Pathological Changes Following Neoadjuvant Endocrine Therapy (NAET): A Multicentre Study of 391 Breast Cancers.

Oestrogen receptor (ER)-positive breast cancer (BC) is generally well responsive to endocrine therapy. Neoadjuvant endocrine therapy (NAET) is increasingly being used for downstaging ER-positive tumours. This study aims to analyse the effect of NAET on a well-characterised cohort of ER-positive BC with particular emphasis on receptor expression. This is a retrospective United Kingdom (UK) multicentre study of 391 patients who received NAET between October 2012 and October 2020. Detailed analyses of the paired pre- and post-NAET morphological changes and hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) expression were performed. The median duration of NAET was 86 days, with median survival and overall survival rates of 380 days and 93.4%, respectively. A total of 90.3% of cases achieved a pathological partial response, with a significantly higher rate of response in the HER2-low cancers. Following NAET, BC displayed some pathological changes involving the tumour stroma including central scarring and an increase in tumour infiltrating lymphocytes (TILs) and tumour cell morphology. Significant changes associated with the duration of NAET were observed in tumour grade (30.6% of cases), with downgrading identified in 19.3% of tumours (p < 0.001). The conversion of ER status from positive to low or negative was insignificant. The conversion of progesterone receptor (PR) and HER2 status to negative status was observed in 31.3% and 38.1% of cases, respectively (p < 0.001). HER2-low breast cancer decreased from 63% to 37% following NAET in the paired samples. Significant morphological and biomarker changes involving PR and HER2 expression occurred following NAET. The findings support biomarker testing on pre-treatment core biopsies and post-treatment residual carcinoma.

Evaluation of the diagnostic utility of NCOA3, Maspin and VHL protein expression in pancreatic ductal adenocarcinoma: An immunohistochemical study.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal tumor with a high mortality rate. The distinction between PDAC and chronic pancreatitis is sometimes challenging on routine histopathological examination, highlighting the need to identify biomarkers that can facilitate this distinction. This retrospective study was conducted to evaluate the diagnostic utility of nuclear receptor co-activator 3 (NCOA3), Maspin and Von Hippel-Lindau protein (VHL) immunostaining in PDAC. Eighty cases of PDAC, 46 cases of chronic pancreatitis and 53 normal pancreatic tissue were immunohistochemically assessed using NCOA3, Maspin and VHL antibodies on sections from a tissue microarray. NCOA3, Maspin and VHL were positive in 90 %, 100 % and 35 %, of PDAC cases respectively, whereas NCOA3, Maspin and VHL expressions were positive in 3.8 %, 0 and 100 % of normal pancreatic tissue and in 15.2 %, 21.7 % and 97.8 % of chronic pancreatitis cases respectively. Significant differences were observed between PDAC and other groups regarding NCOA3, Maspin and VHL expression (p < 0.001). The H scores of NCOA3, Maspin and VHL could significantly distinguish between PDAC and normal cases with high sensitivity (90 %, 100 % and 98.75 % respectively) and specificity (100 %, 100 % and 96.23 % respectively). Similar findings were found in the distinction between PDAC and chronic pancreatitis (Sensitivity: 90 %, 95.25 % and 98.75 %; specificity: 100 %, 100 % and 93.48 % for NCOA3, Maspin and VHL respectively). In conclusion, NCOA3 and Maspin were found to be significantly expressed in PDAC compared to non-tumorous tissue while VHL was significantly expressed in non-tumorous tissue. A panel of NCOA3, Maspin and VHL could potentially distinguish PDAC from non-tumorous pancreatic tissue.

Concordance between ER, PR, Ki67, and HER2-low expression in breast cancer by MammaTyper RT-qPCR and immunohistochemistry: implications for the practising pathologist.

