Clinicopathological Features, Tumor Mutational Burden, and Tumour-Infiltrating Lymphocyte Interplay in ERBB2-Mutated Breast Cancer: In Silico Analysis.

Recent evidence suggests that somatic mutations in ERBB2 activate ERBB2 signaling. These mutations occur at a frequency of approximately 3% in breast cancer (BC). ERBB2 mutations indicate poor prognosis as they are associated with recurrence and metastasis. This study aimed to evaluate the clinicopathological features, immune infiltration levels, tumor mutational burden (TMB), and tumor-infiltrating lymphocytes (TILs) in ERBB2-mutated breast cancer (ERBB2-mutated BC) using a bioinformatic approach and publicly available datasets (i.e., TCGA-BRCA and TIMER2.0). ERBB2-mutated BCs were associated with a high histological grade. ERBB2-mutated BCs comprised invasive breast carcinoma of no special type (21/35, 60%), classic invasive lobular carcinoma (12/35, 34.3%), and pleomorphic invasive lobular carcinoma (2/35, 5.7%). A Kaplan-Meier survival curve demonstrated that ERBB2-mutated BC was associated with a significantly worse prognosis compared to ERBB2 non-mutated BC (p < 0.01). Furthermore, 40% (14/35) of the patients with ERBB2-mutated BC harbored CDH1 mutations. Mutations at L755 and V777 accounted for 30.5% of these mutations in ERBB2-mutated BC, suggesting that these sites are mutational hot spots in BC, particularly in invasive lobular carcinoma. Of the ERBB2-mutated BCs, 8.6% were classified as TIL-high, whereas 77.1% were TILs-low; TMB significantly correlated with TILs (p < 0.05). CD8+ T cell infiltration levels were significantly higher in ERBB2 non-mutated BC. Among ERBB2-mutated BCs, 22.9% were classified as TMB-high, which was significantly higher than the rate in the ERBB2 non-mutated BC (p < 0.01). These findings provide evidence for a link between ERBB2 mutations and high TMB in BC.

Enhanced humoral immunity in breast cancer patients with high serum concentration of anti-HER2 autoantibody.

Humoral immunity plays a substantial role in the suppression of breast cancer. We have revealed that a high serum concentration of anti-HER2 autoantibody (HER2-AAb) is associated with favorable outcomes in patients with invasive breast cancer. Thus, we aimed to clarify the association between high serum concentration of HER2-AAb and humoral immune response in the tumor microenvironment. Out of 500 consecutive patients with invasive breast cancer, we selected those whose HER2-AAb values were high (n = 33) or low (n = 20) based on the distribution of HER2-AAb values of 100 healthy individuals. Tumor and regional lymph node formalin-fixed paraffin-embedded samples prepared from the surgical specimens were subjected to immunohistochemistry. We confirmed that the recurrence-free survival of the high HER2-AAb group was significantly longer than that of the low HER2-AAb group (p = 0.015). The numbers of tumor-infiltrating CD20+ immune cells (ICs) (p < 0.001), IGKC+ICs (p = 0.023), and CXCL13+ ICs (p = 0.044) were significantly higher in the high HER2-AAb group than in the low HER2-AAb group. The number of CD4+ ICs in the B-cell follicles of the regional lymph nodes was also significantly greater in the high HER2-AAb group than in the low HER2-AAb group (p = 0.026). Our findings indicate that a high level of HER2-AAb is associated with enhanced humoral immunity against breast cancer and thus may provide a rationale for the association of HER2-AAb with favorable prognosis.

Heat Shock Protein 90 Family Isoforms as Prognostic Biomarkers and Their Correlations with Immune Infiltration in Breast Cancer.

