Reliability and Variability of Ki-67 Digital Image Analysis Methods for Clinical Diagnostics in Breast Cancer.

Ki-67 is a nuclear protein associated with proliferation, and a strong potential biomarker in breast cancer, but is not routinely measured in current clinical management owing to a lack of standardization. Digital image analysis (DIA) is a promising technology that could allow high-throughput analysis and standardization. There is a dearth of data on the clinical reliability as well as intra- and interalgorithmic variability of different DIA methods. In this study, we scored and compared a set of breast cancer cases in which manually counted Ki-67 has already been demonstrated to have prognostic value (n = 278) to 5 DIA methods, namely Aperio ePathology (Lieca Biosystems), Definiens Tissue Studio (Definiens AG), Qupath, an unsupervised immunohistochemical color histogram algorithm, and a deep-learning pipeline piNET. The piNET system achieved high agreement (interclass correlation coefficient: 0.850) and correlation (R = 0.85) with the reference score. The Qupath algorithm exhibited a high degree of reproducibility among all rater instances (interclass correlation coefficient: 0.889). Although piNET performed well against absolute manual counts, none of the tested DIA methods classified common Ki-67 cutoffs with high agreement or reached the clinically relevant Cohen's κ of at least 0.8. The highest agreement achieved was a Cohen's κ statistic of 0.73 for cutoffs 20% and 25% by the piNET system. The main contributors to interalgorithmic variation and poor cutoff characterization included heterogeneous tumor biology, varying algorithm implementation, and setting assignments. It appears that image segmentation is the primary explanation for semiautomated intra-algorithmic variation, which involves significant manual intervention to correct. Automated pipelines, such as piNET, may be crucial in developing robust and reproducible unbiased DIA approaches to accurately quantify Ki-67 for clinical diagnosis in the future.

Accuracy of genotype MTBDRplus line probe assay in patients with tuberculous pleural effusion: comparison with clinical and culture based diagnosis.

Background: Pleural tuberculosis (TB) diagnosis is challenging due to paucibacillary disease. Diagnostic accuracy of GenoType MTBDRplus Line Probe Assay (MTBDRplus) has been evaluated in this study for pleural TB diagnosis.Objective: To evaluate diagnostic accuracy of MTBDRplus for pleural TB diagnosis compared to clinical and microbiological diagnosis.Methods: This prospective study was conducted at the Aga Khan University, Pakistan. Pleural fluid from 203 suspected pleural TB patients was collected and tested for smear, culture and MTBDRplus. Diagnostic accuracy of MTBDRplus was determined using clinical diagnosis and culture as the gold standard.Results: Out of 203 TB suspect patients, MTBDRplus, culture and smear were positive in 14 (6. 9%), 27 (13.3%) and 4 (1.9%) cases, respectively. A total of 106/203 patients (27 culture positive and 79 culture negative) successfully completed TB treatment. Considering clinical diagnosis as gold standard, sensitivity of MTBDRplus was 13.2%; 95% CI (7.4-21.2%) and specificity was 100%; 95% CI (96.1-100%). The sensitivity and specificity of MTBDRplus in culture positive samples were 44.4%; 95% CI (25.5-64.7%) and 98.9%; 95% CI (95.9-99.9%), respectively. Excluding indeterminate results, MTBDRplus accurately detected isoniazid sensitivity in 5/6 and rifampicin sensitivity in 6/6 cases.Conclusion: MTBDRplus had a low sensitivity of 13.2% in clinically diagnosed and 44% in culture-confirmed pleural TB patients and therefore could not be included in most diagnostic algorithms. Due to a higher sensitivity than smear, MTBDRplus may have a role in tuberculous pleural effusion diagnosis if it is positive pending culture results and pleural biopsy.

Esophageal Variceal Band Ligation Interval and Number Required for the Obliteration of Varices: A Multi-center Study from Karachi, Pakistan.