BACKGROUND: There are limited data on the role of multigene tests and their correlation with immunohistochemistry (IHC), especially on core biopsy. MammaTyper is a quantitative conformite Europeeanne (CE) marked, National Institute for Health and Care excellence (NICE) approved, in in vitro diagnostic quantitative real-time polymerase chain reaction (RT-qPCR) test for assessment of mRNA expression of four biomarkers (ESR1, PGR, ERBB2, MKI67). METHODS: We evaluated the concordance of MammaTyper with oestrogen receptor (ER), progesterone receptor (PR), HER2, and Ki67 by IHC on 133 core needle biopsies of breast cancer. HER2 was positive if IHC 3+ or 2+ and fluorescence in situ hybridization (FISH)-amplified. Global and hotspot Ki67 expression was analysed using a cutoff of ≥20% assessed manually and by digital image analysis. Agreements were expressed as overall percent agreement (OPA), positive percent agreement (PPA), negative percent agreement (NPA), and Cohen's kappa. RESULTS: RT-qPCR results of ESR1 were highly concordant with IHC with OPA of 94.7% using 1% cutoff and 91.7% when the low ER-positive category was included. The PPA and NPA between RT-qPCR and IHC for PR was 91.5% and 88.0%, respectively, when using the 1% cutoff. For ERBB2/HER2, the OPA was 95% and the PPA was 84.6%. 40 of 72 HER2 IHC score 0 tumours were classified as ERBB2 low. Best concordance between MKI67 by MammaTyper and Ki67 IHC was achieved using hotspot digital image analysis (OPA: 87.2%, PPA: 90.6%, NPA: 80%). CONCLUSION: RT-qPCR-based assessment of the mRNA expression of ESR1, PGR, ERBB2, and MKI67 showed high concordance with IHC, suggesting that the MammaTyper test on core needle biopsies represents a reliable, efficient, and reproducible alternative for breast cancer classification and refining HER2 low categorisation.

Tspan6 stimulates the chemoattractive potential of breast cancer cells for B cells in an EV- and LXR-dependent manner.

The immune microenvironment in breast cancer (BCa) is controlled by a complex network of communication between various cell types. Here, we find that recruitment of B lymphocytes to BCa tissues is controlled via mechanisms associated with cancer cell-derived extracellular vesicles (CCD-EVs). Gene expression profiling identifies the Liver X receptor (LXR)-dependent transcriptional network as a key pathway that controls both CCD-EVs-induced migration of B cells and accumulation of B cells in BCa tissues. The increased accumulation oxysterol ligands for LXR (i.e., 25-hydroxycholesterol and 27-hydroxycholesterol) in CCD-EVs is regulated by the tetraspanin 6 (Tspan6). Tspan6 stimulates the chemoattractive potential of BCa cells for B cells in an EV- and LXR-dependent manner. These results demonstrate that tetraspanins control intercellular trafficking of oxysterols via CCD-EVs. Furthermore, tetraspanin-dependent changes in the oxysterol composition of CCD-EVs and the LXR signaling axis play a key role in specific changes in the tumor immune microenvironment.

Inter- and Intra-Observer Agreement of PD-L1 SP142 Scoring in Breast Carcinoma-A Large Multi-Institutional International Study.

The assessment of PD-L1 expression in TNBC is a prerequisite for selecting patients for immunotherapy. The accurate assessment of PD-L1 is pivotal, but the data suggest poor reproducibility. A total of 100 core biopsies were stained using the VENTANA Roche SP142 assay, scanned and scored by 12 pathologists. Absolute agreement, consensus scoring, Cohen's Kappa and intraclass correlation coefficient (ICC) were assessed. A second scoring round after a washout period to assess intra-observer agreement was carried out. Absolute agreement occurred in 52% and 60% of cases in the first and second round, respectively. Overall agreement was substantial (Kappa 0.654-0.655) and higher for expert pathologists, particularly on scoring TNBC (6.00 vs. 0.568 in the second round). The intra-observer agreement was substantial to almost perfect (Kappa: 0.667-0.956), regardless of PD-L1 scoring experience. The expert scorers were more concordant in evaluating staining percentage compared with the non-experienced scorers (R2 = 0.920 vs. 0.890). Discordance predominantly occurred in low-expressing cases around the 1% value. Some technical reasons contributed to the discordance. The study shows reassuringly strong inter- and intra-observer concordance among pathologists in PD-L1 scoring. A proportion of low-expressors remain challenging to assess, and these would benefit from addressing the technical issues, testing a different sample and/or referring for expert opinions.

Characterization of the Immune Microenvironment in Inflammatory Breast Cancer Using Multiplex Immunofluorescence.

