Dynamic Changes in the NK-, Neutrophil-, and B-cell Immunophenotypes Relevant in High Metastatic Risk Post Neoadjuvant Chemotherapy-Resistant Early Breast Cancers.

PURPOSE: To identify potential immune targets in post-neoadjuvant chemotherapy (NAC)-resistant triple-negative breast cancer (TNBC) and ER+HER2- breast cancer disease. EXPERIMENTAL DESIGN: Following pathology review, 153 patients were identified as having residual cancer burden (RCB) II/III disease (TNBC n = 80; ER+HER2-n = 73). Baseline pre-NAC samples were available for evaluation for 32 of 80 TNBC and 36 of 73 ER+HER2- cases. Bright-field hematoxylin and eosin assessment allowed for tumor-infiltrating lymphocyte (TIL) evaluation in all cases. Multiplexed immunofluorescence was used to identify the abundance and distribution of immune cell subsets. Levels of checkpoints including PD-1/PD-L1 expression were also quantified. Findings were then validated using expression profiling of cancer and immune-related genes. Cytometry by time-of-flight characterized the dynamic changes in circulating immune cells with NAC. RESULTS: RCB II/III TNBC and ER+HER2- breast cancer were immunologically "cold" at baseline and end of NAC. Although the distribution of immune cell subsets across subtypes was similar, the mRNA expression profiles were both subtype- and chemotherapy-specific. TNBC RCB II/III disease was enriched with genes related to neutrophil degranulation, and displayed strong interplay across immune and cancer pathways. We observed similarities in the dynamic changes in B-cell biology following NAC irrespective of subtype. However, NAC induced changes in the local and circulating tumor immune microenvironment (TIME) that varied by subtype and response. Specifically, in TNBC residual disease, we observed downregulation of stimulatory (CD40/OX40L) and inhibitory (PD-L1/PD-1) receptor expression and an increase in NK cell populations (especially non-cytolytic, exhausted CD56dimCD16-) within both the local TIME and peripheral white cell populations. CONCLUSIONS: This study identifies several potential immunologic pathways in residual disease, which may be targeted to benefit high-risk patients.

Assessment of the Molecular Heterogeneity of E-Cadherin Expression in Invasive Lobular Breast Cancer.

Mutations and loss of E-cadherin protein expression define the vast majority of invasive lobular carcinomas. In a subset of these cases, the heterogeneous expression of E-cadherin is observed either as wild-type (strong membranous) expression or aberrant expression (cytoplasmic expression). However, it is unclear as to whether the two components would be driven by distinct genetic or epigenetic alterations. Here, we used whole genome DNA sequencing and methylation array profiling of two separately dissected components of nine invasive lobular carcinomas with heterogeneous E-cadherin expression. E-cadherin negative and aberrant/positive components of E-cadherin heterogeneous tumours showed a similar mutational, copy number and promoter methylation repertoire, suggesting they arise from a common ancestor, as opposed to the collision of two independent tumours. We found that the majority of E-cadherin heterogeneous tumours harboured CDH1 mutations in both the E-cadherin negative and aberrant/positive components together with somatic mutations in additional driver genes known to be enriched in both pure invasive carcinomas of no special type and invasive lobular breast cancers, whereas these were less commonly observed in CDH1 wild-type tumours. CDH1 mutant tumours also exhibited a higher mutation burden as well as increased presence of APOBEC-dependent mutational signatures 2 and 13 compared to CDH1 wild-type tumours. Together, our results suggest that regardless of E-cadherin protein expression, tumours showing heterogeneous expression of E-cadherin should be considered as part of the spectrum of invasive lobular breast cancers.

Proteomics of REPLICANT perfusate detects changes in the metastatic lymph node microenvironment.