BACKGROUND: The heat shock protein 90 (HSP90s) family is composed of molecular chaperones composed of four isoforms in humans, which has been widely reported as unregulated in various kinds of cancers. Nevertheless, the role of each HSP90s isoform in prognosis and immune infiltration in distinct subtypes of breast cancer (BRAC) remains unclear. METHODS: Public online databases including the Oncomine, UALCAN, Kaplan-Meier Plotter, Tumor IMmune Estimation Resource (TIMER), Gene Expression Profiling Interactive Analysis (GEPIA), GeneMANIA, and Database for Annotation, Visualization, and Integrated Discovery (DAVID) were integrated to perform bioinformatic analyses and to explore the possible associations among HSP90s gene expression, prognosis, and immune infiltration in BRAC. RESULTS: The mRNA expression of all HSP90s members was elevated in distinct clinical stages and subtypes of BRAC, compared with the normal breast tissue (P < 0.05). Overexpressed HSP90AA1 was associated with poor prognosis, particularly, both short overall survival (OS) and release-free survival (RFS) in Basal-like BRAC patients; overexpressed HSP90AB1 and HSP90B1 were both associated with poor RFS in Luminal A BRAC patients, while overexpressed TRAP1 was associated with favorable RFS in Luminal A BRAC patients. Moreover, HSP90s gene expression in BRAC showed correlations with the infiltration of CD8+ T cells, neutrophils, macrophages, and dendritic cells (DCs), as well as the activation of tumor-associated macrophages (TAMs), DCs, and CD4+ helper T (Th) cells. The underlying mechanisms of HSP90s modulating tumor-infiltrating immune cells (TIICs) might be related with their functions in antigen processing and presentation, major histocompatibility complex (MHC) binding, and assisting client proteins. CONCLUSION: This study demonstrated that HSP90s family genes were overexpressed and might be serve as prognostic biomarkers in subtypes of BRAC. It might be a novel breakthrough point of BRAC treatment to regulate immune infiltration in BRAC microenvironment for more effective anticancer immunity through pharmacological intervention of HSP90s.

Predictive and prognostic role of tumour-infiltrating lymphocytes in breast cancer patients with different molecular subtypes: a meta-analysis.

BACKGROUND: Whether tumour-infiltrating lymphocytes (TILs) play different roles in different molecular subtypes of breast cancer remains unknown. Additionally, their prognostic and predictive value in different molecular subtypes of breast cancer is still controversial. The aim of our meta-analysis was to assess the prognostic and predictive value of TILs in different molecular subtypes of breast cancer by summarizing all relevant studies performing multivariate analysis. METHODS: PubMed, Embase, EBSCO, ScienceDirect, the Cochrane Database and Web of Science were comprehensively searched (until March 2020). Hazard ratios (HRs), odds ratios (ORs) and their 95% confidence intervals (CIs) were used as effect measures to perform our meta-analysis. A random effect model was used. Stata software, version 15 (2017) (StataCorp, College Station, TX, USA) was used to perform the statistical analysis. RESULTS: Thirty-three studies including 18,170 eligible breast cancer patients were analysed. The meta-analysis showed that high TIL expression was significantly associated with increased pathological complete response (pCR) rates after neoadjuvant chemotherapy in patients with the HER2-enriched molecular subtype (OR = 1.137, 95% CI [1.061 ~ 1.218], p < 0.001) and triple-negative breast cancer (TNBC) subtype (OR = 1.120, 95% CI [1.061 ~ 1.182], p < 0.001). However, high TIL expression was not significantly associated with high pCR rates after neoadjuvant chemotherapy in patients with the luminal molecular subtype of breast cancer (OR = 1.154, 95% CI [0.789 ~ 1.690], p = 0.460). We carried out a meta-analysis on the HRs of overall survival (OS) and disease-free survival (DFS) to assess the prognostic value of TILs in breast cancer with different molecular subtypes more deeply. Our meta-analysis confirmed that high TILs were associated with significantly improved DFS in patients with the HER2-enriched molecular subtype [HR = 0.940, 95% CI (0.903 ~ 0.979), p = 0.003] and TNBC molecular subtype [HR = 0.907, 95% CI (0.862 ~ 0.954), p < 0.001]. However, high TILs were not associated with significantly better DFS in patients with the luminal molecular subtype of breast cancer [HR = 0.998, 95% CI (0.977 ~ 1.019), p = 0.840]. Furthermore, the results confirmed that high TILs were significantly related to better OS in patients with the HER2-enriched molecular subtype [HR = 0.910, 95% CI (0.866 ~ 0.957), p < 0.001] and TNBC molecular subtype [HR = 0.869, 95% CI (0.836 ~ 0.904), p < 0.001]. Conversely, the summarized results indicated that high TILs were significantly associated with poor OS in patients with the luminal molecular subtype of breast cancer [HR = 1.077, 95% CI (1.016 ~ 1.141), p = 0.012]. CONCLUSIONS: Our meta-analysis confirms that high TILs are associated with favourable survival and predicts pCR in breast cancer patients with the TNBC and HER2-enriched molecular subtypes.

Insights for the application of TILs and AR in the treatment of TNBC in routine clinical practice.

