Search

Background Breast Cancer (BC) patients who do not obtain pathologic complete response (pCR) after neoadjuvant chemotherapy (NACT) present higher rate of relapse and worse overall survival. Recent evidence suggests that chemotherapy (CT) efficacy relies on the capacity of chemotherapeutic agents to interact with the immune system. BC features a unique tumor microenvironment (TME) comprising of multiple immune cell types including Macrophages that share a double-edged relationship with cancer as they get polarised from M1 (anti-tumour) to M2 (Pro-tumour). Pro-tumour M2 macrophages are referred to as tumor-associated macrophages (TAMs) and are implicated extensively in angiogenesis, metastasis and therapy resistance. Establishing role of TAMs, facilitates the emergence of novel strategies that exploit them as theranostic targets/tools of interest for treating cancer. Utility of using Drugs like Zoledronic Acid, Trabectedin, Rebastinib in combination with 5-fu have shown promising anti-TAM activity and improved response to CT in clinical trials. In this study, we have characterized presence of M2-TAM and its correlation to NACT response in matched primary and residual tumours by examining expression of several biomarkers. Methods Treatment naïve primary and their matched residual tumour specimens from 45 women treated at a single center were accessed through IERB approved protocols. The study included locally advanced HR+HER2-ve and TNBC tumours treated with standard NACT regimes between 2018 to 2020. To determine the association of TAM population with response to NACT, expression levels of CD68 (pan macrophage marker) & CD163 (marker of М2 macrophages), were detected by immunohistochemistry (IHC) and represented as combined H score. We also performed gene expression of chemokines, inflammatory cytokines and interleukins involved in M1-M2 polarization by q-RT-PCR. Residual Cancer Burden Scoring was used to assess response and patients were divided into three groups (complete responders, partial responders and non-responders). Univariate and multi variate analysis were performed between gene expression groups and IHC groups with clinicopathological parameters. Findings from this study was validated on public data bases like TCGA, METABRIC and GEO. Results: 20% of all patients included in the study were complete responders. We arrived at a Macrophage Polarisation Score (MPS) by Gene expression and also a combined H score by IHC. MPS and H-score had a positive correlation (p=0.083) overall. Interestingly, Combined analysis of H-Score and MPS with response to treatment showed a greater and statistically significant correlation with residual tumours as compared to treatment naïve tumours (p=0.009). We also observed that high MPS and high combined H score in residual tumours were associated with increased tumour size and LVI (p=0.055 and p=0.03). Other clinicopathological parameters like receptor status, grade and stage at diagnosis were not significantly associated with H score or MPS. Taken together we found that 11% of patients who exhibited high TAM score by both H sascore and MPS fell into the non-responders category. We therefore report TAMs in residual tumours being more indicative for response to therapy compared to primary tumours. Conclusion Although primary tumours are useful for building predictive models to therapy response, we have demonstrated that there is utility in examining residual tumours as well for choice to adjuvant chemotherapy, since the tumour is constantly evolving through the NACT period. More work to arrive at a “TAM score” that could aid in choice of additional adjuvant treatment strategies is underway. We believe our work is helping us to move one step closer to Precision Medicine in a low and middle income country like India that has a higher burden of locally advanced disease. Our analysis also lends itself to becoming a clinical test since it is performed on an FFPE specimen Citation Format: Lohita Krishna, Aruna Korlimarla, B S Srinath, Anugnya Ranjolkar, Sudipta Nascar, Hari PS, Durga Devi, Nidhi l, Rekha V Kumar. Tumour associated macrophages in Breast Cancer - Are they critical players in response to Neo Adjuvant Chemotherapy? [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-21-10.

