Uracil as a biomarker for spatial pyrimidine metabolism in the development of gingivobuccal oral squamous cell carcinoma.

No biomarker has yet been identified that allows accurate diagnosis and prognosis of oral cancers. In this study, we investigated the presence of key metabolites in oral cancer using proton nuclear magnetic resonance (NMR) spectroscopy to identify metabolic biomarkers of gingivobuccal oral squamous cell carcinoma (GB-OSCC). NMR spectroscopy revealed that uracil was expressed in 83.09% of tumor tissues and pyrimidine metabolism was active in GB-OSCC; these results correlated well with immunohistochemistry (IHC) and RNA sequencing data. Based on further gene and protein analyses, we proposed a pathway for the production of uracil in GB-OSCC tissues. Uridinetriphosphate (UTP) is hydrolyzed to uridine diphosphate (UDP) by CD39 in the tumor microenvironment (TME). We hypothesized that UDP enters the cell with the help of the UDP-specific P2Y6 receptor for further processing by ENTPD4/5 to produce uracil. As the ATP reserves diminish, the weakened immune cells in the TME utilize pyrimidine metabolism as fuel for antitumor activity, and the same mechanism is hijacked by the tumor cells to promote their survival. Correspondingly, the differential expression of ENTPD4 and ENTPD5 in immune and tumor cells, respectively, indicatedtheir involvement in disease progression. Furthermore, higher uracil levels were detected in patients with lymph node metastasis, indicating that metastatic potential is increased in the presence of uracil. The presence of uracil and/or expression patterns of intermediate molecules in purine and pyrimidine pathways, such asCD39, CD73, and P2Y6 receptors together with ENTPD4 and ENTPD5, hold promise as biomarker(s) for oral cancer diagnosis and prognosis.

Detection of Axillary Lymph Node Involvement in Early-Stage Breast Cancer: Comparison between Staging 18F-2-Fluoro-2-Deoxy-D-Glucose Positron Emission Tomography-Computed Tomography Scans, Mammography, and Sentinel Lymph Node Biopsy.

Aims: The aim of this study was to evaluate the role of 18F-2-fluoro-2-deoxy-D-glucose (18F-FDG) positron emission tomography-computed tomography (PET-CT) scan in the detection of axillary lymph node (ALN) involvement and comparison with sentinel lymph node biopsy (SLNB) in operable early-stage breast cancer (EBC). Settings and Design: It is a retrospective analysis of staging PET-CT scan of EBC. Methods: A total of 128 patients with histopathologically proven breast cancer (BC) were included in the study. Preoperative mammography supplemented with ultrasonography and staging 18F-FDG PET-CT scan was done for all patients. Surgery was done within 30 (mean ± standard deviation = 13.8 ± 10.5) days of staging. SLNB was performed in patients without PET-positive ALNs. All patients with positive sentinel nodes and PET-positive ALNs underwent axillary lymph node dissection (ALND). Statistical Analysis Used: The comparison between categorical variables was made by Chi-square/Fisher's exact test as applicable. For continuous variables comparisons, Student's t-test and one-way analysis of variance tests were used. Results: Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of PET-CT scan for detection of ALN involvement were 41.7%, 93.2%, 92.1%, and 45.6%, respectively. Sensitivity, specificity, PPV, and NPV of mammography were 84.5%, 54.5%, 78.0%, and 68.6%, respectively. Sixteen out of 46 (34.7%) patients with negative ALNs in PET-CT scan finally showed involvement in histopathology report after SLNB resulting in upstage of the disease. The size of tumor deposits in sentinel nodes was significantly smaller than PET-positive ALNs (P = 0.01). Our observations correlate with the results of earlier studies published in the literature. Conclusions: 18F-FDG PET-CT scan cannot substitute SLNB for ALN screening in EBC. The limitations are most marked in smaller and micrometastatic tumor deposits in ALNs and may be attributed to limitations of PET resolution. However, PET-positive nodes showed good specificity for disease involvement in our study. Therefore, ALND can safely be performed by omitting SLNB in such cases.

Overexpression of CD73 is associated with recurrence and poor prognosis of gingivobuccal oral cancer as revealed by transcriptome and deep immune profiling of paired tumor and margin tissues.

