Timing evolution of lobular breast cancer through phylogenetic analysis.

BACKGROUND: Late distant recurrence is a challenge for the treatment of invasive lobular carcinoma (ILC) of the breast. Despite in-depth characterisation of primary ILC, the molecular landscape of metastatic ILC is still only partially understood. METHODS: We retrospectively identified 38 ILC patients from the tissue banks of six European institutions. DNA extracted from patient matched primary and metastatic FFPE tissue blocks was whole genome sequenced to compute somatic copy number aberrations. This, in turn, was used to infer the evolutionary history of these patients. FINDINGS: The data show different metastatic seeding patterns, with both an early and late divergence of the metastatic lineage observed in ILC. Additionally, cascading dissemination from a metastatic precursor was a dominant rule. Alterations in key cancer driver genes, such as TP53 or CCND1, were acquired early while additional aberrations were present only in the metastatic branch. In about 30% of the patients, the metastatic lineage harboured less aberrations than the primary tumour suggesting a period of tumour dormancy or prolonged adaptation at the distant site. This phenomenon was mostly observed in tumours from de novo metastatic patients. INTERPRETATION: Our results provide insights into ILC evolution and offer potential paths for optimised ILC care. FUNDING: This work has received financial support from Les Amis de l'Institut Bordet, MEDIC, the Breast Cancer Research Foundation (BCRF) and the Belgian Fonds National de la Recherche Scientifique (F.R.S-FNRS).

Functional Th1-oriented T follicular helper cells that infiltrate human breast cancer promote effective adaptive immunity.

We previously demonstrated that tumor-infiltrating lymphocytes (TIL) in human breast cancer sometimes form organized tertiary lymphoid structures (TLS) characterized by CXCL13-producing T follicular helper (Tfh) cells. The present study found that CD4+ Tfh TIL, CD8+ TIL, and TIL-B, colocalizing in TLS, all express the CXCL13 receptor CXCR5. An ex vivo functional assay determined that only activated, functional Th1-oriented Tfh TIL (PD-1hiICOSint phenotype) provide help for immunoglobulin and IFN-γ production. A functional Tfh TIL presence signals an active TLS, characterized by humoral (immunoglobulins, Ki-67+ TIL-B in active germinal centers) and cytotoxic (GZMB+CD8+ and GZMB+CD68+ TIL plus Th1 gene expression) immune responses. Analysis of active versus inactive TLS in untreated patients revealed that the former are associated with positive clinical outcomes. TLS also contain functional T follicular regulatory (Tfr) TIL, which are characterized by a CD25+CXCR5+GARP+FOXP3+ phenotype and a demethylated FOXP3 gene. Functional Tfr inhibited functional Tfh activities via a glycoprotein A repetitions predominant (GARP)-associated TGF-ß-dependent mechanism. The activity of tumor-associated TLS was dictated by the relative balance between functional Tfh TIL and functional Tfr TIL. These data provide mechanistic insight into TLS processes orchestrated by functional Th1-oriented Tfh TIL, including TIL-B and CD8+ TIL activation and immunological memory generation. Tfh TIL, regulated by functional Tfr TIL, are an expected key target of PD-1/PD-L1 blockade.

Inhibition of RANK signaling in breast cancer induces an anti-tumor immune response orchestrated by CD8+ T cells.

Most breast cancers exhibit low immune infiltration and are unresponsive to immunotherapy. We hypothesized that inhibition of the receptor activator of nuclear factor-κB (RANK) signaling pathway may enhance immune activation. Here we report that loss of RANK signaling in mouse tumor cells increases leukocytes, lymphocytes, and CD8+ T cells, and reduces macrophage and neutrophil infiltration. CD8+ T cells mediate the attenuated tumor phenotype observed upon RANK loss, whereas neutrophils, supported by RANK-expressing tumor cells, induce immunosuppression. RANKL inhibition increases the anti-tumor effect of immunotherapies in breast cancer through a tumor cell mediated effect. Comparably, pre-operative single-agent denosumab in premenopausal early-stage breast cancer patients from the Phase-II D-BEYOND clinical trial (NCT01864798) is well tolerated, inhibits RANK pathway and increases tumor infiltrating lymphocytes and CD8+ T cells. Higher RANK signaling activation in tumors and serum RANKL levels at baseline predict these immune-modulatory effects. No changes in tumor cell proliferation (primary endpoint) or other secondary endpoints are observed. Overall, our preclinical and clinical findings reveal that tumor cells exploit RANK pathway as a mechanism to evade immune surveillance and support the use of RANK pathway inhibitors to prime luminal breast cancer for immunotherapy.