Triple negative breast cancer (TNBC), usually presenting with a very aggressive phenotype, is a heterogeneous entity. We aim to discuss new biomarkers, suitable for prognostic and predictive purposes. We retrospectively collected clinical variables and immunohistochemical characteristics of early TNBCs, specifically focusing on the prognostic and predictive significance of tumor infiltrating lymphocytes (TILs) and androgen receptor (AR) expression, assessing their correlation with clinical variables. Among 159 patients, TILs were significantly higher in younger patients and with lower BMI, and in tumors with higher ki-67 and greater nodal involvement; conversely, AR was significantly higher in older patients and in tumors with lower ki-67. Interestingly and in line with literature, both TILs level and ARs expression were lower within metastatic sites, in patients who developed distant metastases, compared to those found in the primary site. Small (pT1) and node negative tumors were highly represented and no correlation of either TILs or AR with prognosis could be observed. Our findings support the use of stromal TILs to identify a more aggressive, but chemo-sensitive phenotype, mostly represented in younger women, while AR may identify a less aggressive, slow-growing luminal TNBC subtype, more common among older patients. TILs and AR are worth implementing in routine clinical practice to refine prognosis even if, in our case series, we couldn't identify a significant correlation of the two variables with either disease-free and overall survival.

Characterization of Stromal Tumor-infiltrating Lymphocytes and Genomic Alterations in Metastatic Lobular Breast Cancer.

PURPOSE: Invasive lobular carcinoma (ILC) represents the second most common histologic breast cancer subtype after invasive ductal carcinoma (IDC). While primary ILC has been extensively studied, metastatic ILC has been poorly characterized at the genomic and immune level. EXPERIMENTAL DESIGN: We retrospectively assembled the multicentric EuroILC series of matched primary and metastatic samples from 94 patients with estrogen receptor (ER)-positive ILC. Stromal tumor-infiltrating lymphocytes (sTILs) were assessed by experienced pathologists. Targeted sequencing and low pass whole-genome sequencing were conducted to detect mutations and copy-number aberrations (CNAs). We compared the frequencies of the alterations in EuroILC with those from patients with ER-positive metastatic ILC (n = 135) and IDC (n = 563) from MSK-IMPACT. RESULTS: Low sTIL levels were observed in ILC metastases, with higher levels in the mixed nonclassic histology. Considering ILC metastases from EuroILC and MSK-IMPACT, we observed that >50% of tumors harbor genomic alterations that have previously been associated with endocrine resistance. A matched primary/metastasis comparison in EuroILC revealed mutations (AKT1, ARID1A, ESR1, ERBB2, or NF1) and CNAs (PTEN or NF1 deletion, CYP19A1 amplification) associated with endocrine resistance that were private to the metastasis in 22% (7/32) and 19% (4/21) of patients, respectively. An increase in CDH1, ERBB2, FOXA1, and TBX3 mutations, in CDH1 deletions and a decrease in TP53 mutations was observed in ILC as compared with IDC metastases. CONCLUSIONS: ILC metastases harbor genomic alterations that may potentially explain endocrine resistance in a large proportion of patients, and present genomic differences as compared with IDC metastases.

A subset of activated fibroblasts is associated with distant relapse in early luminal breast cancer.

BACKGROUND: Early luminal breast cancer (BC) represents 70% of newly diagnosed BC cases. Among them, small (under 2 cm) BC without lymph node metastasis (classified as T1N0) have been rarely studied, as their prognosis is generally favorable. Nevertheless, up to 5% of luminal T1N0 BC patients relapse with distant metastases that ultimately prove fatal. The aim of our work was to identify the mechanisms involved in metastatic recurrence in these patients. METHODS: Our study addresses the role that autonomous and non-autonomous tumor cell features play with regard to distant recurrence in early luminal BC patients. We created a cohort of T1N0 luminal BC patients (tumors between 0.5-2 cm without lymph node metastasis) with metastatic recurrence ("cases") and corresponding "controls" (without relapse) matched 1:1 on main prognostic factors: age, grade, and proliferation. We deciphered different characteristics of cancer cells and their tumor micro-environment (TME) by deep analyses using immunohistochemistry. We performed in vitro functional assays and highlighted a new mechanism of cooperation between cancer cells and one particular subset of cancer-associated fibroblasts (CAF). RESULTS: We found that specific TME features are indicative of relapse in early luminal BC. Indeed, quantitative histological analyses reveal that "cases" are characterized by significant accumulation of a particular CAF subset (CAF-S1) and decrease in CD4+ T lymphocytes, without any other association with immune cells. In multivariate analysis, TME features, in particular CAF-S1 enrichment, remain significantly associated with recurrence, thereby demonstrating their clinical relevance. Finally, by performing functional analyses, we demonstrated that CAF-S1 pro-metastatic activity is mediated by the CDH11/osteoblast cadherin, consistent with bones being a major site of metastases in luminal BC patients. CONCLUSIONS: This study shows that distant recurrence in T1N0 BC is strongly associated with the presence of CAF-S1 fibroblasts. Moreover, we identify CDH11 as a key player in CAF-S1-mediated pro-metastatic activity. This is independent of tumor cells and represents a new prognostic factor. These results could assist clinicians in identifying luminal BC patients with high risk of relapse. Targeted therapies against CAF-S1 using anti-FAP antibody or CDH11-targeting compounds might help in preventing relapse for such patients with activated stroma.