BACKGROUND: For various cancers, differences in response to treatment and subsequent survival period have been reported to be associated with variation in immune contextures. AIM: We sought to identify whether such association exists in respect of gingivobuccal oral cancer. MATERIALS AND METHODS: We performed deep immune profiling of tumor and margin tissues collected from 46 treatment naïve, Human Papillomavirus (HPV) negative, patients. Each patient was followed for 24 months and prognosis (recurrence/death) noted. Key findings were validated by comparing with TCGA-HNSC cohort data. RESULTS: About 28% of patients showed poor post-treatment prognosis. These patients exhibited a high probability of recurrence even within 1 year and death within 2 years. There was restricted immune cell infiltration in tumor, but not in margin, among these patients. Reduced expression of eight immune-related genes (IRGs) (NT5E, THRA, RBP1, TLR4, ITGA6, BMPR1B, ITGAV, SSTR1) in tumor strongly predicted better quality of prognosis, both in our patient cohort and in TCGA-HNSC cohort. Tumors of patients with better prognosis were associated with (a) lower CD73+ cells with concomitant lower expression level of NT5E/CD73, (b) higher proportions of CD4+ and CD8+ T cells, B cells, NK cells, M1 macrophages, (c) higher %Granzyme+ cells, (d) higher TCR and BCR repertoire diversities. CD73 expression in tumor was associated with low CD8+ and CD4+ T cells, low immune repertoire diversity, and advanced cancer stage. DISCUSSION AND CONCLUSION: High infiltration of anti-tumor immune cells in both tumors and margins results in good prognosis, while in patients with minimal infiltration in tumors in spite of high infiltration in margins results in poor prognosis. Targeted CD73 immune-checkpoint inhibition may improve clinical outcome.

Oncologic safety of breast conservation following NACT in women with locally advanced breast cancer.

Introduction: Breast conservation surgery (BCS) is the accepted standard of treatment for early breast cancer, with evidence from randomized controlled and population-based studies. The oncological outcome of BCS in locally advanced breast cancer (LABC) is mainly available from retrospective series with a small sample size and a shorter follow-up duration. Methods: A retrospective observational study of 411 non-metastatic LABC patients who received neoadjuvant chemotherapy (NACT) followed by surgery from 2011 to 2016. We retrieved the data from a prospectively maintained database and electronic medical records. Survival data were analyzed by Kaplan-Meier curves and Cox regression using Statistical Package for the Social Sciences 25 and STATA 14. Results: 146/411 (35.5%) women had BCS with a margin positivity rate of 3.42%. With a median follow-up of 64 months (IQR 61, 66), the local relapse rate was 8.9% in BCS and 8.3% after mastectomy. The estimated 5-year locoregional recurrence-free survival (LRFS), recurrence-free survival (RFS), distant disease-free survival (DDFS) and overall survival (OS) rates of BCS were 86.9%, 63.9%, 71% and 79.3%, and 90.1%, 57.9%, 58.3% and 71.5% in the mastectomy group. On univariate analysis, BCS showed superior survival outcomes compared to mastectomy (unadjusted HR (95% CI) for RFS: 0.70 (0.50-1), DDFS: 0.57 (0.39-0.84), OS: 0.58 (0.36-0.93)). After adjusting for age, cT stage, cN stage, poorer chemotherapy response (ypT0/is, N0) and radiotherapy, BCS and mastectomy groups were found comparable in terms of LRFS (HR: 1.1, 0.53-2.3), DDFS (HR: 0.67, 0.45-1.01), RFS (HR: 0.80, 0.55-1.17) and OS (HR: 0.69, 0.41-1.14). Conclusion: BCS is technically feasible in LABC patients. LABC patients who respond well to NACT can be offered BCS without compromising survival outcomes.

Subcategorization of Perineural Invasion and Its Impact on Survival in Patients with Oral Squamous Cell Carcinoma.