Introduction: miRNA-based regulation has been implicated in tumor evolution and progression. miR-18a belonging to the miR-17-92 polycistronic cluster has also been reported to have oncogenic effects across multiple cancer types including breast cancer. We have previously demonstrated the epigenetic regulation of ER by miR-18a and its high levels as a poor prognostic marker in ER-positive breast tumors (Nair et al, Cancer Med. 2016). Here, we have examined the effects of high expression of miR-18a in the ER-negative subtype of breast cancer. Methods: 275 surgically excised specimens of primary breast cancers were analyzed. Samples were segregated into ER-positive and ER-negative tumors based on ER positivity as determined by Immunohistochemistry. Relative abundance of hsa-miR-18a-5p in these samples was assessed using a TaqMan qRT-PCR and used to correlate with a probability distribution of proliferation that was derived by fitting a binomial logistic regression model using 4 genes - FOXM1, UBE2C, BIRC5 & ANLN. miR-18a was inhibited using synthetic inhibitors in ER-negative breast cancer cell lines - MDA-MB-468 AND MDA-MB-231. Migratory ability was assessed using wound-healing assays. The expression of miR-18a was further analyzed in ER-negative breast cancer samples from the TCGA (n= 116) and the METABRIC cohort (n=107). ER-negative tumors with higher than the third quartile and lower than the first quartile expression of miR-18a were segregated into tumors with high and low expression, respectively. Functional enrichment of differentially expressed genes (DEGs) between these groups was performed using the G: profiler to identify the deregulated pathways. Result: Evaluation of the levels of miR-18a in 275 breast tumor samples showed that the microRNA was highly expressed (p Citation Format: Madhumathy G Nair, Chandrakala M, Apoorva D, Snijesh VP, Jyothi S Prabhu, Savitha Rajarajan, Aruna Korlimarla, Rakesh S Ramesh, Srinath BS, Sridhar TS. High levels of miR-18a is associated with increased proliferation but suppression of EMT phenotype in ER negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-10-06.

Introduction:Triple-Negative Breast Cancer (TNBC) comprises approximately 30% of all breast cancers in Indian women. Given their aggressive nature, TNBCs have high rates of systemic metastasis and mortality with only chemotherapy available for treatment. The success of immunotherapy in solid tumors has raised the hope for their utility in TNBCs as well. But only a subset of patients have a clinical response to check-point inhibitors. The cellular and molecular mechanisms that mediate the immunological response or tolerance are just beginning to be understood. Compared to ER/PR+ breast cancer, TNBC features a unique tumor microenvironment (TME) characterized by a large number of tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs). The density of TILs in and of themselves do not accurately predict response to neoadjuvant chemotherapy or survival. M2 tumor-associated macrophages (M2-TAMs) have been reported to associate with solid tumors to facilitate epithelial to mesenchymal transition (EMT), tumor invasiveness, metastasis, and resistance to therapy. In this study, we have characterized presence of M2-TAM in the TNBC immune environment by examining expression of several biomarkers. Methods:Surgically excised tumor specimens from 88 Indian women with TNBC were accessed from the longitudinal observational series of SJNAHS tissue bank under IERB approved protocols and assayed on nCounter® PanCancer Immune Profiling Panel which comprised of 740 genes and characterises 14 different immune cell types. Transcriptome analysis using Non-negative Matrix factorization (NMF) based unsupervised clustering was done to arrive at stable subtypes characterized by different tumour microenvironments within TNBC. CIEBERSORT tool was used to analyse for distribution of cell types. Identification of macrophages was done by Immunohistochemistry (IHC) for CD68 and CD163 markers in TNBCs and a control group of ER+HER2-.Results:NMF analysis with an enriched immune gene signature of 111 genes, yielded 3 subtypes (ST) (37%, 27% and 36% with relative proportions of ST1, ST2 and ST3) within the TNBC. The three subtypes had distinct survival patterns with ST1 having the best prognosis with enriched TH1 gene signature and ST3 had poorest (log rank p=0.5). On application of this gene signature to TNBC groups in METABRIC and TCGA data, similar pattern emerged with a significant survival pattern of ST 3 having the poorest outcome (p=0.005). A closer examination of ST 3 revealed a signature enriched for M2 macrophages also known as TAM. Immunohistochemistry for Pan macrophage marker CD68 and an M2 specific marker CD163 between TNBC and ER/PR+ revealed difference in the two groups was significantly different (Mann Whitney p=0.03), with TNBC exhibiting 2.4 fold higher expression of CD163. CD 68, was enriched in ST2 and ST3. CD163 which is an M2 specific marker was highest in ST3 as compared to ST 1& 2 (p=0.04). This group also correlated with signal molecules secreted by macrophages containing growth factors, cytokines and chemokines, such as TGF-β, VEGF, IL-10 and CXCL and interleukins like IL4 and IL6 suggestive of TAM recruitment and polarization. It is likely that the TAM enriched subgroup is likely to be unresponsive to PD1/PDL1 inhibitors but could be targeted by novel therapeutic strategies to directly target M2-TAM. In vitro and In vivo analysis to target M2-TAM to evaluate the response to these therapeutic compounds in cell lines as well as a mouse syngeneic model is underway.Conclusions and future directions: As a cell type within the tumor microenvironment, that promotes invasion, M2-TAMs makes an ideal therapeutic target in TNBC. In addition, this sub-type lends itself to easy identification by a simple IHC assay. Citation Format: Aruna Korlimarla, Jyothi Prabhu, Chantirika Ragulan, Gnanapriya Shivakumar, Krisha Desai, Maggie Cheang, Srinath BS, Ravi Diwakar, Sandhya Apachu, Rekha Kumar, TS Sridhar, Savitha RajaRajan, Rohini Kaluve, Annie Alexander, Anguraj Sadanandam. A pro-tumorigenic mechanism of M2 tumor-associated macrophages (TAM) in triple-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-37.