Comparison of three scoring methods using the FDA-approved 22C3 immunohistochemistry assay to evaluate PD-L1 expression in breast cancer and their association with clinicopathologic factors.

BACKGROUND: In the evaluation of PD-L1 expression to select patients for anti-PD-1/PD-L1 treatment, uniform guidelines that account for different immunohistochemistry assays, different cell types and different cutoff values across tumor types are lacking. Data on how different scoring methods compare in breast cancer are scant. METHODS: Using FDA-approved 22C3 diagnostic immunohistochemistry assay, we retrospectively evaluated PD-L1 expression in 496 primary invasive breast tumors that were not exposed to anti-PD-1/PD-L1 treatment and compared three scoring methods (TC: invasive tumor cells; IC: tumor-infiltrating immune cells; TCIC: a combination of tumor cells and immune cells) in expression frequency and association with clinicopathologic factors. RESULTS: In the entire cohort, positive PD-L1 expression was observed in 20% of patients by TCIC, 16% by IC, and 10% by TC, with a concordance of 87% between the three methods. In the triple-negative breast cancer patients, positive PD-L1 expression was observed in 35% by TCIC, 31% by IC, and 16% by TC, with a concordance of 76%. Associations between PD-L1 and clinicopathologic factors were investigated according to receptor groups and whether the patients had received neoadjuvant chemotherapy. The three scoring methods showed differences in their associations with clinicopathologic factors in all subgroups studied. Positive PD-L1 expression by IC was significantly associated with worse overall survival in patients with neoadjuvant chemotherapy and showed a trend for worse overall survival and distant metastasis-free survival in triple-negative patients with neoadjuvant chemotherapy. Positive PD-L1 expression by TCIC and TC also showed trends for worse survival in different subgroups. CONCLUSIONS: Our findings indicate that the three scoring methods with a 1% cutoff are different in their sensitivity for PD-L1 expression and their associations with clinicopathologic factors. Scoring by TCIC is the most sensitive way to identify PD-L1-positive breast cancer by immunohistochemistry. As a prognostic marker, our study suggests that PD-L1 is associated with worse clinical outcome, most often shown by the IC score; however, the other scores may also have clinical implications in some subgroups. Large clinical trials are needed to test the similarities and differences of these scoring methods for their predictive values in anti-PD-1/PD-L1 therapy.

Tumor-infiltrating lymphocytes are associated with poor prognosis in invasive lobular breast carcinoma.

The prognostic impact of tumor-infiltrating lymphocytes (TILs) within invasive lobular carcinoma (ILC) remains to be better characterized. In estrogen receptor (ER)-negative invasive ductal carcinomas of no special type (IDC-NST), TILs are associated with good prognosis. The aim of this study was to examine TILs in ILC, with particular focus on prognostic and clinicopathologic features. A cohort comprising 459 consecutive ILCs diagnosed in a single institution from 2005 to 2008 met the eligibility criteria for this study. The percentage of tumor area occupied by TILs was quantified by two breast pathologists and categorized into three groups: no TILs, ≤5%, >5%. Clinicopathologic features were tested by Fisher's exact tests or Chi2 tests. Overall survival (OS) and invasive disease-free survival (iDFS) were estimated by Kaplan-Meier and Cox proportional hazard statistics. There were 239 TIL-negative cases, 185 cases with ≤5% TILs, and 35 cases with >5% TILs. TILs were associated with younger age, larger tumors, lymph node involvement, poor Nottingham prognostic index, HER2 amplification, multinucleation, and prominent nucleoli (p < 0.05). Poor OS was significantly associated with increasing TILs in the univariate Cox proportional hazards model (p < 0.001) and Kaplan-Meier estimator (p < 0.05, log-rank test). Similar results were observed for iDFS (p = 0.004 for Cox univariate and p = 0.005 for log-rank test). Notably, TILs can identify a subset of ILC patients with poor OS independently of molecular subtype and lymph node metastases (multivariate Cox, p < 0.001, OS hazard ratio (HR) = 4.38 and HR = 6.15, for ≤5% and >5% TILs, respectively, vs. absence of TILs). Prominent nucleoli was the only nuclear feature associated with poor OS (p = 0.05) and iDFS (p = 0.05) in univariate Cox survival analysis. TILs represent a promising new morphologic biomarker associated with poor outcome of ILC, in contrast with that observed in ER-negative IDC-NST.