BACKGROUND: Perineural invasion (PNI) is  recognized as a poor prognostic factor in oral squamous cell carcinoma (OSCC). However, the prognostic significance of further histologic  subcategorization of PNI is inconclusive. In this study, we determined the prognostic relevance of histologic subcategories of PNI and their correlation with the presence of other clinical and pathological parameters METHODS: This is a retrospective study of 207 homogeneously treated OSCC patients with histologically documented PNI from a single center. Univariate and multivariate survival outcomes, namely, local recurrence-free survival (LRFS), disease-free survival (DFS), and overall survival (OS) of patients with various subcategories of PNI,namely- number of foci, size of the involved nerve, extratumoral or intratumoral extent, and intraneural or perineural location-were determined. RESULTS: Within the histologic subcategories of PNI, tongue primary and presence of lymph node metastasis correlated significantly with the number of nerves involved with PNI. Larger size of involved nerve correlated with advanced tumor stage. Number of foci, extent, and location of PNI were not prognostically significant except size of the involved nerve which showed an inverse correlation with disease outcome as involvement of larger nerves displayed better outcomes in terms of DFS and LRFS but not of OS on multivariate analysis. Addition of adjuvant chemotherapy to radiotherapy emerged as a significant predictor of improved LRFS, DFS, and OS. CONCLUSIONS: Histologic subcategorization of PNI did not have prognostic relevance in our study. Involvement of even small nerves was associated with poor prognosis. Addition of chemoradiation was seen to improve prognosis.

Impact of review of histopathology specimens at a tertiary oncology hospital in Eastern India-lessons learnt.

A central review of histopathology specimens at tertiary oncology hospitals is important for optimum patient care in the modern era of personalised medicine. The challenges of healthcare delivery and access to ancillary investigations faced by a pathologist from the Indian subcontinent are different from the western world. We undertook an audit to analyse the differences of opinion between the diagnosis offered at peripheral hospitals and a tertiary oncology hospital in Eastern India. By analysing the differences, common pitfalls and diagnostic discrepancies are identified which need to be addressed in future. This audit also highlights the need of setting up of tertiary oncology diagnostic centres to help both peripheral pathologists and cancer care clinicians like a hub and spoke model. This is most needed for haematopathology, soft tissue and gynaecologic oncology where the need of ancillary investigations is high.

Diagnostic performance and survival outcome following sentinel lymph node biopsy in breast cancer patients from a tertiary cancer centre in India.

Background: Sentinel lymph node biopsy (SLNB) has replaced axillary lymph node dissection (ALND) for axillary staging in early node-negative breast cancer (BC) patients in developed countries. However, in resource-constrained developing countries, adoption of SLNB is slow due to logistic issues and lack of outcome data from non-screened BC cohort. Therefore, we aim to report diagnostic performance, surgical morbidity and survival outcome of SLNB in BC patients from a tertiary care cancer centre in India. Methodology: 1,521 consecutive early node-negative T1-3N0 BC patients having SLNB from 2011 to 2020 were included in the study. Data were retrieved from the institutional Redcap database and electronic medical records. Analysis was done using Stata14. Results: SLNB was done by dual dye (methylene blue (MB) + radioisotope (RI)/indo cyanine green (ICG)) in 57.7%, MB only in 39.3%, and RI alone in 3% of patients. The identification rate (IR) and SLNB positivity rate were 96% and 27.7%, respectively. IR was highest (98%) with MB + ICG and lowest (94%) with MB alone SLNB. UltraSonoGraphy guided fine needle aspiration cytology of radiological suspicious nodes has significantly reduced the SLNB positivity rate from 34.6% to 26.4% (p < 0.01). One patient had skin necrosis, and 16 had persistent blue staining of the skin in the MB injection site. All were managed conservatively. The lymphedema rate was significantly higher (5.2%) in the ALND versus 0.5% in the SLNB alone patients (p < 0.05). In a median follow up of 27 months, the axillary recurrence rate was 0.04% (4/1,023), and false-negative rate was 0.9% in SLNB negative patients. There were 35 recurrences and 25 deaths in SLNB negative patients, with 10 years predicted disease-free survival of 81% (95% CI 66% to 89%) and overall survival of 79% (95% CI 59% to 90%). Conclusions: SLNB should be offered as an axillary staging procedure to all eligible BC patients from developing countries to avoid the morbidity associated with ALND. Fluorescent dye can be used as an alternative for RI in a resource-constrained setup.