BackgroundTriple-negative breast cancer (TNBC) is a heterogeneous disease with a significant clinical challenge. TNBC alarmingly comprises 25-30% of breast cancers in India compared with only 10-15% in the West. However, immunotherapy was approved for high-risk early-stage TNBCs in the West. Hence, a long-standing question is whether Indian TNBCs immunologically and clinically resemble Western TNBCs such that they respond similarly to immunotherapies. Here we sought to elucidate the immune landscape of Indian TNBCs for the first time, compare them to Western disease and associate them with clinical parameters, cellular types/signaling, and immunotherapy response.MethodsWe profiled 730 immune genes in 88 retrospective Indian TNBC samples using NanoString platform, clustered them into subtypes using a machine-learning approach, and compared them with Western TNBCs (n=422; public datasets). Subtype-specific gene signatures were identified, followed by clinicopathological, immune cell type, and pathway (multiomics) analyses. We also assessed responses to (cross-cancer) immunotherapy. Tumor-infiltrating lymphocytes (TILs) and pan-macrophage marker were evaluated using hematoxilin-eosin staining and immunohistochemistry, respectively.ResultsWe identified three robust and similarly distributed TNBC immune transcriptome subtypes (Subtypes-1-3) in Indian women, and they are represented correspondingly in Western TNBCs and associated with well-known TNBC subtypes. Irrespective of the ethnicity, Subtype-1 harbored tumor microenvironmental and anti-tumor immune events associated with smaller tumors, younger age, and a better prognosis. Subtype-1 mainly represented basal-like/claudin-low breast cancer and immunomodulatory TNBC subtypes. Subtype-1 showed an increase in a cascade of events, including damage-associated molecular patterns, acute inflammation, Th1 responses, T-cell receptor-related and chemokine-specific signaling, antigen presentation, and viral-mimicry pathways. Subtype-1 was significantly (p+ regulatory cells, basal-like/mesenchymal subtypes, and an intermediate prognosis. Subtype-3 patients expressed innate immune genes/proteins, including those representing macrophages and neutrophils, and had poor survival.ConclusionWe identified three immune-specific TNBC subtypes in Indian patients with differential clinical and immune behaviors, which largely overlapped with the Western TNBC cohorts. This study suggests cancer immunotherapy in TNBC may work similarly in both populations. Hence, this may expedite pharmaceutical adoption of immunotherapy to Indian TNBC patients.

Despite the established benefits of long‐term endocrine therapy, women with hormone receptor‐positive breast cancer remain at risk for late relapse. The basis of this is multi‐factorial including genetic, epigenetic, and host factors. In this study we have explored the epigenetic regulation of estrogen receptor (ER)‐dependent molecular and cellular phenotype by hsa‐miR‐18a‐5p using well‐established human ER‐positive (ER+) breast cancer cell lines. miR‐18a was overexpressed in MCF7 and ZR‐75‐1 and this led to an increase in the proliferative ability of the cells and concurrently resulted in decreased expression of luminal markers and higher expression of the basal marker, cytokeratin 14. The cells became more migratory with a significant repression of E‐cadherin and activation of the Wnt noncanonical pathway. We observed an activation of the planar cell polarity (PCP) pathway with increased activation of JNK pathway and eventually change in actin dynamics. There was increased F‐actin polymerization in cells with higher expression of miR‐18a. Examination of miR‐18a expression in a set of human ER+ breast cancer specimens showed a negative correlation between miR‐18a and ESR1 transcripts as well as ER protein. Kaplan‐Meier survival analysis of the cohort stratified by tumor hsa‐miR‐18a‐5p levels produced significant differences in disease‐free survival (log rank P, Through the manuscript, we have tried to explore the epigenetic regulation of estrogen receptor (ER) dependent molecular and cellular phenotype by hsa‐miR‐18a‐5p. High levels of miR‐18a activates the epigenetic pathway of repression of the luminal phenotype through activation of Wnt pathway.