Pitfalls in assessing stromal tumor infiltrating lymphocytes (sTILs) in breast cancer.

Stromal tumor-infiltrating lymphocytes (sTILs) are important prognostic and predictive biomarkers in triple-negative (TNBC) and HER2-positive breast cancer. Incorporating sTILs into clinical practice necessitates reproducible assessment. Previously developed standardized scoring guidelines have been widely embraced by the clinical and research communities. We evaluated sources of variability in sTIL assessment by pathologists in three previous sTIL ring studies. We identify common challenges and evaluate impact of discrepancies on outcome estimates in early TNBC using a newly-developed prognostic tool. Discordant sTIL assessment is driven by heterogeneity in lymphocyte distribution. Additional factors include: technical slide-related issues; scoring outside the tumor boundary; tumors with minimal assessable stroma; including lymphocytes associated with other structures; and including other inflammatory cells. Small variations in sTIL assessment modestly alter risk estimation in early TNBC but have the potential to affect treatment selection if cutpoints are employed. Scoring and averaging multiple areas, as well as use of reference images, improve consistency of sTIL evaluation. Moreover, to assist in avoiding the pitfalls identified in this analysis, we developed an educational resource available at www.tilsinbreastcancer.org/pitfalls.

Reactive stroma and trastuzumab resistance in HER2-positive early breast cancer.

We investigated the value of reactive stroma as a predictor for trastuzumab resistance in patients with early HER2-positive breast cancer receiving adjuvant therapy. The pathological reactive stroma and the mRNA gene signatures that reflect reactive stroma in 209 HER2-positive breast cancer samples from the FinHer adjuvant trial were evaluated. Levels of stromal gene signatures were determined as a continuous parameter, and pathological reactive stromal findings were defined as stromal predominant breast cancer (SPBC; ≥50% stromal) and correlated with distant disease-free survival. Gene signatures associated with reactive stroma in HER2-positive early breast cancer (N = 209) were significantly associated with trastuzumab resistance in estrogen receptor (ER)-negative tumors (hazard ratio [HR] = 1.27 p interaction = 0.014 [DCN], HR = 1.58, p interaction = 0.027 [PLAU], HR = 1.71, p interaction = 0.019 [HER2STROMA, novel HER2 stromal signature]), but not in ER-positive tumors (HR = 0.73 p interaction = 0.47 [DCN], HR = 0.71, p interaction = 0.73 [PLAU], HR = 0.84; p interaction = 0.36 [HER2STROMA]). Pathological evaluation of HER2-positive/ER-negative tumors suggested an association between SPBC and trastuzumab resistance. Reactive stroma did not correlate with tumor-infiltrating lymphocytes (TILs), and the expected benefit from trastuzumab in patients with high levels of TILs was pronounced only in tumors with low stromal reactivity (SPBC <50%). In conclusion, reactive stroma in HER2-positive/ER-negative early breast cancer tumors may predict resistance to adjuvant trastuzumab therapy.

The Relationship Between Tumor-Infiltrating Lymphocytes, PD-L1 Expression, Driver Mutations and Clinical Outcome Parameters in Non-Small Cell Lung Cancer Adenocarcinoma in Patients with a Limited to no Smoking History.