In breast cancer (BC), detecting low volumes of axillary lymph node (ALN) metastasis pre-operatively is difficult and novel biomarkers are needed. We recently showed that patient-derived ALNs can be sustained ex-vivo using normothermic perfusion. We now compare reactive (tumour-free; n = 5) and macrometastatic (containing tumour deposits >2 mm; n = 4) ALNs by combining whole section multiplex immunofluorescence with TMT-labelled LC-MS/MS of the circulating perfusate. Macrometastases contained significantly fewer B cells and T cells (CD4+/CD8+/regulatory) than reactive nodes (p = 0.02). Similarly, pathway analysis of the perfusate proteome (119/1453 proteins significantly differentially expressed) showed that immune function was diminished in macrometastases in favour of 'extracellular matrix degradation'; only 'neutrophil degranulation' was preserved. Qualitative comparison of the perfusate proteome to that of node-positive pancreatic and prostatic adenocarcinoma also highlighted 'neutrophil degranulation' as a contributing factor to nodal metastasis. Thus, metastasis-induced changes in the REPLICANT perfusate proteome are detectable, and could facilitate biomarker discovery.

3D Functional Genomics Screens Identify CREBBP as a Targetable Driver in Aggressive Triple-Negative Breast Cancer.

Triple-negative breast cancers (TNBC) are resistant to standard-of-care chemotherapy and lack known targetable driver gene alterations. Identification of novel drivers could aid the discovery of new treatment strategies for this hard-to-treat patient population, yet studies using high-throughput and accurate models to define the functions of driver genes in TNBC to date have been limited. Here, we employed unbiased functional genomics screening of the 200 most frequently mutated genes in breast cancer, using spheroid cultures to model in vivo-like conditions, and identified the histone acetyltransferase CREBBP as a novel tumor suppressor in TNBC. CREBBP protein expression in patient tumor samples was absent in 8% of TNBCs and at a high frequency in other tumors, including squamous lung cancer, where CREBBP-inactivating mutations are common. In TNBC, CREBBP alterations were associated with higher genomic heterogeneity and poorer patient survival and resulted in upregulation and dependency on a FOXM1 proliferative program. Targeting FOXM1-driven proliferation indirectly with clinical CDK4/6 inhibitors (CDK4/6i) selectively impaired growth in spheroids, cell line xenografts, and patient-derived models from multiple tumor types with CREBBP mutations or loss of protein expression. In conclusion, we have identified CREBBP as a novel driver in aggressive TNBC and identified an associated genetic vulnerability in tumor cells with alterations in CREBBP and provide a preclinical rationale for assessing CREBBP alterations as a biomarker of CDK4/6i response in a new patient population. SIGNIFICANCE: This study demonstrates that CREBBP genomic alterations drive aggressive TNBC, lung cancer, and lymphomas and may be selectively treated with clinical CDK4/6 inhibitors.

Real-time ex vivo perfusion of human lymph nodes invaded by cancer (REPLICANT): a feasibility study.

Understanding how breast cancer (BC) grows in axillary lymph nodes (ALNs), and refining how therapies might halt that process, is clinically important. However, modelling the complex ALN microenvironment is difficult, and no human models exist at present. We harvested ALNs from ten BC patients, and perfused them at 37 °C ex vivo for up to 24 h. Controlled autologous testing showed that ALNs remain viable after 24 h of ex vivo perfusion: haematoxylin and eosin-stained histological appearance and proliferation (by Ki67 immunohistochemistry) did not change significantly over time for any perfused ALN compared with a control from time-point zero. Furthermore, targeted gene expression analysis (NanoString PanCancer IO360 panel) showed that only 21/750 genes were differentially expressed between control and perfused ALNs (|log2 FC| > 1 and q < 0.1): none were involved in apoptosis and metabolism, but rather all 21 genes were involved in immune function and angiogenesis. During perfusion, tissue acid-base balance remained stable. Interestingly, the flow rate increased (p < 0.001) in cancer-replaced (i.e. metastasis occupied more than 90% of the surface area on multiple levels) compared to cancer-free nodes (i.e. nodes with no metastasis on multiple sections). CXCL11 transcripts were significantly more abundant in cancer-replaced nodes, while CXCL12 transcripts were significantly more abundant in cancer-free nodes. These cytokines were also detected in the circulating perfusate. Monoclonal antibodies (nivolumab and trastuzumab) were administered into a further three ALNs to confirm perfusion efficacy. These drugs saturated the nodes; nivolumab even induced cancer cell death. Normothermic ALN perfusion is not only feasible but sustains the tumour microenvironment ex vivo for scientific investigation. This model could facilitate the identification of actionable immuno-oncology targets. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Rational treatment of chemotherapy-induced peripheral neuropathy with capsaicin 8% patch: from pain relief towards disease modification.