Introduction: Triple-Negative Breast Cancer (TNBC) comprises approximately 30% of all breast cancers in Indian women. Given their aggressive nature, TNBCs have high rates of systemic metastasis and mortality with only chemotherapy available for treatment. Compelling evidence has demonstrated the prognostic value of tumor-infiltrating lymphocytes (TILs), in many cancers especially in Breast Cancer and play a critical role in tumour progression, response to therapeutics and prognosis. They are typically measured by H&E staining and immunohistochemistry for research purpose and degree of infiltrate is also known to differ among subtypes. Nonetheless, the literature regarding the types of immune cells characterizing TILs and their prognostic utility in TNBC has been conflicting for lack of accurate “functional” TIL assessments. Herein we have used a combination signature of TILs and a 2 gene immune function expression based signature to develop an unique classifier, to identify a subgroup within TNBC that has better clinical outcome, such as survival and response to treatment. Our findings also suggest a possible use of the score as a predictive biomarker for response to immune checkpoint therapy Patients and Methods: Surgically excised TNBC tumor specimens from 44 women from a single treating hospital in Bangalore, India were accessed under IERB approved protocols and assayed on nCounter® PanCancer Immune Profiling Panel which comprised of 740 genes and characterises 14 different immune cell types. Analysis using Non-negative Matrix factorization (NMF) based unsupervised clustering was done to arrive at stable subtypes characterized by different tumor microenvironments within TNBC. SSGSEA analysis was done to identify immune cell types. TILs were characterised by a pathologist on H&E sections according to guidelines from TIL working group. The Immune classifier was validated on Breast Cancer data sets from TCGA. Results: NMF followed by SAM and PAM analysis yielded 2 stable subtypes (ST1 and ST2) of proportions 59% and 41% respectively. TIL groups also were divided into Dense and Mild groups which were 52% and 48% respectively. Despite similar distributions of the groups, ST1 and ST2 had distinct survival with ST2 having poorer outcome (p=0.023) while the dense and mild TIL groups did not separate as distinct groups. On a closer examination using SSGSEA analysis with Rooney et al signature, ST1 was enriched with T-cells, Dendritic cells, MHC-class 1 and very significantly high cytolytic score (CYT), which was defined by Granzyme A and Perforin expressions, proteins secreted by cytotoxic T cells (p=0.0074). The CYT Score ranged from -35 to 170 and cutoff was based on 75th percentile. We next hypothesized that a combined score of TIL and CYT would represent a functional TIL group with high immune activity. We arrived at a CYT+TIL score based on 75th percentile cut off of CYT and 2 groups of TIL. Of the resultant four groups, TIL High-CYT high that constituted about 20% of all TNBCs indicated an elevated immune response because of their microenvironment constitution and this can be identified using our simple immune classifier. On the other hand, in the TIL-high and CYT-low group, despite lymphocyte migration, outcome was not favorable. The classifier was applied to TNBC from TCGA (n=96) and similar results seen, with ST1 and ST2 which had distinctly separate survival and had similar patterns on CYT high groups. Conclusion: We developed a simple immune classifier with 2 gene signature c and H&E slide TIL assessment for identifying a group with TNBC that can be better targeted with therapies. Our signature is predictive for selecting TNBCs which are potential responders to Immune therapies and has the potential for quick clinical adoption though it requires validation on a larger set. Citation Format: Aruna Korlimarla, Hari PS, Jyothi S Prabhu, Chantirika Ragulan, Ravi B Diwakar, Sandhya Apachu, Maggie Cheang, Rekha V Kumar, BS Srinath, TS Sridhar, Savitha Rajarajan, Annie Alexander, Anguraj Sadanandam. A novel combination of a 2 gene score & TIL as a predictive Biomarker for responders to novel therapies in Indian TNBC - A population with greater proportion of TNBCs [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-14.