Tumor infiltrating lymphocytes (TIL), programmed death 1 (PD-1) and programmed death-ligand 1 (PD-L1) expression are important prognostic markers. This study aimed to investigate these markers in lung adenocarcinoma (ADC) biopsies from patients with stage IIIB or IV ADC with little or no smoking history, to investigate their prognostic value and to correlate these results with the presence of driver mutations in the tumors. TIL were retrospectively evaluated on hematoxylin and eosin stained slides from 152 tumor samples. PD-1/PD-L1 expression was retrospectively evaluated with PD-1/PD-L1 immunohistochemistry (IHC) double staining on 74 tumor samples with sufficient residual tissue. PD-L1 expression was analysed on stromal cells of the tumor compartment, the tumor cells and TIL and PD-1 on TIL. Median overall survival (OS) was longer in patients with high stromal TIL infiltration compared to patients with low stromal TIL infiltration (68 weeks vs. 35 weeks respectively; p = 0.003). This was observed most prominently in KRAS mutant tumors (95 weeks vs. 12 weeks; p = 0.003). Only PD-L1 expression on tumor stromal cells influenced OS and indicated a worse prognosis (77 weeks vs 25 weeks; p = 0.013). Stromal TIL counts nor PD-1/PD-L1 expression levels were associated with the presence of driver mutations. The results of the current study reinforce the prognostic role of TIL in lung ADC, which is most prominent in KRAS mutant cancers. The results of the PD-1/PD-L1 analysis suggest that stromal cells can effectively suppress the anti-tumor immune response via the PD-L1 pathway.

Comprehensive evaluation of methods to assess overall and cell-specific immune infiltrates in breast cancer.

BACKGROUND: Breast cancer (BC) immune infiltrates play a critical role in tumor progression and response to treatment. Besides stromal tumor infiltrating lymphocytes (sTILs) which have recently reached level 1B evidence as a prognostic marker in triple negative BC, a plethora of methods to assess immune infiltration exists, and it is unclear how these compare to each other and if they can be used interchangeably. METHODS: Two experienced pathologists scored sTIL, intra-tumoral TIL (itTIL), and 6 immune cell types (CD3+, CD4+, CD8+, CD20+, CD68+, FOXP3+) in the International Cancer Genomics Consortium breast cancer cohort using hematoxylin and eosin-stained (n = 243) and immunohistochemistry-stained tissue microarrays (n = 254) and whole slides (n = 82). The same traits were evaluated using transcriptomic- and methylomic-based deconvolution methods or signatures. RESULTS: The concordance correlation coefficient (CCC) between pathologists for sTIL was very good (0.84) and for cell-specific immune infiltrates slightly lower (0.63-0.66). Comparison between tissue microarray and whole slide pathology scores revealed systematically higher values in whole slides (ratio 2.60-5.98). The Spearman correlations between microscopic sTIL and transcriptomic- or methylomic-based assessment of immune infiltrates were highly variable (r = 0.01-0.56). Similar observations were made for cell type-specific quantifications (r = 0.001-0.54). We observed a strong inter-method variability between the omics-derived estimations, which is further cell type dependent. Finally, we demonstrated that most methods more accurately identify highly infiltrated (sTIL ≥ 60%; area under the curve, AUC, 0.64-0.99) as compared to lowly infiltrated tumors (sTIL ≤ 10%; AUC 0.52-0.82). CONCLUSIONS: There is a lower inter-pathologist concordance for cell-specific quantification as compared to overall infiltration quantification. Microscopic assessments are underestimated when considering small cores (tissue microarray) instead of whole slides. Results further highlight considerable differences between the microscopic-, transcriptomic-, and methylomic-based methods in the assessment of overall and cell-specific immune infiltration in BC. We therefore call for extreme caution when assessing immune infiltrates using current methods and emphasize the need for standardized immune characterization beyond TIL.

Tumor infiltrating B-cells signal functional humoral immune responses in breast cancer.

Tumor-infiltrating B-cells (TIL-B) in breast cancer (BC) have previously been associated with improved clinical outcomes; however, their role(s) in tumor immunity is not currently well known. This study confirms and extends the correlation between higher TIL-B densities and positive outcomes through an analysis of HER2-positive and triple-negative BC patients from the BIG 02-98 clinical trial (10yr mean follow-up). Fresh tissue analyses identify an increase in TIL-B density in untreated primary BC compared to normal breast tissues, which is associated with global, CD4+ and CD8+ TIL, higher tumor grades, higher proliferation and hormone receptor negativity. All B-cell differentiation stages are detectable but significant increases in memory TIL-B are consistently present. BC with higher infiltrates are specifically characterized by germinal center TIL-B, which in turn are correlated with TFH TIL and antibody-secreting TIL-B principally located in tertiary lymphoid structures. Some TIL-B also interact directly with tumor cells. Functional analyses reveal TIL-B are responsive to BCR stimulation ex vivo, express activation markers and produce cytokines and immunoglobulins despite reduced expression of the antigen-presenting molecules HLA-DR and CD40. Overall, these data support the concept that ongoing humoral immune responses are generated by TIL-B and help to generate effective anti-tumor immunity at the tumor site.