Systematically higher Ki67 scores on core biopsy samples compared to corresponding resection specimen in breast cancer: a multi-operator and multi-institutional study.

Ki67 has potential clinical importance in breast cancer but has yet to see broad acceptance due to inter-laboratory variability. Here we tested an open source and calibrated automated digital image analysis (DIA) platform to: (i) investigate the comparability of Ki67 measurement across corresponding core biopsy and resection specimen cases, and (ii) assess section to section differences in Ki67 scoring. Two sets of 60 previously stained slides containing 30 core-cut biopsy and 30 corresponding resection specimens from 30 estrogen receptor-positive breast cancer patients were sent to 17 participating labs for automated assessment of average Ki67 expression. The blocks were centrally cut and immunohistochemically (IHC) stained for Ki67 (MIB-1 antibody). The QuPath platform was used to evaluate tumoral Ki67 expression. Calibration of the DIA method was performed as in published studies. A guideline for building an automated Ki67 scoring algorithm was sent to participating labs. Very high correlation and no systematic error (p = 0.08) was found between consecutive Ki67 IHC sections. Ki67 scores were higher for core biopsy slides compared to paired whole sections from resections (p ≤ 0.001; median difference: 5.31%). The systematic discrepancy between core biopsy and corresponding whole sections was likely due to pre-analytical factors (tissue handling, fixation). Therefore, Ki67 IHC should be tested on core biopsy samples to best reflect the biological status of the tumor.

The tale of TILs in breast cancer: A report from The International Immuno-Oncology Biomarker Working Group.

The advent of immune-checkpoint inhibitors (ICI) in modern oncology has significantly improved survival in several cancer settings. A subgroup of women with breast cancer (BC) has immunogenic infiltration of lymphocytes with expression of programmed death-ligand 1 (PD-L1). These patients may potentially benefit from ICI targeting the programmed death 1 (PD-1)/PD-L1 signaling axis. The use of tumor-infiltrating lymphocytes (TILs) as predictive and prognostic biomarkers has been under intense examination. Emerging data suggest that TILs are associated with response to both cytotoxic treatments and immunotherapy, particularly for patients with triple-negative BC. In this review from The International Immuno-Oncology Biomarker Working Group, we discuss (a) the biological understanding of TILs, (b) their analytical and clinical validity and efforts toward the clinical utility in BC, and (c) the current status of PD-L1 and TIL testing across different continents, including experiences from low-to-middle-income countries, incorporating also the view of a patient advocate. This information will help set the stage for future approaches to optimize the understanding and clinical utilization of TIL analysis in patients with BC.

Determinants of prognosis in patients with oral squamous cell carcinoma metastasizing to a single cervical lymph node.

OBJECTIVES: Metastasis in a single lymph node without adverse tumour factors and perineural invasion has been assigned to a lower risk category by the ASCO recommendations of 2019. We analyzed patients with a single positive node with a view to identifying high risk features and their impact on prognosis within this subgroup. MATERIALS AND METHODS: The study retrospectively analyzed 707 patients with OSCC. Descriptive statistics were used to compare distribution of clinicopathologic risk features between 323 N0 and 121 single node positive (Ns) patients. The Ns group was further analyzed for the impact of clinicopathologic factors on disease free (DFS) and overall survival (OS) using univariate and multivariate models. RESULTS: The Ns group exhibited greater depth of invasion compared to the N0 group and significantly higher proportion of lymphovascular invasion (LVI), perineural invasion (PNI) and poorly differentiated tumors. Within the Ns group, primary tumor localised to the gingivobuccal subsite (HR 2.55, 1.18-5.52 95%CI, p = 0.02) and PNI (HR 2.55, 1.14-5.62, 95%CI p = 0.02) exhibited poor DFS. PNI also contributed to poor OS (HR 2.86, 1.27-6.47 95%CI, p = 0.01). Uninvolved margins (HR 0.46, 0.22-0.96 95%CI, p = 0.04) and chemoradiation (HR 0.18,0.05-0.68 95% CI, p = 0.01) improved OS. CONCLUSION: Significant differences are noted in the prevalence of pathologic risk factors between the single node positive and node negative groups. Within the single node positive group, tumour factors like the gingivobuccal subsite, PNI and margin positivity impacted survival. Among nodal factors, deposit size of 12 mm or more and presence of ENE are pointers to poor prognosis. These patients would benefit from adjuvant treatment.