Tumour-infiltrating lymphocytes and response to neoadjuvant letrozole in patients with early oestrogen receptor-positive breast cancer: analysis from a nationwide phase II DBCG trial.

BACKGROUND: The presence of tumour-infiltrating lymphocytes (TILs) is associated with response to neoadjuvant chemotherapy among patients with triple-negative and HER2-positive breast cancer. However, the significance of TILs is less clear in luminal breast cancer. Here, we in postmenopausal patients with primary oestrogen receptor-positive (ER+), HER2 normal, operable breast cancer assessed the importance of inducing TILs during 4 months of letrozole on response in a neoadjuvant phase II study. METHODS: Participants were postmenopausal women with ER+, HER2 normal operable breast cancer assigned to 4 months of neoadjuvant letrozole. Pretreatment core biopsies and surgical specimens were assessed centrally for the percentage of TILs on haematoxylin and eosin-stained slides according to the International Immuno-Oncology Biomarker Working Group on Breast Cancer guidelines. Pathological response was assessed by the Residual Cancer Burden (RCB) index and a modified Miller-Payne grading system and was analysed according to change in TILs. RESULTS: Tumour specimens were available from 106 of the 112 patients treated per protocol. TIL concentration increased with mean 6.8 percentage point (p < 0.0001) during treatment (range - 39 to 60). An increase in TILs was significantly associated with pathological response with OR = 0.71 (95% CI 0.53-0.96; p = 0.02) per 10% absolute increase for pathological response and correspondingly OR = 0.56 (95% CI 0.40-0.78; p = 0.0007) for lower RCB index per 10% increase. CONCLUSION: Increasing TILs during letrozole was significantly associated with a poor treatment response. An increase in TILs during endocrine therapy might imply immunogenicity, and these patients could be targetable by immunotherapy. TRIAL REGISTRATION: ClinicalTrials.govNCT00908531, registered 27 May 2009.

Targeting ANXA1 abrogates Treg-mediated immune suppression in triple-negative breast cancer.

BACKGROUND: Regulatory T (Treg) cells play a negative role in anti-tumor immunity against triple-negative breast cancer, so it is of great significance to find the potential therapeutic target of Treg cells. METHODS: First, Annexin A1 (ANXA1) expression and survival of patients with breast cancer were analyzed using TCGA data. Then plasma ANXA1 levels in patients with malignant and benign breast tumors were detected by ELISA. Next, the effect of ANXA1 on Treg cells was studied through suppressive assays, and how ANXA1 regulates the function of Treg cells was detected by RNA sequencing. Finally, the in vivo experiment in balb/c mice was conducted to test whether the ANXA1 blocker Boc1 could shrink tumors and affect the function of Treg cells. RESULTS: Our data suggest that ANXA1 expression is associated with lower survival and a higher risk of breast malignancy. Suppressive assays show that ANXA1 can enhance the inhibition function of Treg cells. RNA-Sequencing results indicate that Boc1 could reduce the expression of granzyme A mRNA in Treg cells. Animal experiments have been done to show that Boc1 can reduce tumor size and down regulate Treg cell function. CONCLUSIONS: ANXA1 can enhance the function of Treg cells and reduce the survival rate of patients with breast cancer. Targeting ANXA1 can reduce Treg cell function and shrink breast tumors.

Prospective, randomized, single-blinded, multi-center phase II trial of two HER2 peptide vaccines, GP2 and AE37, in breast cancer patients to prevent recurrence.