ESR1 mutations in metastatic lobular breast cancer patients.

Invasive lobular breast cancer (ILC) represents the second most common histology of breast cancer after invasive ductal breast cancer (IDC), accounts for up to 15% of all invasive cases and generally express the estrogen receptor (ER, coded by the ESR1 gene). ESR1 mutations have been associated with resistance to endocrine therapy, however these have not been specifically evaluated in ILC. We assessed the frequency of ESR1 mutations by droplet digital PCR in a retrospective multi-centric series of matched primary tumor and recurrence samples (n = 279) from 80 metastatic ER-positive ILC patients. We further compared ESR1 mutations between IDC and ILC patients in metastatic samples from MSKCC-IMPACT (n = 595 IDC and 116 ILC) and in ctDNA from the SoFEA and PALOMA-3 trials (n = 416 IDC and 76 ILC). In the retrospective series, the metastases from seven patients (9%) harbored ESR1 mutations, which were absent from the interrogated primary samples. Five patients (6%) had a mutation in the primary tumor or axillary metastasis, which could not be detected in the matched distant metastasis. In the MSKCC-IMPACT cohort, as well as in the SoFEA and PALOMA-3 trials, there were no differences in prevalence and distribution of the mutations between IDC and ILC, with D538G being the most frequent mutation in both histological subtypes. To conclude, no patient had an identical ESR1 mutation in the early and metastatic disease in the retrospective ILC series. In the external series, there was no difference in terms of prevalence and type of ESR1 mutations between ILC and IDC.

BRCA gene mutations do not shape the extent and organization of tumor infiltrating lymphocytes in triple negative breast cancer.

This study investigated the prevalence of TIL subpopulations, TLS, PD-1 and PD-L1 in tumors from TNBC patients harboring wild-type or mutated BRCA1 or BRCA2 germline genes. This TNBC cohort included 85% TIL-positive (≥10%) tumors with 21% classified as TILhi (≥50%). Interestingly, the BRCAmut group had a significantly higher incidence of TILpos tumors compared to the BRCAwt group (P = 0.037). T cells were dominant in the infiltrate but no statistically significant differences were detected between BRCAwt and BRCAmut for CD3+, CD4+ and CD8+ T cells or CD20+ B cells. TLS were detected in 74% of tumors but again no significant differences between the BRCA groups. PD-1 expression was observed in 33% and PD-L1 in 53% (any cell, cut-off ≥1%) tumors for the entire TNBC cohort. PD-1 expression correlated with PD-L1 and both with TIL and TLS but was not associated with BRCA mutational status. Our analyses reveal that BRCAwt and BRCAmut TNBC are similar except for a significant increase of TILpos tumors in the BRCAmut group. While BRCA gene mutations may not directly drive immune infiltration, the greater number of TILpos tumors could signal greater immunogenicity in this group.

Spatially distinct tumor immune microenvironments stratify triple-negative breast cancers.

Understanding the tumor immune microenvironment (TIME) promises to be key for optimal cancer therapy, especially in triple-negative breast cancer (TNBC). Integrating spatial resolution of immune cells with laser capture microdissection gene expression profiles, we defined distinct TIME stratification in TNBC, with implications for current therapies including immune checkpoint blockade. TNBCs with an immunoreactive microenvironment exhibited tumoral infiltration of granzyme B+CD8+ T cells (GzmB+CD8+ T cells), a type 1 IFN signature, and elevated expression of multiple immune inhibitory molecules including indoleamine 2,3-dioxygenase (IDO) and programmed cell death ligand 1 (PD-L1), and resulted in good outcomes. An "immune-cold" microenvironment with an absence of tumoral CD8+ T cells was defined by elevated expression of the immunosuppressive marker B7-H4, signatures of fibrotic stroma, and poor outcomes. A distinct poor-outcome immunomodulatory microenvironment, hitherto poorly characterized, exhibited stromal restriction of CD8+ T cells, stromal expression of PD-L1, and enrichment for signatures of cholesterol biosynthesis. Metasignatures defining these TIME subtypes allowed us to stratify TNBCs, predict outcomes, and identify potential therapeutic targets for TNBC.

The circular RNome of primary breast cancer.