Introduction Esophageal variceal band ligation (EVBL) is the best form of treatment for variceal bleeding. The frequency of EVBL for the eradication of esophageal varices has no consensus. We evaluated the number and interval of EVBL sessions required for the obliteration of esophageal varices. Methods Esophagogastric varices were treated endoscopically with band ligation on initial presentation and then every after three weeks till the obliteration of the varices. Endoscopic band ligation consists of placing rubber elastic bands on large varices. Frequencies were calculated for qualitative variables and mean ± standard deviations for continuous variables. Results A total of 107 cases with esophagogastric varices were enrolled. Out of them, seven patients with small esophageal varices and large fundal varices were excluded. The remaining 100 with large esophageal varices had EVBL performed. The second session of EVBL was done in 46 patients with large esophageal varices. The third session of EVBL for the obliteration of esophageal varices required in 20 patients with large esophageal varices and the fourth session was required in only two patients. The total sessions required for the complete obliteration for esophageal varices were 2±1. Only one patient developed post-EVBL bleeding one week after band ligation. Conclusion Esophageal variceal ligation was a safe and well-tolerated procedure performed at three-week intervals in patients with large esophageal varices. On average, two to three sessions of EVBL are required for the complete obliteration of esophageal varices.

Epidemiology, Clinical Characteristics, and Management Status of Hepatitis B: A Cross-sectional Study in a Tertiary Care Hospital at Karachi, Pakistan.

Background Hepatitis B virus (HBV) infection is a serious health problem in Pakistan. In view of the serious socioeconomic consequences, identifying patient characteristics and the current treatment for the disease will enhance HBV regulation and its medical management. Aims To describe the epidemiology, clinical characteristics, and current management status of patients infected by HBV. Methods We undertook an observational, cross-sectional, and epidemiological study at the Jinnah Postgraduate Medical Centre, Karachi, during the period from January 2014 to November 2017. Male and female patients of any age and with documentation for an HBV infection were eligible for inclusion in the study. An HBV infection was defined as a positive hepatitis B surface antigen test. Results A total of 500 patients were analyzed. The mean age at presentation was 29.86±13.68 years. The majority of the patients (25.6%) were ethnically Sindhi followed by Pathan (24.4%), indicating a high prevalence among the rural-based population of Pakistan. The mean duration of the disease was 3.51±4.46 years. The most common cause for the spread was positive family history (40.4%) followed by roadside barbers (30.0%). Most patients were Child-Pugh (CP) class A (84.6%) and the median Modified End-Stage Liver Disease (MELD) score was 7. Upper gastrointestinal bleeding was the most frequent hepatic complication (6.2%). Antiviral medications had been received by 18.6% of patients previously. Peg-interferon (6.0%) was the major antiviral medication prescribed to treatment-experienced patients. Conclusions This observational, real-life study has identified some gaps between clinical practice and guideline recommendations in Pakistan. To achieve better health outcomes, several improvements, such as disease monitoring and optimizing antiviral regimens, should be made to improve disease management.

Efficacy and Safety of Sofosbuvir and Ribavirin for Treating Chronic Hepatitis C, Genotype 3: Experience of a Tertiary Care Hospital at Karachi, Pakistan.