Outcomes of diffuse large B-cell lymphoma in elderly patients-real-world experience from a middle-income country setting.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is the commonest subtype of lymphoma in the elderly and poses unique challenges in this group of patients. There is a need for more information on real-world outcomes across economic disparities. METHODS: Electronic Medical Record of 3,087 lymphomas (>18 years) were evaluated retrospectively, of which 842 (27%) patients were ≥65 years. Two hundred and twelve patients who were ≥65 years received first line treatment for DLBCL between May 2011 and Dec 2016. Demography, clinical features, associated co-morbidities, first line treatment outcomes and hospital costs were analysed. Patients were followed up till March 2020. RESULTS: The median age at presentation was 71 years. Gender ratio was 2.5:1. 38% patients presented with early-stage disease, 37% with low and low-intermediate International prognostic index, 49% with nodal disease. One or more co-morbidities were present in 58%. The commonest extra nodal site was gastro-intestinal (29%). Two-thirds of the patients presented with non-Germinal centre B subtype. The overall response (OR) to treatment was 72.5%. Patients who received anthracycline-based therapy (n = 124) and rituximab-based therapy (n = 159) had a median progression free survival (PFS), not reached and 47.0 months, respectively, versus 10 months and 7.9 months, respectively, for patients receiving non-anthracycline and non-rituximab therapies. At a median follow-up of 24 months, the 5-year overall survival and PFS are 44% and 41%, respectively, for the entire cohort. CONCLUSIONS: DLBCL is a curable lymphoma in elderly patients with standard anthracycline and rituximab-based therapies. Improvement in outcomes largely depends on social and financial support to complete the scheduled treatments.

Reliability of Ki67 visual scoring app compared to eyeball estimate and digital image analysis and its prognostic significance in hormone receptor-positive breast cancer.

We analysed the reproducibility of Ki67 labelling index (LI) between two scorers using the International Ki67 Working Group (IKWG) global methods on an Android application (APP), correlated the APP and eyeball estimate (EBE) with digital image analysis (DIA) scores and determined the prognostic significance of Ki67LI. Global weighted (GW) and global unweighted (GUW) Ki67 app scores of hormone receptor-positive and HER2 (human epidermal growth factor receptor 2)-negative breast cancer patients were obtained. Reproducibility of Ki67LI between 2 scorers and correlation of APP and EBE scores with DIA scores were performed. The prognostic significance of APP scores and its correlation with other clinico-pathologic variables were evaluated. The intra-class correlation coefficient (ICC) between 2 scorers showed excellent reliability with both GW and GUW methods. ICC between DIA and APP scores was significantly greater than DIA versus EBE. The three categories of APP scores based on median value and cut points of 10%, 18% and 38% were significantly associated with poor DFS. On multivariate analysis, significant association between Ki67LI, tumour size, nodal involvement and DFS was noted. Our study shows that the visual Ki67 scoring app is effective in bringing consistency to KI67LI and APP scores showed significant correlation with DFS.

Relapsed plasmablastic lymphoma in a HIV-negative patient: Pushing the envelope.

This case emphasizes that, with the availability of novel immunotherapy agents (Daratumumab), and repurposed use of bortezomib, a patient with HIV-negative relapsed PBL can be treated successfully and consolidated with an allogeneic haploidentical hematopoietic cell transplantation.

Nodal yield and topography of nodal metastases from oral cavity squamous cell carcinoma - An audit of 1004 cases undergoing primary surgical resection.