Circular RNAs (circRNAs) are a class of RNAs that is under increasing scrutiny, although their functional roles are debated. We analyzed RNA-seq data of 348 primary breast cancers and developed a method to identify circRNAs that does not rely on unmapped reads or known splice junctions. We identified 95,843 circRNAs, of which 20,441 were found recurrently. Of the circRNAs that match exon boundaries of the same gene, 668 showed a poor or even negative (R < 0.2) correlation with the expression level of the linear gene. In silico analysis showed only a minority (8.5%) of circRNAs could be explained by known splicing events. Both these observations suggest that specific regulatory processes for circRNAs exist. We confirmed the presence of circRNAs of CNOT2, CREBBP, and RERE in an independent pool of primary breast cancers. We identified circRNA profiles associated with subgroups of breast cancers and with biological and clinical features, such as amount of tumor lymphocytic infiltrate and proliferation index. siRNA-mediated knockdown of circCNOT2 was shown to significantly reduce viability of the breast cancer cell lines MCF-7 and BT-474, further underlining the biological relevance of circRNAs. Furthermore, we found that circular, and not linear, CNOT2 levels are predictive for progression-free survival time to aromatase inhibitor (AI) therapy in advanced breast cancer patients, and found that circCNOT2 is detectable in cell-free RNA from plasma. We showed that circRNAs are abundantly present, show characteristics of being specifically regulated, are associated with clinical and biological properties, and thus are relevant in breast cancer.

Tumor-Infiltrating Lymphocytes in Patients Receiving Trastuzumab/Pertuzumab-Based Chemotherapy: A TRYPHAENA Substudy.

Background: There is an urgent requirement to identify biomarkers to tailor treatment in human epidermal growth factor receptor 2 (HER2)-amplified early breast cancer treated with trastuzumab/pertuzumab-based chemotherapy. Methods: Among the 225 patients randomly assigned to trastuzumab/pertuzumab concurrently or sequentially with an anthracycline-containing regimen or concurrently with an anthracycline-free regimen in the Tryphaena trial, we determined the percentage of tumor-infiltrating lymphocytes (TILs) at baseline in 213 patients, of which 126 demonstrated a pathological complete response (pCR; ypT0/is ypN0), with 28 demonstrating event-free survival (EFS) events. We investigated associations between baseline TIL percentage and either pCR or EFS after adjusting for clinicopathological characteristics using logistic and Cox regression models, respectively. To understand TIL biology, we evaluated associations between baseline TILs and baseline tumor gene expression data (800 gene set by NanoString) in a subset of 173 patients. All statistical tests were two-sided. Results: Among the patients with measurable TILs at baseline, the median level was 14.1% (interquartile range = 7.1%-32.4%). After adjusting for clinicopathological characteristics, baseline percentage TIL was not associated with pCR (adjusted odds ratio [aOR] for every 10-percentage unit increase in TILs = 1.12, 95% confidence interval [CI] = 0.95 to 1.31, P = .17). At a median follow-up of 4.7 years, for every increase in baseline TILs of 10%, there was a 25% reduction in the hazard for an EFS event (aOR = 0.75, 95% CI = 0.56 to 1.00, P = .05) after adjusting for baseline clinicopathological characteristics and pCR. Additionally, genes associated with epithelial-mesenchymal transition, angiogenesis, and T-cell inhibition such as SNAIL1, ZEB1, NOTCH3, and B7-H3 were statistically significantly inversely correlated with percentage TIL. Conclusions: Baseline TIL percentage provides independent prognostic information in patients treated with trastuzumab/pertuzumab-based neoadjuvant chemotherapy. However, further validation is required.

TP53 Outperforms Other Androgen Receptor Biomarkers to Predict Abiraterone or Enzalutamide Outcome in Metastatic Castration-Resistant Prostate Cancer.