INTRODUCTION: Inflammatory breast cancer (IBC) is an aggressive form of breast cancer with a poorly characterized immune microenvironment. METHODS: We used a five-colour multiplex immunofluorescence panel, including CD68, CD4, CD8, CD20, and FOXP3 for immune microenvironment profiling in 93 treatment-naïve IBC samples. RESULTS: Lower grade tumours were characterized by decreased CD4+ cells but increased accumulation of FOXP3+ cells. Increased CD20+ cells correlated with better response to neoadjuvant chemotherapy and increased CD4+ cells infiltration correlated with better overall survival. Pairwise analysis revealed that both ER+ and triple-negative breast cancer were characterized by co-infiltration of CD20 + cells with CD68+ and CD4+ cells, whereas co-infiltration of CD8+ and CD68+ cells was only observed in HER2+ IBC. Co-infiltration of CD20+, CD8+, CD4+, and FOXP3+ cells, and co-existence of CD68+ with FOXP3+ cells correlated with better therapeutic responses, while resistant tumours were characterized by co-accumulation of CD4+, CD8+, FOXP3+, and CD68+ cells and co-expression of CD68+ and CD20+ cells. In a Cox regression model, response to therapy was the most significant factor associated with improved patient survival. CONCLUSION: Those results reveal a complex unique pattern of distribution of immune cell subtypes in IBC and provide an important basis for detailed characterization of molecular pathways that govern the formation of IBC immune landscape and potential for immunotherapy.

Clinicopathological features and androgen receptor expression in triple negative breast cancer at Lagos, Nigeria.

Introduction: Androgen receptor (AR) is one of the predominant nuclear hormone receptors in invasive breast cancer and can be explored as a biomarker of response for targeted anti-androgen therapy, especially in the setting of triple negative breast cancer (TNBC). Luminal AR is a distinct subtype amongst TNBC cases following gene expression studies. TNBC is higher in Africans (23%-82%) and African-Americans (29.8%) compared to Caucasian (10%-15%) breast cancer patients; however, there is a paucity of data on AR expression in this population. The aim of this study is to determine the expression of AR and the proportion of AR positive cancers in TNBCs at the Lagos University Teaching Hospital, Lagos, Nigeria. Methodology: Out of 99 reviewed cases, 78 formalin fixed, paraffin embedded TNBC cases were assembled into a tissue microarray, stained and analysed for AR expression using immunohistochemistry. Results: The mean age of the TNBC patients was 49.3 years (range: 20-80 years). The histologic types in this study were invasive carcinoma (no special type) 75.4%; metaplastic carcinoma 21.4%; lobular carcinoma and mucinous carcinoma 1.6% each. Of 61 TNBC cases analysed, 37.7% were AR positive and 62.3% were AR negative, making the latter to become quadruple negative breast cancers. There was a significant association between age and AR expression (p = 0.02). In the subjects that expressed AR positivity, patients below 50 years accounted for 34.8% (8 of 23) while 65.2% (15 of 23) were above 50 years. There was no significant association between AR expression and histologic type or tumour grade. Conclusion: Over a third of this Nigerian TNBC cohort study is AR+. This warrants further exploration of the predictive and prognostic significance of its expression amongst TNBC and the potential for targeted therapy, specifically androgen antagonists to improve the outcome of this disease with limited therapeutic options.

Retrospective observational study of HER2 immunohistochemistry in borderline breast cancer patients undergoing neoadjuvant therapy, with an emphasis on Group 2 (HER2/CEP17 ratio ≥2.0, HER2 copy number <4.0 signals/cell) cases.

BACKGROUND: The ASCO/CAP guidance on HER2 testing in breast cancer (BC) has recently changed. Group 2 tumours with immunohistochemistry score 2+ and HER2/CEP17 ratio ≥2.0 and HER2 copy number <4.0 signals/cell were re-classified as HER2 negative. This study aims to examine the response of Group 2 tumours to neoadjuvant chemotherapy (NACT). METHODS: 749 BC cases were identified from 11 institutions. The association between HER2 groups and pathological complete response (pCR) was assessed. RESULTS: 54% of immunohistochemistry HER2 positive (score 3+) BCs showed pCR, compared to 19% of immunohistochemistry 2+ FISH amplified cases. 27% of Group 2 treated with HER2 targeted therapy achieved pCR, compared to 19 and 11% in the combined Groups 1 + 3 and Groups 4 + 5, respectively. No difference in pCR rates was identified between Group 2 and Group 1 or combined Groups 1 + 3. However, Group 2 response rate was higher than Groups 4 + 5 (p = 0.017). CONCLUSION: No difference in pCR was detected in tumours with a HER2/CEP17 ratio ≥2.0 and a HER2 score 2+ by IHC when stratified by HER2 gene copy number. Our data suggest that ASCO/CAP HER2 Group 2 carcinomas should be evaluated further with respect to eligibility for HER2 targeted therapy.

Predictors of pathological complete response to neoadjuvant treatment and changes to post-neoadjuvant HER2 status in HER2-positive invasive breast cancer.