PURPOSE: Chemotherapy-induced peripheral neuropathy (CIPN) with associated chronic pain is a common and disabling condition. Current treatments for neuropathic pain in CIPN are largely ineffective, with unfavorable side-effects. The capsaicin 8% patch (capsaicin 179 mg patch) is approved for the treatment of neuropathic pain: a single topical cutaneous application can produce effective pain relief for up to 12 weeks. We assessed the therapeutic potential of capsaicin 8% patch in patients with painful CIPN, and its mechanism of action. PATIENTS AND METHODS: 16 patients with chronic painful CIPN (mean duration 2.5 years), in remission for cancer and not receiving chemotherapy, were treated with 30 min application of capsaicin 8% patch to the feet. Symptoms were monitored using the 11-point numerical pain rating scale (NPRS), and questionnaires. Investigations were performed at baseline and three months after patch application, including skin biopsies with a range of markers, and quantitative sensory testing (QST). RESULTS: Patients reported significant reduction in spontaneous pain (mean NPRS: -1.27; 95% CI 0.2409 to 2.301; p=0.02), touch-evoked pain (-1.823; p=0.03) and cold-evoked pain (-1.456; p=0.03). Short-Form McGill questionnaire showed a reduction in neuropathic (p=0.0007), continuous (p=0.01) and overall pain (p=0.004); Patient Global Impression of Change showed improvement (p=0.001). Baseline skin biopsies showed loss of intra-epidermal nerve fibers (IENF), and also of sub-epidermal nerve fibers quantified by image analysis. Post-patch application skin biopsies showed a significant increase towards normalization of intra-epidermal and sub-epidermal nerve fibers (for IENF: structural marker PGP9.5, p=0.009; heat receptor TRPV1, p=0.027; regenerating nerve marker GAP43, p=0.04). Epidermal levels of Nerve Growth Factor (NGF), Neurotrophin-3 (NT-3), and Langerhans cells were also normalized. QST remained unchanged and there were no systemic side-effects, as in previous studies. CONCLUSION: Capsaicin 8% patch provides significant pain relief in CIPN, and may lead to regeneration and restoration of sensory nerve fibers ie, disease modification.

Optimised ARID1A immunohistochemistry is an accurate predictor of ARID1A mutational status in gynaecological cancers.

ARID1A is a tumour suppressor gene that is frequently mutated in clear cell and endometrioid carcinomas of the ovary and endometrium and is an important clinical biomarker for novel treatment approaches for patients with ARID1A defects. However, the accuracy of ARID1A immunohistochemistry (IHC) as a surrogate for mutation status has not fully been established for patient stratification in clinical trials. Here we tested whether ARID1A IHC could reliably predict ARID1A mutations identified by next-generation sequencing. Three commercially available antibodies - EPR13501 (Abcam), D2A8U (Cell Signaling), and HPA005456 (Sigma) - were optimised for IHC using cell line models and human tissue, and screened across a cohort of 45 gynaecological tumours. IHC was scored independently by three pathologists using an immunoreactive score. ARID1A mutation status was assessed using two independent sequencing platforms and the concordance between ARID1A mutation and protein expression was evaluated using Receiver Operating Characteristic statistics. Overall, 21 ARID1A mutations were identified in 14/43 assessable tumours (33%), the majority of which were predicted to be deleterious. Mutations were identified in 6/17 (35%) ovarian clear cell carcinomas, 5/8 (63%) ovarian endometrioid carcinomas, 2/5 (40%) endometrial carcinomas, and 1/7 (14%) carcinosarcomas. ROC analysis identified greater than 95% concordance between mutation status and IHC using a modified immunoreactive score for all three antibodies allowing a definitive cut-point for ARID1A mutant status to be calculated. Comprehensive assessment of concordance of ARID1A IHC and mutation status identified EPR13501 as an optimal antibody, with 100% concordance between ARID1A mutation status and protein expression, across different gynaecological histological subtypes. It delivered the best inter-rater agreement between all pathologists, as well as a clear cost-benefit advantage. This could allow patients to be accurately stratified based on their ARID1A IHC status into early phase clinical trials.

Histological scoring of immune and stromal features in breast and axillary lymph nodes is prognostic for distant metastasis in lymph node-positive breast cancers.