PURPOSE: To infer the prognostic value of simultaneous androgen receptor (AR) and TP53 profiling in liquid biopsies from patients with metastatic castration-resistant prostate cancer (mCRPC) starting a new line of AR signaling inhibitors (ARSi).Experimental Design: Between March 2014 and April 2017, we recruited patients with mCRPC (n = 168) prior to ARSi in a cohort study encompassing 10 European centers. Blood samples were collected for comprehensive profiling of CellSearch-enriched circulating tumor cells (CTC) and circulating tumor DNA (ctDNA). Targeted CTC RNA sequencing (RNA-seq) allowed the detection of eight AR splice variants (ARV). Low-pass whole-genome and targeted gene-body sequencing of AR and TP53 was applied to identify amplifications, loss of heterozygosity, mutations, and structural rearrangements in ctDNA. Clinical or radiologic progression-free survival (PFS) was estimated by Kaplan-Meier analysis, and independent associations were determined using multivariable Cox regression models. RESULTS: Overall, no single AR perturbation remained associated with adverse prognosis after multivariable analysis. Instead, tumor burden estimates (CTC counts, ctDNA fraction, and visceral metastases) were significantly associated with PFS. TP53 inactivation harbored independent prognostic value [HR 1.88; 95% confidence interval (CI), 1.18-3.00; P = 0.008], and outperformed ARV expression and detection of genomic AR alterations. Using Cox coefficient analysis of clinical parameters and TP53 status, we identified three prognostic groups with differing PFS estimates (median, 14.7 vs. 7.51 vs. 2.62 months; P < 0.0001), which was validated in an independent mCRPC cohort (n = 202) starting first-line ARSi (median, 14.3 vs. 6.39 vs. 2.23 months; P < 0.0001). CONCLUSIONS: In an all-comer cohort, tumor burden estimates and TP53 outperform any AR perturbation to infer prognosis.See related commentary by Rebello et al., p. 1699.

Cell-free DNA profiling of metastatic prostate cancer reveals microsatellite instability, structural rearrangements and clonal hematopoiesis.

BACKGROUND: There are multiple existing and emerging therapeutic avenues for metastatic prostate cancer, with a common denominator, which is the need for predictive biomarkers. Circulating tumor DNA (ctDNA) has the potential to cost-efficiently accelerate precision medicine trials to improve clinical efficacy and diminish costs and toxicity. However, comprehensive ctDNA profiling in metastatic prostate cancer to date has been limited. METHODS: A combination of targeted and low-pass whole genome sequencing was performed on plasma cell-free DNA and matched white blood cell germline DNA in 364 blood samples from 217 metastatic prostate cancer patients. RESULTS: ctDNA was detected in 85.9% of baseline samples, correlated to line of therapy and was mirrored by circulating tumor cell enumeration of synchronous blood samples. Comprehensive profiling of the androgen receptor (AR) revealed a continuous increase in the fraction of patients with intra-AR structural variation, from 15.4% during first-line metastatic castration-resistant prostate cancer therapy to 45.2% in fourth line, indicating a continuous evolution of AR during the course of the disease. Patients displayed frequent alterations in DNA repair deficiency genes (18.0%). Additionally, the microsatellite instability phenotype was identified in 3.81% of eligible samples (≥ 0.1 ctDNA fraction). Sequencing of non-repetitive intronic and exonic regions of PTEN, RB1, and TP53 detected biallelic inactivation in 47.5%, 20.3%, and 44.1% of samples with ≥ 0.2 ctDNA fraction, respectively. Only one patient carried a clonal high-impact variant without a detectable second hit. Intronic high-impact structural variation was twice as common as exonic mutations in PTEN and RB1. Finally, 14.6% of patients presented false positive variants due to clonal hematopoiesis, commonly ignored in commercially available assays. CONCLUSIONS: ctDNA profiles appear to mirror the genomic landscape of metastatic prostate cancer tissue and may cost-efficiently provide somatic information in clinical trials designed to identify predictive biomarkers. However, intronic sequencing of the interrogated tumor suppressors challenges the ubiquitous focus on coding regions and is vital, together with profiling of synchronous white blood cells, to minimize erroneous assignments which in turn may confound results and impede true associations in clinical trials.

Immune Infiltration in Invasive Lobular Breast Cancer.