PURPOSE: AE37 and GP2 are HER2 derived peptide vaccines. AE37 primarily elicits a CD4+ response while GP2 elicits a CD8+ response against the HER2 antigen. These peptides were tested in a large randomized trial to assess their ability to prevent recurrence in HER2 expressing breast cancer patients. The primary analyses found no difference in 5-year overall disease-free survival (DFS) but possible benefit in subgroups. Here, we present the final landmark analysis. METHODS: In this 4-arm, prospective, randomized, single-blinded, multi-center phase II trial, disease-free node positive and high-risk node negative breast cancer patients enrolled after standard of care therapy. Six monthly inoculations of vaccine (VG) vs. control (CG) were given as the primary vaccine series with 4 boosters at 6-month intervals. Demographic, safety, immunologic, and DFS data were evaluated. RESULTS: 456 patients were enrolled; 154 patients in the VG and 147 in CG for AE37, 89 patients in the VG and 91 in CG for GP2. The AE37 arm had no difference in DFS as compared to CG, but pre-specified exploratory subgroup analyses showed a trend towards benefit in advanced stage (p = 0.132, HR 0.573 CI 0.275-1.193), HER2 under-expression (p = 0.181, HR 0.756 CI 0.499-1.145), and triple-negative breast cancer (p = 0.266, HR 0.443 CI 0.114-1.717). In patients with both HER2 under-expression and advanced stage, there was significant benefit in the VG (p = 0.039, HR 0.375 CI 0.142-0.988) as compared to CG. The GP2 arm had no significant difference in DFS as compared to CG, but on subgroup analysis, HER2 positive patients had no recurrences with a trend toward improved DFS (p = 0.052) in VG as compared to CG. CONCLUSIONS: This phase II trial reveals that AE37 and GP2 are safe and possibly associated with improved clinical outcomes of DFS in certain subgroups of breast cancer patients. With these findings, further evaluations are warranted of AE37 and GP2 vaccines given in combination and/or separately for specific subsets of breast cancer patients based on their disease biology.

Rare Breast Carcinoma with Paradoxical Plasma Cell Immunoprofile: A Case Report.

Plasma cell features are encountered in a variety of non-plasma cell neoplasias, especially carcinomas of a discohesive type, such as those occurring in the digestive tract and breast. Lobular carcinomas of the breast present themselves in a variety of architectural patterns and many cell morphologies, including plasmacytoid types. A matching plasma cell phenotype is sometimes an associated feature. We report a case of a moderate grade invasive lobular carcinoma with focal plasmacytoid morphology and aberrant expression of plasma cell markers in a patient previously diagnosed with multiple myeloma. Paradoxical plasma cell immunoprofiles can be encountered in many malignancies, causing serious diagnostic problems, even more so with those occurring in discohesive carcinomas in multiple myeloma patients.

Tumour-infiltrating lymphocytes (TILs) and BRCA-like status in stage III breast cancer patients randomised to adjuvant intensified platinum-based chemotherapy versus conventional chemotherapy.

BACKGROUND: The prognostic value of tumour-infiltrating lymphocytes (TILs) differs by breast cancer (BC) subtype. The aim of this study was to evaluate TILs in stage III BC in the context of BRCA1/2-like phenotypes and association with outcome and benefit of intensified platinum-based chemotherapy. PATIENTS AND METHODS: Patients participated in a randomised controlled trial of adjuvant intensified platinum-based chemotherapy versus conventional anthracycline-based chemotherapy carried out between 1993 and 1999 in stage III BC. Stromal TILs were scored according to International guidelines in these human epidermal growth factor receptor 2 (HER2)-negative tumours. BRCA-profiles were determined using Comparative Genomic Hybridization. RESULTS: TIL levels were evaluated in 248 BCs. High TILs were associated with Triple Negative BC (TNBC). BRCA-like tumours harboured higher TILs compared to non-BRCA-like tumours (median TILs of 20% versus 10%, p < 0.01). TIL levels in BRCA1-like tumours were higher compared to BRCA2-like tumours (median TILs of 20% versus 10%, p < 0.001). These correlations remained significant within the oestrogen (ER)-positive subgroup, however not within the TNBC subgroup. In this stage III BC cohort, high TIL level was associated with favourable outcome (TILs per 10% increment, recurrence-free survival (RFS): multivariate hazard ratio (HR) 0.82, 95% confidence interval (CI) 0.71-0.94, p = 0.01; overall survival (OS): multivariate HR 0.80, 95% CI 0.68-0.94, p = 0.01). There was no significant interaction between TILs and benefit of intensified platinum-based chemotherapy. CONCLUSION: In this high-risk breast cancer cohort, high TILs were associated with TNBC and BRCA1-like status. Within the ER-positive subgroup, TIL levels were higher in BRCA1-like compared to BRCA2-like tumours. When adjusted for clinical characteristics, TILs were significantly associated with a more favourable outcome in stage III BC patients.

Immune Profiles of Tumor Microenvironment and Clinical Prognosis among Women with Triple-Negative Breast Cancer.