The response of human epidermal growth factor receptor2 (HER2)- positive breast cancer (BC) patients to anti-HER2 targeted therapy is significant. However, the response is not uniform and a proportion of HER2-positive patients do not respond. This study aims to identify predictors of response in the neoadjuvant treatment and to assess the discordance rate of HER2 status between pre- and post-treatment specimens in HER2-positive BC patients. The study group comprised 500 BC patients treated with neoadjuvant chemotherapy (NACT) and/or neoadjuvant anti-HER2 therapy and surgery who had tumours that were 3+ or 2+ with HER2 immunohistochemistry (IHC). HER2 IHC 2+ tumours were classified into five groups by fluorescence in situ hybridisation (FISH) according to the 2018 ASCO/CAP guidelines of which Groups 1, 2 and 3 were considered HER2 amplified. Pathological complete response (pCR) was more frequent in HER2 IHC 3+ tumours than in HER2 IHC 2+/HER2 amplified tumours, when either in receipt of NACT alone (38% versus 13%; p = 0.22) or neoadjuvant anti-HER2 therapy (52% versus 20%; p < 0.001). Multivariate logistic regression analysis showed that HER2 IHC 3+ and histological grade 3 were independent predictors of pCR following neoadjuvant anti-HER2 therapy. In the HER2 IHC 2+/HER2 amplified tumours or ASCO/CAP FISH Group 1 alone, ER-negativity was an independent predictor of pCR following NACT and/or neoadjuvant anti-HER2 therapy. In the current study, 22% of HER2-positive tumours became HER2-negative by IHC and FISH following neoadjuvant treatment, the majority (74%) HER2 IHC 2+/HER2 amplified tumours. Repeat HER2 testing after neoadjuvant treatment should therefore be considered.

Morphological and molecular changes following neoadjuvant endocrine therapy of oestrogen receptor-positive breast cancer: implications for clinical practice.

AIMS: Neoadjuvant endocrine therapy (NAET) is used in the management of oestrogen receptor (ER)-positive breast cancer. The optimal method for histological assessment of response and the effect of NAET on the tumour morphology, grade and molecular profile remain unclear. The aim of this study is to investigate the NAET effect on tumour type, grade and molecular profile by analysing a well-characterised cohort of breast cancer samples in a single large UK tertiary referral centre, and to provide guidance on the pathological assessment of those lesions to inform adjuvant management and prognosis. METHODS AND RESULTS: A single large-institution cohort of 132 patients who received NAET over a 13-year period was identified. Comprehensive clinical, histopathological and follow-up data were collected. A detailed histological review of a subset with residual post-treatment carcinoma was undertaken. Two carcinomas (both of the lobular type) achieved complete pathological response. Central scarring was seen in 49.3% of tumours post-treatment. Significant changes in tumour type (41.6%), tumour grade (downgrading in one-third of tumours), and progesterone receptor (PR) expression (22.3%), with a switch to PR-negative status in 17.6% of cases, were observed. The last of these was associated with an absence of tumour-infiltrating lymphocytes (P = 0.005). Ten per cent of cases showed a change in HER2 expression (P = 0.002). The median patient survival was 60 months, and downgrading of tumours was associated with better overall survival (P = 0.05). CONCLUSIONS: We propose a histological method for assessment of residual carcinoma following NAET, and recommend repeat ER/PR/HER2 testing to inform management and prognosis.

The CD151-midkine pathway regulates the immune microenvironment in inflammatory breast cancer.

The immune microenvironment in inflammatory breast cancer (IBC) is poorly characterised, and molecular and cellular pathways that control accumulation of various immune cells in IBC tissues remain largely unknown. Here, we discovered a novel pathway linking the expression of the tetraspanin protein CD151 in tumour cells with increased accumulation of macrophages in cancerous tissues. It is notable that elevated expression of CD151 and a higher number of tumour-infiltrating macrophages correlated with better patient responses to chemotherapy. Accordingly, CD151-expressing IBC xenografts were characterised by the increased infiltration of macrophages. In vitro migration experiments demonstrated that CD151 stimulates the chemoattractive potential of IBC cells for monocytes via mechanisms involving midkine (a heparin-binding growth factor), integrin α6ß1, and production of extracellular vesicles (EVs). Profiling of chemokines secreted by IBC cells demonstrated that CD151 increases production of midkine. Purified midkine specifically stimulated migration of monocytes, but not other immune cells. Further experiments demonstrated that the chemoattractive potential of IBC-derived EVs is blocked by anti-midkine antibodies. These results demonstrate for the first time that changes in the expression of a tetraspanin protein by tumour cells can affect the formation of the immune microenvironment by modulating recruitment of effector cells to cancerous tissues. Therefore, a CD151-midkine pathway can be considered as a novel target for controlled changes of the immune landscape in IBC. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The Immune Microenvironment in Breast Carcinoma: Predictive and Prognostic Role in the Neoadjuvant Setting.