Background: Invasive lobular breast cancer (ILC) is the second most common histological subtype of breast cancer after invasive ductal cancer (IDC). Here, we aimed at evaluating the prevalence, levels, and composition of tumor-infiltrating lymphocytes (TILs) and their association with clinico-pathological and outcome variables in ILC, and to compare them with IDC. Methods: We considered two patient series with TIL data: a multicentric retrospective series (n = 614) and the BIG 02-98 study (n = 149 ILC and 807 IDC). We compared immune subsets identified by immuno-histochemistry in the ILC (n = 159) and IDC (n = 468) patients from the Nottingham series, as well as the CIBERSORT immune profiling of the ILC (n = 98) and IDC (n = 388) METABRIC and The Cancer Genome Atlas patients. All ILC/IDC comparisons were done in estrogen receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative tumors. All statistical tests were two-sided. Results: TIL levels were statistically significantly lower in ILC compared with IDC (fold-change = 0.79, 95% confidence interval = 0.70 to 0.88, P < .001). In ILC, high TIL levels were associated with young age, lymph node involvement, and high proliferative tumors. In the univariate analysis, high TIL levels were associated with worse prognosis in the retrospective and BIG 02-98 lobular series, although they did not reach statistical significance in the latter. The Nottingham series revealed that the levels of intratumoral but not total CD8+ were statistically significantly lower in ILC compared with IDC. Comparison of the CIBERSORT profiles highlighted statistically significant differences in terms of immune composition. Conclusions: This study shows differences between the immune infiltrates of ER-positive/HER2-negative ILC and IDC in terms of prevalence, levels, localization, composition, and clinical associations.

Circulating tumor cells and survival in abiraterone- and enzalutamide-treated patients with castration-resistant prostate cancer.

BACKGROUND: The outcome to treatment administered to patients with metastatic castration-resistant prostate cancer (mCRPC) greatly differs between individuals, underlining the need for biomarkers guiding treatment decision making. OBJECTIVE: To investigate the prognostic value of circulating tumor cell (CTC) enumeration and dynamics, in the context of second-line endocrine therapies (ie, abiraterone acetate or enzalutamide), irrespective of prior systemic therapies. DESIGN, SETTINGS, AND PARTICIPANTS: In a prospective, multicentre study blood samples for CTC enumeration were collected from patients with mCRPC at baseline (n = 174). In patients who responded for minimally 10-12 weeks a follow-up sample was collected. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: For baseline analysis, patients were stratified in <5 or ≥5 CTCs/7.5 mL, whereas for the analysis of CTC dynamics at 10-12 weeks, in patients with stable, increasing or decreasing CTC counts. Progression-free survival (PFS), overall survival (OS), and PSA changes at 10-12 weeks were compared between groups. RESULTS: Patients demonstrating increasing CTCs on therapy had a shorter median PFS (4.03 vs 12.98 vs 13.67 months, HR 3.6, 95%CI 1.9-6.8; P < 0.0001) and OS (11.2 months vs not reached, HR 9.5, 95%CI 3.7-24; P < 0.0001), compared to patients with decreasing or stable CTCs. Multivariable Cox regression showed that prior chemotherapy (HR 4.1, 95%CI 1.9-8.9; P = 0.0003), a high baseline CTC count (HR 1.5, 95%CI 1.2-1.9; P = 0.002) and increasing CTCs at follow-up (HR 3.3, 95%CI 1.4-7.6; P = 0.005) were independent predictors of worse PFS. Previous chemotherapy (HR 7, 95%CI 1.9-25; P = 0.003), high baseline CTC counts (HR 2.2, 95%CI 1.4-3.7; P = 0.002) and increasing CTCs during therapy (HR 4.6, 95%CI 1.4-15; P = 0.01) were independently associated with shorter OS. ≥30% and ≥50% PSA responses less frequently occurred in patients with CTC inclines at 10-12 weeks on therapy (χ2 test: P < 0.01). CONCLUSIONS: CTC dynamics during therapy are associated with PSA response and provide independent clinical prognostication over PSA declines. Hence the study demonstrates the pharmacodynamic properties of CTCs.

p-STAT3 in luminal breast cancer: Integrated RNA-protein pooled analysis and results from the BIG 2-98 phase III trial.