Purpose: To understand the role played by the immediate family in treatment decision and support in patients diagnosed with breast cancer, the influence of demographic factors on psychosocial roles of women within the family. Methods: A mixed method design used for data collection on family support, financial arrangement and psychosocial impact of cancer from 378 women with breast cancer recruited at first diagnosis between 2008 and 2012, during multiple counseling sessions. The median follow-up is 7 years with only 2% lost to follow-up. Results: Most patients (99%) had support from family members. 57% of patients met the costs of treatment through personal savings and health insurance. The rest (43%) had difficulty and had to resort to desperate measures such as selling their property or taking on high-interest personal loans. Patients with higher education and urban settings had better financial management. A male member of the family (husband or son) was the main decision maker in half of the cases. Concerns over women's responsibilities within the family varied by the age of the patient. The vast majority of women (90%) experienced social embarrassment in dealing with the disease and its aftermath. Conclusion: In India, it is the family that provides crucial support to a woman with breast cancer during her ordeal with the disease and its treatment. This study has implications on the psychosocial support beyond the cancer patients alone, to include the immediate family and consider aspects of finance and social adjustments as critical in addition to the routine medical aspects of the disease.

Background The study assessed the epigenetic regulation and the role of microRNA (miR) expression in locally advanced triple negative breast cancers (TNBC) and comparison with the clinico-pathological variables and survival. Methods Fifty patients of locally advanced TNBC during the period 2011–2013 were included. Expression level of test microRNA (miR-182 and miR-18a) was determined using Taqman quantitative Real time polymerase chain reaction (qRT-PCR) from formalin fixed paraffin embedded biopsy blocks. Clinical and demographic information and survival data was retrieved from the Hospital medical records. Results An improved clinical complete response (cCR) was observed in patients with age ≥ 45 years (80%), premenopausal status (70%), tumor size < 6 cms (80%), nodal status N0-N1 (95%) and grade II-III tumor (80%). A statistically significant correlation was observed on comparison of cCR with menopausal status (p-value 0.020), T category (p-value 0.018) and the clinical nodal status (p-value 0.003). pCR also correlated with clinical nodal status (p-value 0.008). Epigenetically, miR-18a under expression (< 8.84) was most commonly associated with tumor size < 6 cms (76.7%), clinical nodal status N0-N1 (90%), cCR (60%) and pCR (53.3%). A similar trend was observed with miR-182. Statistical significance was observed with T category (p-values 0.003 and 0.004), clinical nodal status (p-values 0.001 and 0.001), clinical response (p-values 0.002 and 0.002) and pathological response (p-values 0.007 and 0.006) with respect to miR-18a and miR-182, respectively. Also, the menopausal status significantly correlated with the miR-182 expression (p-value 0.009). miR-182 overexpression (≥ 6.32) was not observed in any of the postmenopausal patients. A univariate cox proportional hazard regression model also showed statistical interactions (p-values Conclusion miR-182 and miR-18a overexpression correlates with worse clinical and pathological tumor characteristics in locally advanced TNBC and hence could be used to predict the outcomes and prognosis in these patients.

Cystic neutrophilic granulomatous mastitis (CNGM) is a histologically characterized variant of granulomatous lobular mastitis that is associated with lipophilic Corynebacterium species. It remains a largely underrecognized entity in India. Our aim was to study CNGM in the Asian Indian population and explore if 16s rRNA sequencing could be used on formalin-fixed paraffin-embedded (FFPE) tissue to identify the causative organism. We studied 24 cases with histological features of CNGM with hematoxylin and eosin, Gram, Ziehl-Neelsen, and Periodic acid–Schiff stains. Tuberculosis-polymerase chain reaction and 16s rRNA gene sequencing on DNA extracted from FFPE was attempted (N = 23). Gram-positive bacilli were seen in 20/24 cases. Routine culture with prolonged incubation yielded Corynebacterium species in 8 cases; 7 of these cases were evaluated by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for species identification. C matruchotti was identified in one case by BD Phoenix. MALDI-TOF MS identified the remaining 7 cases as C kroppenstedtii (N = 4) and C tuberculostearicum (N = 2), with no identification in one. Corynebacteria were identified by 16s rRNA sequencing on DNA extracted from FFPE in 12/23 cases using a primer targeting the V5-V6 region that was found to be more conserved in Corynebacterium species. All cases were negative for the diagnosis of tuberculosis. CNGM can be identified by routine stains. Culture using routine media with prolonged incubation is often adequate to isolate the organism. 16s rRNA sequencing on DNA extracted from FFPE tissue can help make an etiological diagnosis in some cases where only paraffin blocks are available.