OBJECTIVES: Nodal metastasis is an important prognostic factor in oral squamous cell carcinoma (OSCC). Detailed topographic study of metastasis can guide surgical and adjuvant radiation treatment protocols. METHODS: Retrospective analysis of distribution of nodal spread was done by auditing pathology records of 1004 patients who underwent primary surgical management at our center. RESULTS: The median nodal yield was 41 (range of 9-166) nodes, per patient. Metastasis was present in 42.9% patients, of which 52.3% demonstrated extranodal extension. Reclassification by AJCC8 criteria resulted in up-staging in 35.6% patients (pN1, pN2a, pN2b, pN2c, pN3a and pN3b in 13.1%, 3.7%, 6.9%, 0.9%, 0%, 18.1% respectively). Ipsilateral levels Ib and IIa were involved in a quarter of patients each, while IIb, IV and V were involved in < 4%, 3% and 1% of patients, respectively. Contralateral nodal metastasis was present in 5.4%. Skip metastases to level IV were 2.2% and 1.2% for tongue and gingivobuccal primaries. Tongue primaries had a lower likelihood of involving level Ib, but higher of level IIa and III, compared to gingivobuccal primaries, and a lower likelihood of extranodal extension. Primary site did not influence nodal metastasis to levels IIb, IV or V, but other factors like lymphovascular invasion, pT stage and margin status had an influence. CONCLUSION: This large series with high nodal yield, shows low level of metastasis to level IIb, IV and V, which can help modify future guidelines for extent of surgery and avoid targeted adjuvant radiation to specific levels.

Relapsed Refractory Hodgkin Lymphoma and Brentuximab Vedotin-Bendamustine Combination Therapy as a Bridge to Transplantation: Real-World Evidence From a Middle-Income Setting and Literature Review.

INTRODUCTION: Despite high cure rates with standard treatment, 30% patients with Hodgkin lymphoma develop relapsed or refractory (R/R) disease. Salvage therapy followed by autologous hematopoietic cell transplantation (HCT) is considered standard of care. Brentuximab Vedotin (Bv) in combination with Bendamustine (B) has been tested in the salvage setting with promising results. MATERIALS AND METHODOLOGY: We conducted a single centre retrospective chart review of patients who received BBv salvage therapy to determine its activity and safety in patients with R/R classical Hodgkin lymphoma (HL). Between May 2011- December 2019, 179 patients were diagnosed with R/R HL. RESULTS: Thirty patients received BBv [median age: 30 (15-59) years, females (n=15)]. Primary refractory disease in 19 patients (63%), and 26 patients (87%) had advanced stage at treatment. Most patients received BBv after 2 prior lines of therapy [n=16 (53%)]. The median number of cycles of BBv were 3 (1-6). The number of BBv cycles delivered as outpatient was 63%. The most common Grade III/IV hematological adverse event was neutropenia [n=21, (70%)], while grade III/IV non-hematological toxicities included infections in 4 (13%), neuropathy in 4(13%), skin rash in 2 (7%), GI toxicities in 3 (10%) and liver dysfunction in 2 (7%) patients. The ORR and CR rates were 79% and 62%, respectively. Seventeen patients (57%) underwent an autologous HCT and 8 (26%) underwent an Allogeneic HCT (all haploidentical). The median follow up time from BBv administration was 12 months. Six patients died: 2 = disease progression, and 4 = non-relapse causes (Infection and sepsis = 2, GVHD=2). In addition to this, one patient progressed soon after HCT and another patient relapsed 22 months post HCT. Three year Overall survival (OS) and Event free survival (EFS) probability post-BBv treatment was 75% and 58%, respectively. OS and EFS analysis based on response (viz., CMR) to BBv demonstrated that patients in CMR had better survival probability [93% (p=0.0022) 3yr-OS and 72% (p=0.038) 3yr-EFS probability]. CONCLUSIONS: BBv is an active and well-tolerated salvage treatment for patients with R/R HL, even in refractory and advanced settings. In middle-income settings, cost constraints and access determine patient uptake of this regimen.

Lymph node characteristics and their prognostic significance in oral squamous cell carcinoma.

BACKGROUND: The prognostic significance of various histopathologic lymph node-based biomarkers in oral squamous cell carcinoma (OSCC) needs further evaluation. METHODS: Retrospective analysis of 212 OSCC patients with regional metastasis to determine the association of extranodal extension (ENE), extent of ENE, size of metastatic deposit, lymph node yield (LNY), lymph node ratio (LNR), and topography of involvement with survival outcomes. RESULTS: The presence of ENE, larger nodal deposit, higher pN stage, lymph nodes in the lower levels, and patients who did not receive adjuvant treatment had poor disease-free survival (DFS). In addition, more positive nodes and high LNR showed worse overall survival (OS). ENE beyond 5 mm resulted in poorer outcomes. Larger sizes of metastatic deposit predisposed to ENE. Multivariate analyses showed only lower level of neck involvement to affect both DFS and OS. CONCLUSIONS: Lymph node metastasis to lower levels and other lymph node characteristics affect prognosis and must be considered in the evolution of staging systems for OSCC.