BACKGROUND: The impact of the immune landscape of the microenvironment on cancer progression is not well understood for triple-negative breast cancer (TNBC). We, therefore, aimed to examine the association of immune cell enrichment scores as a proxy for immune profiles of tumor microenvironment with TNBC prognosis. METHODS: We included 76 patients with TNBC diagnosed between 2008 to 2016 in West China Hospital and 158 patients with TNBC from The Cancer Genome Atlas. On the basis of transcriptome data, we calculated the overall ImmuneScore and type-specific enrichment scores for 34 types of immune cells, using xCell, a gene signature-based method. HRs of recurrence-free survival (RFS) and overall survival (OS) were calculated by Cox proportional hazards models. RESULTS: During the median follow-up time of 2.8 (0.1-9.8) years, 42 patients had a recurrence, and 34 patients died. The overall ImmuneScore and most immune cell enrichment scores were relatively higher in tumors than normal tissues. A higher enrichment score of plasma cells was associated with favorable RFS [HR 0.45; 95% confidence interval (CI), 0.27-0.73] and OS (HR 0.32; 95% CI, 0.17-0.61). The score of CD4+ central memory T cell (Tcm) was negatively associated with RFS (HR 1.52; 95% CI, 1.17-1.97). Besides, CD4+ Tcm enrichment score was higher in invasive tumors that were not ductal/lobular carcinoma (OR 1.59; 95% CI, 1.06-2.37). CONCLUSIONS: Our findings suggest that plasma cells and CD4+ Tcm in the tumor microenvironment may play a role in the subsequent progression of TNBC. IMPACT: This study provides evidence of the role of immune cells in TNBC progression that may have clinical utility.

Rabbit monoclonal E-cadherin antibody: A cost-effective alternative to mouse monoclonal antibody in distinguishing ductal carcinoma in situ from lobular carcinoma in situ.

Rabbit monoclonal antibody (RabMAb) demonstrates higher sensitivity without sacrificing specificity than mouse monoclonal antibody (MMAb). MMAb against E-cadherin stain is heavily utilized in distinguishing ductal carcinoma in situ (DCIS) from lobular carcinoma in situ (LCIS). We aimed to compare the E-cadherin stain using RabMAb vs MMAb in distinguishing DCIS from LCIS. One hundred and seventeen in situ breast carcinomas (55 DCIS, 58 LCIS, and 4 DCIS and LCIS) were studied. Sections from a representative block of each were stained with RabMAb [EP700Y] and MMAb [36B5]. Scanned images of stained slides were compared in tandem. All DCIS cases (59/59) showed comparable staining by RabMAb and MMAb. Comparable staining was also observed in all but one case of LCIS (61/62; 98%). One case of pleomorphic LCIS showed mostly complete, weak to moderately intense membranous staining with RabMAb and fragmented, weak membranous staining with MMAb. Consistently better staining quality was observed in slides stained by RabMAb vs MMAb. RabMAb and MMAb against E-cadherin were diagnostically equivalent with the exception of one case where RabMAb may have led to diagnostic misinterpretation. However, the not insignificant cost savings and easier interpretation using RabMAb may justify the risk of misinterpretation of increased staining in rare cases, largely avertable with proper training.

A high-risk luminal A dominant breast cancer subtype with increased mobility.

PURPOSE: Breast cancer is a heterogeneous disease, and although advances in molecular subtyping have been achieved in recent years, most subtyping strategies target individual genes independent of one another and primarily concentrate on proliferative markers. The contributions of biological processes and immune patterns have been neglected in breast cancer subtype stratification. METHODS: We performed a gene set variation analysis to simplify the information on biological processes using hallmark terms and to decompose immune cell data using the immune cell gene terms on 985 breast invasive ductal/lobular carcinoma RNAseq samples in the TCGA database. RESULTS: The samples were gathered into three clusters following implementation of the t-SNE and DBSCAN algorithms and were categorized as 'hallmark-tsne' subtypes. Here, we identified a high-risk luminal A dominant breast cancer subtype (C3) that displayed increased motility, cancer stem cell-like features, a higher expression of hormone/luminal-related genes, a lower expression of proliferation-related genes and immune dysfunction. With regard to immune dysfunction, we observed that the motility-increased C3 subtype exhibited high granulocyte colony stimulating factor (G-CSF) expression accompanied by neutrophil aggregation. Cancer cells that produce high levels of G-CSF can stimulate neutrophils to form neutrophil extracellular traps, which promote cancer cell migration. This finding sheds light on one potential explanation for why the C3 subtype correlates with poor prognosis. CONCLUSIONS: The hallmark-tsne subtypes confirmed again that even the luminal A subtype is heterogeneous and can be further subdivided. The biological processes and immune heterogeneity of breast cancer must be understood to facilitate the improvement of clinical treatments.