Background It is estimated that approximately 10 million individuals in Pakistan are infected with hepatitis C virus (HCV). Historically, it was very difficult not just to cure but even treat HCV as available options did not have desirable outcomes. However, the approval of directly acting antiviral (DAA) drugs has revolutionized treatment and management. These are specific proteases and polymerase inhibitors with profound capability for accomplishing elimination and overtime eradication of the virus. Objective The aim of this study was to evaluate the efficacy and safety of sofosbuvir (SOF) in combination with ribavirin (RIB) for the treatment of chronic hepatitis C virus with genotype 3. Materials and methods This prospective observational study was conducted at the gastroenterology section of Medical Unit IV, Jinnah Post-graduate Medical Center, Karachi and Medical Unit II, Dow University of Health Sciences, Ojha Campus, Karachi from January 2016 to December 2016. Patients aged 18 years or older of either gender having chronic active HCV infection as demonstrated by a positive Anti-HCV (ELISA) test and a qualitative polymerase chain reaction (PCR) analysis along with genotype analysis showing only type 3 were inducted into the study. Treatment was initiated with either 12-week or 24-week regimen of SOF 400 mg once daily along with weight-adjusted RIB orally. Successful treatment was indicated by the elimination of the virus, i.e., undetectable viral load/levels by PCR qualitative analysis. Rapid virological response (RVR), end of treatment response (ETR), and sustained virological response (SVR) were defined as the undetectable viral load at four, 12, and 24 weeks, respectively. Results A total of 300 patients were inducted into the study, predominantly female (57%). The mean age of presentation was 41.14 ± 11.48, and most (70.33%) were treatment naïve. The mean alanine transaminase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (GGT) levels at presentation were 41.89 ± 46.23 IU/l, 68.57 ± 83.62 IU/l, and 54.52 ± 77.57 IU/l, respectively. ALT, AST, and GGT levels at 24 weeks were 33.84 ± 13.60 IU/l, 32.44 ± 16.16 IU/l, and 37.59 ± 22.41 IU/l, respectively, showing significant improvement. ETR was achieved in 99.1% (209) treatment-naïve patients and 98.9% (88) treatment-experienced patients. SVR rates were almost similar with 98% (208) achieving it in the treatment-naïve group and 96.6% (86) achieving it in the treatment-experienced group. Conclusion SOF in combination with RIB is safe and remarkably efficacious in the treatment of chronic HCV, genotype 3. Not only is this regimen associated with the elimination of viral replication but it also improved transaminase levels. Outcomes are rarely, if ever, affected by previous use of antiviral medications.

Accuracy of Specimen Radiography in Assessing Complete Local Excision with Breast-Conservation Surgery

Objective: The aim of this study was to evaluate the accuracy of "X- ray examination of surgically resected specimen'' in assessing complete local excision (CLE). Materials and Methods: In this retrospective cross sectional study, data were collected for all female breast cancer cases who underwent breast-conserving surgery after needle localization of mammographically visible disease. Males, patients with mammographically invisible disease and cases with benign or inconclusive histopathology, those undergoing modified radical mastectomy and individuals with dense breast parenchyma were excluded. We evaluated radiography of resected specimens to assess margin spiculation, distance of mass/microcalcification from the excised margin, presence of a mass, and presence of any adjacent microcalcification, Other features including mass size, nuclear grade and patient's age were also recorded and all were analyzed for any association with CLE. Results: Absence of adjacent microcalcification and the presence of a mass on radiographs showed significant associations with CLE, but no links were evident with other features. Specimen radiography was found to be a sufficient tool to predict CLE with a positive predictive value of 83.3%, a sensitivity of 80.7% and a specificity of 81%. Conclusion: Specimen radiography is an important and sensitive tool to predict CLE.

Evofosfamide for the treatment of human papillomavirus-negative head and neck squamous cell carcinoma.

Evofosfamide (TH-302) is a clinical-stage hypoxia-activated prodrug of a DNA-crosslinking nitrogen mustard that has potential utility for human papillomavirus (HPV) negative head and neck squamous cell carcinoma (HNSCC), in which tumor hypoxia limits treatment outcome. We report the preclinical efficacy, target engagement, preliminary predictive biomarkers and initial clinical activity of evofosfamide for HPV-negative HNSCC. Evofosfamide was assessed in 22 genomically characterized cell lines and 7 cell line-derived xenograft (CDX), patient-derived xenograft (PDX), orthotopic, and syngeneic tumor models. Biomarker analysis used RNA sequencing, whole-exome sequencing, and whole-genome CRISPR knockout screens. Five advanced/metastatic HNSCC patients received evofosfamide monotherapy (480 mg/m2 qw × 3 each month) in a phase 2 study. Evofosfamide was potent and highly selective for hypoxic HNSCC cells. Proliferative rate was a predominant evofosfamide sensitivity determinant and a proliferation metagene correlated with activity in CDX models. Evofosfamide showed efficacy as monotherapy and with radiotherapy in PDX models, augmented CTLA-4 blockade in syngeneic tumors, and reduced hypoxia in nodes disseminated from an orthotopic model. Of 5 advanced HNSCC patients treated with evofosfamide, 2 showed partial responses while 3 had stable disease. In conclusion, evofosfamide shows promising efficacy in aggressive HPV-negative HNSCC, with predictive biomarkers in development to support further clinical evaluation in this indication.