The prognostic importance of lymph node (LN) status and tumour-infiltrating lymphocytes (TILs), is well established, particularly TILs in triple negative breast cancers (TNBCs). So far, few studies have interrogated changes in involved and uninvolved LNs and evaluated if their morphological patterns add valuable information for the prediction of disease progression in breast cancer. In a cohort of 309 patients enriched for TNBCs (170/309), we histologically characterised immune and stromal features in primary tumours and associated involved and uninvolved axillary LNs on routine haematoxylin and eosin stained sections. Of the 309 patients, 143 had LN-positive disease. Twenty-five histopathological features were assessed, including the degree of TIL presence, quantitative and qualitative assessment of germinal centres (GCs) and sinus histiocytosis. Multivariate and cross-validated proportional hazard regression analyses were used to identify optimal covariate sets for prediction of distant metastasis-free survival (DMFS). The degree of intratumoural and peritumoural immune infiltrate was associated with architectural changes in both uninvolved and involved LNs. By including clinicopathological characteristics as well as tumour and LN histopathological features in L2-regularised proportional hazard models, the prediction of 5-year DMFS was improved by 3-15% over the baseline in all cancers and in TNBCs. In LN-positive cancers, the combination of Salgado's classification, lymphocytic lobulitis, size and number of GCs in the uninvolved LNs and location of GCs in the involved LNs carried significant prognostic information. From these features, a multivariate cross-validation-stable risk signature was constructed, which identified low-risk groups within both LN-positive breast cancers and the LN-positive TNBCs group with a 10-year DMFS probability of 78 and 87%, respectively. This study illustrates that, by incorporating histopathological patterns of involved and uninvolved LNs combined with primary tumour immune and stromal features, the prediction of developing distant metastasis in LN-positive breast cancers can be estimated more accurately.

Relevance of Spatial Heterogeneity of Immune Infiltration for Predicting Risk of Recurrence After Endocrine Therapy of ER+ Breast Cancer.

Background: Despite increasing evidence supporting the clinical utility of immune infiltration in the estrogen receptor-negative (ER-) subtype, the prognostic value of immune infiltration for ER+ disease is not well defined. Methods: Quantitative immune scores of cell abundance and spatial heterogeneity were computed using a fully automated hematoxylin and eosin-stained image analysis algorithm and spatial statistics for 1178 postmenopausal patients with ER+ breast cancer treated with five years' tamoxifen or anastrozole. The prognostic significance of immune scores was compared with Oncotype DX 21-gene recurrence score (RS), PAM50 risk of recurrence (ROR) score, IHC4, and clinical treatment score, available for 963 patients. Statistical tests were two-sided. Results: Scores of immune cell abundance were not associated with recurrence-free survival. In contrast, high immune spatial scores indicating increased cell spatial clustering were associated with poor 10-year, early (0-5 years), and late (5-10 years) recurrence-free survival (Immune Hotspot: LR-χ2 = 14.06, P < .001, for 0-10 years; LR-χ2 = 6.24, P = .01, for 0-5 years; LR-χ2 = 7.89, P = .005, for 5-10 years). The prognostic value of spatial scores for late recurrence was similar to that of IHC4 and RS in both populations, but was not as strong as other tests in comparison for recurrence across 10 years. Conclusions: These results provide a missing link between tumor immunity and disease outcome in ER+ disease by examining tumor spatial architecture. The association between spatial scores and late recurrence suggests a lasting memory of protumor immunity that may impact disease progression and evolution of endocrine treatment resistance, which may be exploited for therapeutic advances.

Evaluation of CDK12 Protein Expression as a Potential Novel Biomarker for DNA Damage Response-Targeted Therapies in Breast Cancer.