Interaction of Breast Cancer and Insulin Resistance on PD1 and TIM3 Expression in Peripheral Blood CD8 T Cells.

Epidemiological evidence points to a link between insulin resistance (IR) and breast cancer (BrCA). Insulin plays a role in CD8+ T cells (CD8T) differentiation and function and affects adipocytokines levels. CD8T activity in BrCA is associated with favorable outcome; while PD1 and TIM3 are markers of CD8T exhaustion and play critical roles in the negative regulation of T cell responses. Patients with (BrCA) have high expression levels of PD1 on circulating. Therefore, we hypothesized that BrCA and IR could affect PD1 and/or TIM3 expression on circulating CD8T. We determine PD1 and TIM3 expression on CD8T and analyze the relationship of CD8T phenotype with serum insulin and plasma adipocytokines levels in the different groups. We enrolled four groups of treatment-naive patients: women without neoplasms (Neo-)/without IR (IR-), Neo-/with IR (IR+), BrCa/IR- and BrCa/IR+. We found interactions between BrCA and IR with respect to TIM3 on naïve and central memory (CM) CD8T subsets. Furthermore, BrCA had a greater PD1 + TIM3- CD8T frequency in CD8T subsets than Neo-. IR+ presented a significantly lower PD1 + TIM3- frequency in CD8T subsets compare to Non-IR. In addition, we found a negative correlation between insulin levels, HOMA and frequency of PD1 + TIM3- in CD8T and a positive correlation between adiponectin levels and the frequency PD1 + TIM3- in CD8T. The increased expression of PD1 on different subsets of CD8T from BrCa patients is consistent with immunological tolerance, whereas IR has a contrary effect. IR could have a deleterious role in the activation of CD8T that can be relevant to new BrCa immunotherapy.

Cdh1 and Pik3ca Mutations Cooperate to Induce Immune-Related Invasive Lobular Carcinoma of the Breast.

CDH1 and PIK3CA are the two most frequently mutated genes in invasive lobular carcinoma (ILC) of the breast. Transcription profiling has identified molecular subtypes for ILC, one of which, immune-related (IR), is associated with gene expression linked to lymphocyte and macrophage infiltration. Here, we report that deletion of Cdh1, together with activation of Pik3ca in mammary epithelium of genetically modified mice, leads to formation of IR-ILC-like tumors with immune cell infiltration, as well as gene expression linked to T-regulatory (Treg) cell signaling and activation of targetable immune checkpoint pathways. Interestingly, these tumors show enhanced Rac1- and Yap-dependent transcription and signaling, as well as sensitivity to PI3K, Rac1, and Yap inhibitors in culture. Finally, high-dimensional immunophenotyping in control mouse mammary gland and IR-ILC tumors by mass cytometry shows dramatic alterations in myeloid and lymphoid populations associated with immune suppression and exhaustion, highlighting the potential for therapeutic intervention via immune checkpoint regulators.

Hot Spot and Whole-Tumor Enumeration of CD8+ Tumor-Infiltrating Lymphocytes Utilizing Digital Image Analysis Is Prognostic in Triple-Negative Breast Cancer.

BACKGROUND: CD8+ tumor-infiltrating lymphocytes (TILs) have emerged as a prognostic indicator in triple-negative breast cancer (TNBC). There is debate surrounding the prognostic value of hot spots for CD8+ TIL enumeration. METHODS: We compared hot spot versus whole-tumor CD8+ TIL enumeration in prognosticating TNBC using immunohistochemistry on whole tissue sections and quantification by digital image analysis (Halo imaging analysis software; Indica Labs, Corrales, NM). A wide range of clinically relevant hot spot sizes was evaluated. RESULTS: CD8+ TIL enumeration was independently statistically significant for all hot spot sizes and whole-tumor annotations for disease-free survival by multivariate analysis. A 10× objective (2.2 mm diameter) hot spot was found to correlate significantly with overall survival (P = .04), while the remaining hot spots and whole-tumor CD8+ TIL enumeration did not (P > .05). Statistical significance was not demonstrated when comparing between hot spots and whole-tumor annotations, as the groups had overlapping confidence intervals. CONCLUSION: CD8+ TIL hot spot enumeration is equivalent to whole-tumor enumeration for prognostication in TNBC and may serve as a good alternative methodology in future studies and clinical practice.