancy, impacting over 1.5 million women (25% of all women with cancer) every year, accounting for the highest number of cancer-related deaths in women globally. (Sun et al., 2017) (WHO, 2021).Hereditary breast cancer (HBC), an important subset of breast cancer, accounts for 5–10% of total cases. (Sun et al., 2017) These cases are attributed to pathogenic (P) and likely pathogenic (LP) germline variants in genes that have a predisposition for breast cancer. However, in Low Middle-Income Countries (LMICs), the population-specific risk of hereditary breast cancer in different ethnicities and the correlation with clinical characteristics remains unexplored.Objective:• To see the spectrum of variants beyond BRCA1/2 in Pakistani breast cancer patients.• To compare the clinical characteristics of patients who test positive, with patients who have variants of uncertain significance (VUS) and negative results.• To show preliminary population specific data that can guide tailored genetic testing criteria in a resource-limited country.Methods: A retrospective chart review of 284 patients who visited the hereditary breast cancer (HBC) clinic and proceeded with multi-gene panel testing (Invitae Genetics, USA) from May 2017 to April 2020. Descriptive and inferential statistics are used to analyze clinical characteristics of patients with positive results, comparing with patients with VUS and negative test results. Fisher’s exact, Pearson’s chi-squared tests and Logistic regression analysis were used for categorical variables and Wilcoxon rank-sum test for quantitative variables were used. For comparation between two independent groups, Mann-Whitney test was performed. Results were considered significant at a p value of \u3c0.05.Results and Discussion: Positive results included results for patients with pathogenic and likely pathogenic variants. Out of 284 patients, 22% (63/284) tested positive, 37% (104/284) had a VUS and 41% (117/ 284) had a negative result. Fifty-five percent of the positive patients were in either BRCA1 or BRCA2, while the other 45% of the positive results were attributed to other genes. The spectrum of the identified variants is summarized in Table 1. Patients with positive results had a younger age at diagnosis compared with those who had a VUS or a negative result; mean age (in years) = 38, IQR (32–48) vs 45, (37–51) vs 44 (38–50), (p value = 0.002). 73% of the positive results were found in patients under the age of 44. Having a positive result showed a positive association with triple negative breast cancer (TNBC) as well as higher disease grade (p = 0.024), using multi-variate analysis. Patients with TNBC were almost 3 times more likely to harbor a P or LP variant (OR = 2.8, CI = 1.42–5.48 p = 0.003). Of the patients who tested positive for BRCA1 or BRCA2, 67% had a diagnosis of TNBC. Of all patients with positive results, 25% of patients had a negative family history of breast and/or related cancers. The likelihood of the genetic test being positive was two times higher if there was a family history of cancer, as compared to no family history of cancer (OR = 2.13, Cl = 1.12–4.08, p = 0.021).Conclusion: In our HBC clinic, we observed that our rate of positive results was twice of what is reported in populations of Caucasian descent. This could either be due to referral bias, or a true higher rate of hereditary breast cancer in our population. The importance of expanded, multi-gene panel testing is highlighted by the fact that almost half of the patients had P or LP variants in genes other than BRCA1/2, and that our test positivity rate would have only been 10% if only BRCA1/2 testing was done. Consistent with data from other countries, we also found that in our population, having TNBC or a young age at diagnosis increased the likelihood of a positive result. Up to a quarter of patients with positive result had a negative family history. As the database expands and protocol-driven referrals are made across the country, our insight about the genetic architecture of hereditary breast cancer in our population will continue to increase

Delineating Novel and Known Pathogenic Variants in TYR, OCA2 and HPS-1 Genes in Eight Oculocutaneous Albinism (OCA) Pakistani Families