Evaluation of Common Risk Factors for Breast Carcinoma in Females: a Hospital Based Study in Karachi, Pakistan.

BACKGROUND: Breast malignancies are one of the leading causes of deaths in females worldwide. There are a number of risk factors associated with breast cancer but in Karachi Pakistan there are insufficient data available. MATERIALS AND METHODS: A case control study was conducted on females in age group between 30-80 years. This study was accomplished by retrospective data collection in Aga Khan University Hospital Karachi, Pakistan. A total of 108 females with primary malignancy of breast were included along with 108 matched controls. Relationship of various factors with disease was studied using logistic regression to calculate odds ratios with 95 % confidence intervals. RESULTS: A total of 14 variables were analyzed and based on and 7 were found to be risk factors: old age, family history of breast cancer, family history of other carcinomas, personal history of breast carcinoma, early age of menarche, older age of mother at first delivery and lower number of children. Five factors, parity, breast feeding, history of oral contraceptive pills intake, past history of oophorectomy and hysterectomy showed protective associations. One variable, use of hormonal replacement therapy, showed a controversial link and one other, marital status, was not significant in this study. CONCLUSIONS: It is concluded that most of the well-known risk factors for breast cancer are also associated with the disease in the female population of Karachi, Pakistan. High risk patients should be the focus with the help of this study so that screening can be more effective for early diagnosis before clinically evident breast malignancy.

The level and distribution of the GABA(B)R1 and GABA(B)R2 receptor subunits in the rat's inferior colliculus.

The type B γ-aminobutyric acid receptor (GABA(B) receptor) is an important neurotransmitter receptor in the midbrain auditory structure, the inferior colliculus (IC). A functional GABA(B) receptor is a heterodimer consisting of two subunits, GABA(B)R1 and GABA(B)R2. Western blotting and immunohistochemical experiments were conducted to examine the expression of the two subunits over the IC including its central nucleus, dorsal cortex, and external cortex (ICc, ICd, and ICx). Results revealed that the two subunits existed in both cell bodies and the neuropil throughout the IC. The two subunits had similar regional distributions over the IC. The combined level of cell body and neuropil labeling was higher in the ICd than the other two subdivisions. Labeling in the ICc and ICx was stronger in the dorsal than the ventral regions. In spite of regional differences, no defined boundaries were formed between different areas. For both subunits, the regional distribution of immunoreactivity in the neuropil was parallel to that of combined immunoreactivity in the neuropil and cell bodies. The density of labeled cell bodies tended to be higher but sizes of cell bodies tended to be smaller in the ICd than in the other subdivisions. No systematic regional changes were found in the level of cell body immunoreactivity, except that GABA(B)R2-immunoreactive cell bodies in the ICd had slightly higher optic density (OD) than in other regions. Elongated cell bodies existed throughout the IC. Many labeled cell bodies along the outline of the IC were oriented in parallel to the outline. No strong tendency of orientation was found in labeled cell bodies in ICc. Regional distributions of the subunits in ICc correlated well with inputs to this subdivision. Our finding regarding the contrast in the level of neuropil immunoreactivity among different subdivisions is consistent with the fact that the GABA(B) receptor has different pre- and postsynaptic functions in different IC regions.