Integrin αvβ6 is involved in the transition from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) of the breast. In addition, integrin β6 (ITGB6) is of prognostic value in invasive breast cancers, particularly in HER2+ subtype. However, pathways mediating the activity of integrin αvβ6 in clinical progression of invasive breast cancers need further elucidation. We have examined human breast cancer specimens (N = 460) for the expression of integrin β6 (ITGB6) mRNA by qPCR. In addition, we have examined a subset (N = 147) for the expression of αvβ6 integrin by immunohistochemistry (IHC). The expression levels of members of Rho–Rac pathway including downstream genes (ACTR2,ACTR3) and effector proteinases (MMP9,MMP15) were estimated by qPCR in the HER2+ subset (N = 59). There is a significant increase in the mean expression of ITGB6 in HER2+ tumors compared to HR+HER2‐ and triple negative (TNBC) subtypes (P = 0.00). HER2+ tumors with the highest levels (top quartile) of ITGB6 have significantly elevated levels of all the genes of the Rho–Rac pathway (P‐values from 0.01 to 0.0001). Patients in this group have a significantly shorter disease‐free survival compared to the group with lower ITGB6 levels (HR = 2.9 (0.9–8.9), P = 0.05). The mean level of ITGB6 expression is increased further in lymph node‐positive tumors. The increased regional and distant metastasis observed in HER2+ tumors with high levels of ITGB6 might be mediated by the canonical Rho–Rac pathway through increased expression of MMP9 and MMP15.

Background: The 2010 guidelines by ASCO-CAP have mandated that breast cancer specimens with ≥1% positively staining cells by immunohistochemistry should be considered Estrogen Receptor (ER) positive. This has led to a subclass of low-ER positive (1-10%) breast cancers. We have examined the biology and clinical behavior of these low ER staining tumors. Methods: We have developed a probabilistic score of the “ER-positivity” by quantitative estimation of ER related gene transcripts from FFPE specimens. Immunohistochemistry for ER was done on 240 surgically excised tumors of primary breast cancer. Relative transcript abundance of 3 house-keeping genes and 6 ER related genes were determined by q-RT PCR. A logistic regression model using 3 ER associated genes provided the best probability function, and a cut-off value was derived by ROC analysis. 144 high ER (>10%), 75 ER negative and 21 low-ER (1-10%) tumors were evaluated using the probability score and the disease specific survival was compared. Results: Half of the low-ER positive tumors were assigned to the ER negative group based on the probability score; in contrast 95% of ER negative and 92% of the high ER positive tumors were assigned to the appropriate ER group (p

Despite an overall good prognosis, a significant proportion of patients with hormone receptor positive human epidermal growth factor receptor 2 negative breast cancers develop distant metastases. The metastatic potential of epithelial cells is known to be regulated by tumor–stromal interaction and mediated by epithelial-to-mesenchymal transition. Hormone receptor positive human epidermal growth factor receptor 2 negative tumors were used to estimate markers of epithelial-to-mesenchymal transition, and the luminal breast cancer cell line MCF-7 was used to examine the interactions between integrins and growth factor receptors in causation of epithelial-to-mesenchymal transition. A total of 140 primary tumors were sub-divided into groups enriched for the markers of epithelial-to-mesenchymal transition (snail family transcriptional repressor 2 and integrin β6) versus those with low levels. Within the epithelial-to-mesenchymal transition+ tumors, there was a positive correlation between the transcripts of integrin β6 and growth factor receptors—human epidermal growth factor receptor 2 and epidermal growth factor receptor. In tumors enriched for epithelial-to-mesenchymal transition markers, patients with tumors with the highest quartile of growth factor receptor transcripts had a shorter disease-free survival compared to patients with low growth factor receptor expression by Kaplan–Meier analysis (log rank, p = 0.03). Epithelial-to-mesenchymal transition was induced in MCF-7 cells by treatment with transforming growth factor beta 1 and confirmed by upregulation of SNAI1 and SNAI2 transcripts, increase of vimentin and integrin β6 protein, and repression of E-cadherin. Treatment of these cells with the dual-specificity tyrosine-kinase inhibitor lapatinib led to downregulation of epithelial-to-mesenchymal transition as indicated by lower levels of SNAI1 and SNAI2 transcripts, integrin αvβ6, and matrix metalloproteinase 9 protein. The results suggest that synergistic interactions between growth factor receptors and integrin β6 could mediate epithelial-to-mesenchymal transition and migration in a subset of luminal breast cancers and lapatinib might be effective in disrupting this interaction.