Oculocutaneous albinism (OCA) is associated with a wide range of clinical presentations and has been categorized with syndromic and non-syndromic features. The most common causative genes in non-syndromic OCA are TYR and OCA2 and HSP1 is in the syndromic albinism. The objective of this study was to identify pathogenic variants in congenital OCA families from Pakistan. Eight consanguineous families were recruited, and clinical and ophthalmological examination was carried out to diagnose the disease. Whole blood was collected from the participating individuals, and genomic DNA was extracted for sequencing analysis. TruSight one-panel sequencing was carried out on one affected individual of each family, and termination Sanger sequencing was carried out to establish the co-segregation of the causative gene or genes. In silico analysis was conducted to predict the causative pathogenic variants. Two families were found to have novel genetic pathogenic variants, and six families harbored previously reported variants. One novel compound heterozygous pathogenic variant in the TYR gene, c.1002delA; p.Ala335LeufsTer20, a novel frameshift deletion pathogenic variant and c.832C>T; and p.Arg278Ter (a known pathogenic variant) were found in one family, whereas HPS1; c.437G>A; and p.Trp146Ter were detected in another family. The identification of new and previous pathogenic variants in TYR, OCA2, and HPS1 genes are causative of congenital OCA, and these findings are expanding the heterogeneity of OCA

Spectrum of germline pathogenic variants using a targeted next generation sequencing panel and genotype-phenotype correlations in patients with suspected hereditary breast cancer at an academic medical centre in Pakistan

Background: Breast cancer is the most common malignancy in women, affecting over 1.5 million women every year, which accounts for the highest number of cancer-related deaths in women globally. Hereditary breast cancer (HBC), an important subset of breast cancer, accounts for 5-10% of total cases. However, in Low Middle-Income Countries (LMICs), the population-specific risk of HBC in different ethnicities and the correlation with certain clinical characteristics remain unexplored.Methods: Retrospective chart review of patients who visited the HBC clinic and proceeded with multi-gene panel testing from May 2017 to April 2020. Descriptive and inferential statistics were used to analyze clinical characteristics of patients. Fisher\u27s exact, Pearson\u27s chi-squared tests and Logistic regression analysis were used for categorical variables and Wilcoxon rank-sum test were used for quantitative variables. For comparison between two independent groups, Mann-Whitney test was performed. Results were considered significant at a p value of \u3c 0.05.Results: Out of 273 patients, 22% tested positive, 37% had a VUS and 41% had a negative genetic test result. Fifty-five percent of the positive patients had pathogenic variants in either BRCA1 or BRCA2, while the remaining positive results were attributed to other genes. Patients with a positive result had a younger age at diagnosis compared to those having a VUS and a negative result; median age 37.5 years, IQR (Interquartile range) (31.5-48). Additionally, patients with triple negative breast cancer (TNBC) were almost 3 times more likely to have a positive result (OR = 2.79, CI = 1.42-5.48 p = 0.003). Of all patients with positive results, 25% of patients had a negative family history of breast and/or related cancers.Conclusions: In our HBC clinic, we observed that our rate of positive results is comparable, yet at the higher end of the range which is reported in other populations. The importance of expanded, multi-gene panel testing is highlighted by the fact that almost half of the patients had pathogenic or likely pathogenic variants in genes other than BRCA1/2, and that our test positivity rate would have only been 12.8% if only BRCA1/2 testing was done. As the database expands and protocol-driven referrals are made across the country, our insight about the genetic architecture of HBC in our population will continue to increase

Novel nonsense variants in SLURP1 and DSG1 cause palmoplantar keratoderma in Pakistani families

Inherited palmoplantar keratodermas (PPKs) are clinically and genetically heterogeneous and phenotypically diverse group of genodermatoses characterized by hyperkeratosis of the palms and soles. More than 20 genes have been reported to be associated with PPKs including desmoglein 1 (DSG1) a key molecular component for epidermal adhesion and differentiation. Mal de Meleda (MDM) is a rare inherited autosomal recessive genodermatosis characterized by transgrediens PPK, associated with mutations in the secreted LY6/PLAUR domain containing 1 (SLURP1) gene.This article is freely available online via Open Access. Click on the Publisher URL to access the full-text via the publisher's site