In the present study, in order to investigate the role of signal transducer and activator of transcription 3 (STAT3) in estrogen receptor (ER)-positive breast cancer prognosis, we evaluated the phosphorylated STAT3 (p-STAT3) status and investigated its effect on the outcome in a pooled analysis and in a large prospective adjuvant trial. By using the TCGA repository, we developed gene signatures that reflected the level of p-STAT3. Using pooled analysis of the expression data from luminal breast cancer patients, we assessed the effects of the p-STAT3 expression signature on prognosis. We further validated the p-STAT3 prognostic effect using immunohistochemistry (IHC) and immunofluorescence staining of p-STAT3 tissue microarrays from a large randomised prospective trial. Our analysis demonstrated that p-STAT3 expression was elevated in luminal A-type breast cancer (Kruskal-Wallis test, P<10e-10) and was significantly associated with a good prognosis (log-rank, P<10e-10). Notably, the p-STAT3 expression signature identified patients with a good prognosis irrespective of the luminal subtype (log-rank: luminal A, P=0.026; luminal B, P=0.006). p-STAT3 staining by IHC in the stroma or tumour was detected in 174 out of 610 ER-positive samples (28.5%) from the BIG 2-98 randomised trial. With a median follow-up of 10.1 years, p-STAT3 was associated with a reduced risk of recurrence in ER-positive/HER2-negative breast cancer (Cox univariate HR, 0.66; 95% CI, 0.44-0.98; P=0.04). On the whole, our data indicate that p-STAT3 is associated with an improved outcome in ER-positive breast cancer.

Update on tumor-infiltrating lymphocytes (TILs) in breast cancer, including recommendations to assess TILs in residual disease after neoadjuvant therapy and in carcinoma in situ: A report of the International Immuno-Oncology Biomarker Working Group on Breast Cancer.

Morphological evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer is gaining momentum as evidence strengthens the clinical relevance of this immunological biomarker. TILs in the post-neoadjuvant residual disease setting are acquiring increasing importance as a stratifying marker in clinical trials, considering the raising interest on immunotherapeutic strategies after neoadjuvant chemotherapy. TILs in ductal carcinoma in situ, with or without invasive carcinoma, represent an emerging area of clinical breast cancer research. The aim of this report is to update pathologists, clinicians and researchers on TIL assessment in both the post-neoadjuvant residual disease and the ductal carcinoma in situ settings. The International Immuno-Oncology Working Group proposes a method for assessing TILs in these settings, based on the previously published International Guidelines on TIL Assessment in Breast Cancer. In this regard, these recommendations represent a consensus guidance for pathologists, aimed to achieve the highest possible consistency among future studies.

Immune Checkpoint Molecules on Tumor-Infiltrating Lymphocytes and Their Association with Tertiary Lymphoid Structures in Human Breast Cancer.

There is an exponentially growing interest in targeting immune checkpoint molecules in breast cancer (BC), particularly in the triple-negative subtype where unmet treatment needs remain. This study was designed to analyze the expression, localization, and prognostic role of PD-1, PD-L1, PD-L2, CTLA-4, LAG3, and TIM3 in primary BC. Gene expression analysis using the METABRIC microarray dataset found that all six immune checkpoint molecules are highly expressed in basal-like and HER2-enriched compared to the other BC molecular subtypes. Flow cytometric analysis of fresh tissue homogenates from untreated primary tumors show that PD-1 is principally expressed on CD4+ or CD8+ T cells and CTLA-4 is expressed on CD4+ T cells. The global proportion of PD-L1+, PD-L2+, LAG3+, and TIM3+ tumor-infiltrating lymphocytes (TIL) was low and detectable in only a small number of tumors. Immunohistochemically staining fixed tissues from the same tumors was employed to score TIL and tertiary lymphoid structures (TLS). PD-L1+, PD-L2+, LAG3+, and TIM3+ cells were detected in some TLS in a pattern that resembles secondary lymphoid organs. This observation suggests that TLS are important sites of immune activation and regulation, particularly in tumors with extensive baseline immune infiltration. Significantly improved overall survival was correlated with PD-1 expression in the HER2-enriched and PD-L1 or CTLA-4 expression in basal-like BC. PD-1 and CTLA-4 proteins were most frequently detected on TIL, which supports the correlations observed between their gene expression and improved long-term outcome in basal-like and HER2-enriched BC. PD-L1 expression by tumor or immune cells is uncommon in BC. Overall, the data presented here distinguish PD-1 as a marker of T cell activity in both the T and B cell areas of BC associated TLS. We found that immune checkpoint molecule expression parallels the extent of TIL and TLS, although there is a noteworthy amount of heterogeneity between tumors even within the same molecular subtype. These data indicate that assessing the levels of immune checkpoint molecule expression in an individual patient has important implications for the success of therapeutically targeting them in BC.