Estimations of RNA abundance and DNA methylation by quantitative PCR (qPCR) from formalin-fixed, paraffin-embedded (FFPE) tissue specimens are not yet routine in clinical laboratory practice. Excluding specimens with poorly preserved nucleic acids is an important quality-control step for avoiding unreliable results. Because the assays for RNA abundance and DNA methylation have different critical limiting factors, we examined the extent of overlap of excluded specimens for RNA abundance versus methylated DNA. The transcript abundance of three reference genes and of the test gene, estrogen receptor 1 (ESR1), was estimated by SYBR Green qPCR in 250 breast cancer specimens. The estrogen receptor (ER) protein was identified by IHC, and concordance between ESR1 and ER was estimated by Cohen's κ. TaqMan PCR for the ALU-C4 sequence was performed with bisulfite-treated DNA to determine usability in the MethyLight assay. Excluding specimens with mean reference gene CT values exceeding the group mean by >1 SD led to significant improvement of the concordance of ESR1 and ER. Specimens with usable DNA after bisulfite treatment likewise had ALU-C4 CT values of less than the group mean + 1 SD. Samples with low-quality RNA and DNA were partly nonoverlapping. RNA and DNA extracted from the same FFPE block need separate exclusion criteria for qPCR assays of transcript abundance and methylated DNA.

The proportion of estrogen receptor (ER)-negative and triple-negative (TN) breast cancer in Indian women is higher than that reported in the West, and this difference persists even after their migration to the West. The causes for this significant difference are not entirely clear. Hypermethylation of the ER promoter, an epigenetic alteration, is known to be one of the mechanisms by which the expression of ER is suppressed. Two thirds of breast cancer specimens from an Indian center tested, using the highly sensitive, methylation-specific polymerase chain reaction (MSP) technique, were reported positive. We have used a quantitative assay, the MethyLight, to better assess the extent of methylation in the ESR1 promoter region in 98 breast cancer tumor specimens from Indian women. In addition, the amount of ER transcripts was determined by quantitative reverse transcriptase polymerase chain reaction. Using the stringent cutoff of at least 4% of the target sequence being methylated, 27% of TN tumors were methylated. In addition they demonstrated the highest levels of methylation. In contrast less than 2% ER-positive tumors were hypermethylated. While the proportion of hypermethylated tumors are lower in this study than that estimated using MSP, our results support the notion of increased epigenetic deregulations in ER-negative tumors in general and TN tumors in particular. The development of this assay also permits a rational approach to the selection of patients for clinical trials examining the efficacy of demethylating agents in the treatment of ER-negative breast cancer.

p53 Mutation Carcinoma of the larynx Treatment response Prognostic markers Cisplatin resistance Larynx preservation summary p53 Mutations and over expression have been shown to predict treatment response in head and neck cancer patients. Failure of organ sparing therapy has been attributed to cisplatin and radiotherapy resistance in carcinoma of the larynx patients. In this study, we evaluate the relationships between p53 over expression/mutations, bcl2 expression and ploidy status in a retrospective cohort of responder and non-responder carcinoma of the larynx patients. Tissue samples from 22 patients with histopathologically confirmed carcinoma of the larynx and matched for age, stage, node status and treatment regimen, were analysed from our tissue biorepository. Differences in the above molecular markers were analysed between the responders and non-responders to conventional treatment. p53 and bcl2 over expression was checked by IHC and p53 mutation by PCR and direct sequencing. DNA ploidy and S-phase fractions were also analysed. Chi square analysis was used to identify changes in proportions of these markers in responders and non-responders and likelihood ratio test was done to determine the best predictor biological marker for treatment response. Bivariate relationships were determined between these variables using Spearman’s rank correlation. Node negativity at time of diagnosis (p = 0.05), p53 mutation (p = 0.02) and bcl2 negativity (p = 0.05) are some of the factors that are known to influence treatment response in our study. p53 over expression, Sphase fractions and ploidy status did not seem to influence treatment response. There was a significant inverse correlation between stage of cancer (p = 0.03) and node positivity (p = 0.06) with bcl2 positivity. There was an inverse correlation between mutation category to treatment response (p = 0.01). The results suggest p53 mutations to be a promising marker in predicting treatment response in carcinoma of the larynx patients.