Disruption of Cyclin-Dependent Kinase 12 (CDK12) is known to lead to defects in DNA repair and sensitivity to platinum salts and PARP1/2 inhibitors. However, CDK12 has also been proposed as an oncogene in breast cancer. We therefore aimed to assess the frequency and distribution of CDK12 protein expression by IHC in independent cohorts of breast cancer and correlate this with outcome and genomic status. We found that 21% of primary unselected breast cancers were CDK12 high, and 10.5% were absent, by IHC. CDK12 positivity correlated with HER2 positivity but was not an independent predictor of breast cancer-specific survival taking HER2 status into account; however, absent CDK12 protein expression significantly correlated with a triple-negative phenotype. Interestingly, CDK12 protein absence was associated with reduced expression of a number of DDR proteins including ATR, Ku70/Ku80, PARP1, DNA-PK, and γH2AX, suggesting a novel mechanism of CDK12-associated DDR dysregulation in breast cancer. Our data suggest that diagnostic IHC quantification of CDK12 in breast cancer is feasible, with CDK12 absence possibly signifying defective DDR function. This may have important therapeutic implications, particularly for triple-negative breast cancers. Mol Cancer Ther; 17(1); 306-15. ©2017 AACR.

Automated Classification of Breast Cancer Stroma Maturity From Histological Images.

OBJECTIVE: The tumor microenvironment plays a crucial role in regulating tumor progression by a number of different mechanisms, in particular, the remodeling of collagen fibers in tumor-associated stroma, which has been reported to be related to patient survival. The underlying motivation of this work is that remodeling of collagen fibers gives rise to observable patterns in hematoxylin and eosin (H&E) stained slides from clinical cases of invasive breast carcinoma that the pathologist can label as mature or immature stroma. The aim of this paper is to categorise and automatically classify stromal regions according to their maturity and show that this classification agrees with that of skilled observers, hence providing a repeatable and quantitative measure for prognostic studies. METHODS: We use multiscale basic image features and local binary patterns, in combination with a random decision trees classifier for classification of breast cancer stroma regions-of-interest (ROI). RESULTS: We present results from a cohort of 55 patients with analysis of 169 ROI. Our multiscale approach achieved a classification accuracy of 84%. CONCLUSION: This work demonstrates the ability of texture-based image analysis to differentiate breast cancer stroma maturity in clinically acquired H&E-stained slides at least as well as skilled observers.

Micro- and macro-metastasis in the axillary lymph node: A review.

Pathologists typically examine the sentinel lymph nodes excised from patients with invasive breast cancer more thoroughly than they have historically those from axillary lymph node clearance specimens. This, it is thought, increases the chances of detecting small metastatic foci (i.e. macrometastases (>2 mm), micrometastases (0.2-2 mm), or isolated tumour cell clusters (<0.2 mm or <200 cancer cells in one section)). However, the clinical significance of these small metastatic deposits remains unclear. Although an increasing nodal burden is known to proportionally decrease patient survival, the American College of Surgeons Oncology Group (ACOSOG) Z0011 trial has, at the least, raised questions as to how best to manage nodal metastasis in early invasive breast cancer. These issues, and a brief overview of the biology of metastatic spread are presented in this review.

A simple human co-culture model of lymphatic invasion.

True lymphovascular invasion in most carcinomas adversely affects survival, but in vitro human models of lymphatic invasion are lacking. Herein is discussed one such model in which certain mechanistic aspects of lymphatic dissemination can be investigated easily in any laboratory. S100P expression is known to be significantly upregulated in the lymph node metastases of human pancreatic adenocarcinoma. Thus, an S100P overexpressing cell line (S5L) and a vector control (V3L) pancreatic cancer cell lines were used to develop a co-culture model with human dermal lymphatic endothelial cells. Adhesion, the effect of cancer cells on lymphatic permeability and trans-lymphatic endothelial migration could be reproducibly quantified in this model and parametric statistical analysis confirmed significant differences between the two cancer cell lines in these functional assays. This proof-of-principle study demonstrates that this simple model can be used to assess lymphatic invasion in a human cancer context in real-time in vitro.

An audit of residual cancer burden reproducibility in a UK context.