The prognostic value of the immune cell infiltrate in the breast carcinoma microenvironment is still uncertain. We reviewed published articles analysing the infiltration of inflammatorycells in the microenvironment of breast carcinoma. Data revealed the importance of infiltration of these immune cells in the prognosis of breast carcinoma, particularly the triple-negative and HER2-positive phenotypes. Tumour-infiltrating lymphocytes and their subtypes play a fundamental role in predicting the pathological complete response (pCR) to neoadjuvant chemotherapy. More research aiming to dissect a complex network of communication between cancer cells and other cellular components of the tumour microenvironment is necessary to develop more effective therapeutic approaches.

Extramedullary Haematopoiesis in Axillary Lymph Nodes of Breast Carcinoma Patients Receiving Neoadjuvant Chemotherapy: A Potential Diagnostic Pitfall.

Extramedullary haematopoiesis (EMH) in the axillary lymph node of breast cancer patients is extremely rare but when encountered can represent a diagnostic challenge. We aim to highlight this incidental finding as a diagnostic pitfall which can be mistaken for metastatic carcinoma (particularly of the metaplastic type). We report the case of a 68-year-old Caucasian female with a family history of cancer. Core biopsy revealed that she had grade II oestrogen receptor-negative, Her2-positive invasive ductal carcinoma. She was offered neoadjuvant chemotherapy with Herceptin and subsequently underwent breast-conserving surgery. Microscopic examination of the post-treatment breast surgical specimen showed a partial pathological response with large areas of tumour regression. The sentinel lymph node showed frequent large single and multinucleate giant cells with hyperchromatic nuclei located predominantly within the subcapsular and medullary sinuses. The morphological differentials of metastatic carcinoma, sinus histiocytosis and extramedullary haematopoiesis were considered. A panel of immunohistochemistry showed these large cells to be negative for epithelial markers and CD68. They were strongly positive for CD42b (megakaryocyte marker). Smaller myeloperoxidase and factor VIII-positive cells were identified. The findings confirmed EMH. Sentinel nodes are often well scrutinised by pathologists for evidence of metastatic carcinoma as an important prognostic parameter both in the standard and neoadjuvant setting. Nodal megakaryocytes have been described in response to neoadjuvant chemotherapy particularly in association with Herceptin treatment. Pathologists' awareness of this finding in the neoadjuvant setting is crucial to avoid a mistaken diagnosis of malignancy. A relevant immunohistochemical panel together with careful attention to morphology should help establish the correct diagnosis.

The prognostic role of tumor-associated macrophages and dendritic cells in classic Hodgkin's lymphoma.

Tumor-associated macrophages (TAMs) and dendritic cells (DCs) may play a role in tumor progression as a part of the tumor microenvironment in many neoplasms, including those in Hodgkin's lymphoma. The current study investigated the relationship between the presence and density of macrophages and dendritic cells in the background of classic Hodgkin's lymphoma (CHL) and different clinicopathological parameters, including survival and response to therapy. CD68 and CD1a immunohistochemical staining were used to detect and highlight macrophages and dendritic cells, respectively, in 61 cases of CHL. CD68 was expressed in all studied cases, with no significant association with the studied parameters. In total, 54.1% (33/61) of cases showed CD1a expression. High CD1a expression (>7%) was associated with localized lymphadenopathy (p=0.01), early stage (p=0.005), and good revised international prognostic index (R-IPI) (p=0.07). Hodgkin's lymphoma cases that showed high CD68 and low CD1a were associated with adverse prognostic parameters such as advanced stage (p=0.03) and generalized lymphadenopathy (p=0.05). Old age (>60 years) (P=0.005), poor R-IPI (P=0.010), and negative CD1a expression (P=0.045) were significantly associated with poor outcome. Finally, our study demonstrated the importance of the presence and density of DCs in determining progression and prognosis in CHL. A certain interaction between TAMs and DCs may affect the progression of CHL. Further investigation is required to clarify whether TAMs release certain factors that decrease the number or function of DCs.