AutoIHC-Analyzer: computer-assisted microscopy for automated membrane extraction/scoring in HER2 molecular markers.

Human epidermal growth factor receptor 2 (HER2) is one of the widely used Immunohistochemical (IHC) markers for prognostic evaluation amongst the patient of breast cancer. Accurate quantification of cell membrane is essential for HER2 scoring in therapeutic decision making. In modern laboratory practice, expert pathologist visually assesses the HER2-stained tissue sample under the bright field microscope for cell membrane assessment. This manual assessment is time consuming, tedious and quite often results in interobserver variability. Further, the burden of increasing number of patients is a challenge for the pathologists. To address these challenges, there is an urgent need with a rapid HER2 cell membrane extraction method. The proposed study aims at developing an automated IHC scoring system, termed as AutoIHC-Analyzer, for automated cell membrane extraction followed by HER2 molecular expression assessment from stained tissue images. A series of image processing approaches have been used to automatically extract the stained cells and membrane region, followed by automatic assessment of complete and broken membrane. Finally, a set of features are used to automatically classify the tissue under observation for the quantitative scoring as 0/1+, 2+ and 3+. In a set of surgically extracted cases of HER2-stained tissues, obtained from collaborative hospital for the testing and validation of the proposed approach AutoIHC-Analyzer and publicly available open source ImmunoMembrane software are compared for 90 set of randomly acquired images with the scores by expert pathologist where significant correlation is observed [(r = 0.9448; p < 0.001) and (r = 0.8521; p < 0.001)] respectively. The output shows promising quantification in automated scoring. LAY DESCRIPTION: In cancer prognosis amongst the patient of breast cancer, human epidermal growth factor receptor 2 (HER2) is used as Immunohistochemical (IHC) biomarker. The correct assessment of HER2 leads to the therapeutic decision making. In regular practice, the stained tissue sample is observed under a bright microscope and the expert pathologists score the sample as negative (0/1+), equivocal (2+) and positive (3+) case. The scoring is based on the standard guidelines relating the complete and broken cell membrane as well as intensity of staining in the membrane boundary. Such evaluation is time consuming, tedious and quite often results in interobserver variability. To assist in rapid HER2 cell membrane assessment, the proposed study aims at developing an automated IHC scoring system, termed as AutoIHC-Analyzer, for automated cell membrane extraction followed by HER2 molecular expression assessment from stained tissue images. The input image is preprocessed using modified white patch and CMYK and RGB colour space were used in extracting the haematoxylin (negatively stained cells) and diaminobenzidine (DAB) stain observed in the tumour cell membrane. Segmentation and postprocessing are applied to create the masks for each of the stain channels. The membrane mask is then quantified as complete or broken using skeletonisation and morphological operations. Six set of features were assessed for the classification from a set of 180 training images. These features are: complete to broken membrane ratio, amount of stain using area of Blue and Saturation channels to the image size, DAB to haematoxylin ratio from segmented masks and average R, G and B from five largest blobs in segmented DAB-masked image. These features are then used in training the SVM classifier with Gaussian kernel using 5-fold cross-validation. The accuracy in the training sample is found to be 88.3%. The model is then used for 90 set of unknown test sample images and the final labelling of stained cells and HER2 scores (as 0/1+, 2+ and 3+) are compared with the ground truth, that is expert pathologists' score from the collaborative hospital. The test sample images were also fed to ImmunoMembrane software for a comparative assessment. The results from the proposed AutoIHC-Analyzer and ImmunoMembrane software were compared with the expert pathologists' score where significant agreement using Pearson's correlation coefficient [(r = 0.9448; p < 0.001) and (r = 0.8521; p < 0.001) respectively] is observed. The results from AutoIHC-Analyzer show promising quantitative assessment of HER2 scoring.