AIMS: The residual cancer burden score (RCB) is currently the preferred quantification tool for assessing residual disease following neoadjuvant chemotherapy (NACT) in breast cancer clinical trials. This has been shown to be highly reproducible at the MD Anderson Cancer Centre, where it was developed originally. We wanted to evaluate RCB in a UK context, where macroscopic handling of tissue may differ between sites. METHODS AND RESULTS: The pathology slides from 90 post-NACT patients from Guy's and St Thomas' NHS Foundation Trust and the Royal Bournemouth Hospital were reviewed independently by two specialist breast histopathologists who recalculated the RCB for each case. Data were collated and analysed statistically for interobserver reproducibility, for both numerical and categorical RCB. Overall, agreement between pathologists was 'good' [kappa = 0.775; 95% confidence interval (CI) = 0.668-0.882]. The overall concordance for continuous RCB score and for categorical RCB group was statistically significant (Spearman's correlation coefficient = 0.9497; 95% CI = 0.9235-0.9671; P < 0.0001 and Spearman's correlation coefficient = 0.9145; 95% CI = 0.8712-0.9437; P < 0.0001, respectively). Discordance could not be attributed to any one component of the RCB calculation. CONCLUSIONS: These data suggest that the RCB score is reproducible in a UK context. Further data comparing it to other quantification systems is required, however, before any superiority can be established.

Accuracy of classification of invasive lobular carcinoma on needle core biopsy of the breast.

Although the UK National Institute for Health and Care Excellence guidelines recommend that in patients with biopsy-proven invasive lobular carcinoma (ILC), preoperative MRI scan is considered, the accuracy of diagnosis of ILC in core biopsy of the breast has not been previously investigated. Eleven pathology laboratories from the UK and Ireland submitted data on 1112 cases interpreted as showing features of ILC, or mixed ILC and IDC/no special type (NST)/other tumour type, on needle core biopsy through retrieval of histology reports. Of the total 1112 cases, 844 were shown to be pure ILC on surgical excision, 154 were mixed ILC plus another type (invariably ductal/NST) and 113 were shown to be ductal/NST. Of those lesions categorised as pure ILC on core, 93% had an element of ILC correctly identified in the core biopsy sample and could be considered concordant. Of cores diagnosed as mixed ILC plus another type on core, complete agreement between core and excision was 46%, with 27% cases of pure ILC, whilst 26% non-concordant. These data indicate that there is not a large excess of expensive MRIs being performed as a result of miscategorisation histologically.

Immunohistochemistry for Triple-Negative Breast Cancer.

Triple-negative breast cancers are a heterogeneous group of tumors that are, as yet, not entirely understood. Although triple-negative carcinomas are strictly defined as invasive carcinomas lacking expression of estrogen receptor, progesterone receptor, and HER2, some use the terms triple-negative and basal-like cancer synonymously. It should be noted that these are not entirely equivalent. Nevertheless, it has been shown that a panel of immunohistochemical markers can be used as a surrogate for genomic profiling and thus to identify basal-like breast cancers. We describe the panels of immunohistochemical markers that can be applied and how to interpret these markers herein.

Proteome of formalin-fixed paraffin-embedded pancreatic ductal adenocarcinoma and lymph node metastases.

Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer-related death, largely due to metastatic disease. To better understand PDAC metastatic spread and identify novel therapeutic targets, we analysed the proteome of primary tumours and matched lymph node (LN) metastases. As frozen specimens of metastatic lesions are scarce, we examined formalin-fixed paraffin-embedded (FFPE) tissues. This poses technical challenges because of the cross-linkages induced by fixation. Using laser capture microdissection (PALM system), we isolated malignant epithelia from seven FFPE primary PDAC tumours and matched LN metastases. Following dissection, samples were analysed in duplicate using Multidimensional Protein Identification Technology (MudPIT); this resulted in the identification of 1504 proteins, 854 of which were common to all samples analysed. Comparison of the obtained proteins with data from previous proteomics studies on pancreatic tissue, pancreatic juice, serum, and urine resulted in a less than 30% overlap, indicating that our study has substantially expanded the current database of proteins expressed in this malignancy. Statistical analysis further showed that 115/854 proteins (13.5%) were significantly differentially expressed (g-value ≥ 3.8). Two proteins, S100P and 14-3-3 sigma, with highly significant g-values were confirmed to be significantly differentially expressed (S100P: p = 0.05 and 14-3-3 sigma: p < 0.001) in a larger series of 55 cases of matched primary PDAC and LN metastases using immunohistochemistry. Thus, laser capture microdissection of FFPE tissue coupled with downstream proteomic analysis is a valid approach for the investigation of metastatic PDAC. This is the first study to establish and compare the protein composition of primary PDAC and matched LN metastases, and has resulted in the identification of several potential epithelial-specific therapeutic targets, including 14-3-3 sigma and S100P.