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.
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.
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).
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.
Abstract
We retrospectively assessed association of stromal tumor-infiltrating lymphocytes (sTILs) with clinical outcomes and molecular variables reportedly predictive of trastuzumab-benefit in ...National Surgical Adjuvant Breast and Bowel Project B-31 (N = 2130). sTILs were assessed in 1581 eligible B-31 cases utilizing all available hematoxylin and eosin slides. Mean concordance between main reviewer and six other pathologists was 90.8% in 100 cases. Cox regressions were used to calculate hazard ratios (HRs). In chemotherapy and trastuzumab added to chemotherapy arms, increases in sTILs, as a semicontinuous variable (combined arms HR = 0.42, 95% confidence interval = 0.27 to 0.64, two-sided P < .001) or as lymphocyte-predominant breast cancer with more than 50% sTILs (combined arms HR = 0.65, 95% confidence interval = 0.49 to 0.86, two-sided P = .003) were statistically significantly associated with improved disease-free survival. There was no association of sTILs with trastuzumab benefit. However, higher sTILs were statistically significantly associated with higher trastuzumab benefit groups by 8-gene prediction model (two-sided P < .001). Neither PIK3CA mutations nor Fc-gamma-receptor polymorphisms were associated with sTILs. sTILs may have utility as a prognostic biomarker identifying HER2-positive early breast cancer at low recurrence risk.
The enumeration of circulating tumour cells (CTCs) with the EpCAM-based CellSearch system has prognostic significance in patients with metastatic breast cancer (MBC). The aim of this study was to ...explore potential differences in the detection and prognostic significance of CTCs in MBC according to immunohistochemical subtypes of breast cancer.
CellSearch CTC counts were obtained from 154 MBC patients before first-line systemic treatment between November 2007 and August 2012. Patients were categorised in five subgroups according to immunohistochemical surrogate definitions of intrinsic subtypes in breast cancer based on hormone receptor status, HER2/neu status and histological grade. Differences in progression-free (PFS) and overall survival (OS) were assessed relative to the cut-off value of ≥5 CTCs per 7.5 ml blood.
No significant differences were observed in the absolute CTC counts (P=0.120) or in CTC positivity rates according to ≥1 and ≥5 CTCs per 7.5 ml blood detection thresholds (P=0.165 and P=0.651, respectively) between immunohistochemical subtypes. However, very high CTC counts, defined as ≥80 CTCs per 7.5 ml, were observed more frequently in patients with Luminal A and triple negative (TN) breast cancer (P=0.024). In the total study population, the presence of ≥5 CTCs was the single most significant prognostic factor for both PFS and OS in multivariate analysis (P<0.001). A more limited prognostic impact, not reaching statistical significance, was observed in patients with HER2-positive disease as opposed to patients with Luminal A, Luminal B-HER2-negative and TN disease.
The detection of EpCAM+CTCs was not clearly associated with any of the immunohistochemical subtypes of breast cancer in patients with MBC before first-line treatment. Potentially clinically relevant differences were however observed at very high CTC counts. Furthermore, our data suggest a lower prognostic significance of CTC evaluation in HER2-positive patients with MBC.
A recent comprehensive whole genome analysis of a large breast cancer cohort was used to link known and novel drivers and substitution signatures to the transcriptome of 266 cases. Here, we validate ...that subtype-specific aberrations show concordant expression changes for, for example, TP53, PIK3CA, PTEN, CCND1 and CDH1. We find that CCND3 expression levels do not correlate with amplification, while increased GATA3 expression in mutant GATA3 cancers suggests GATA3 is an oncogene. In luminal cases the total number of substitutions, irrespective of type, associates with cell cycle gene expression and adverse outcome, whereas the number of mutations of signatures 3 and 13 associates with immune-response specific gene expression, increased numbers of tumour-infiltrating lymphocytes and better outcome. Thus, while earlier reports imply that the sheer number of somatic aberrations could trigger an immune-response, our data suggests that substitutions of a particular type are more effective in doing so than others.
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 (
< 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
,
, and
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
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,
levels are predictive for progression-free survival time to aromatase inhibitor (AI) therapy in advanced breast cancer patients, and found that
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.
Evidence is strengthening for the morphological evaluation of tumor infiltrating lymphocytes (TILs) in breast cancer. Herein, the concepts for TILs assessment are laid out by pathologists to ...facilitate their wider evaluation and consistent interpretation. The goal of this article is to promote the evaluation of TILs as a biomarker in research, clinical trial settings and day-to-day practice.
The morphological evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer (BC) is gaining momentum as evidence strengthens for the clinical relevance of this immunological biomarker. Accumulating evidence suggests that the extent of lymphocytic infiltration in tumor tissue can be assessed as a major parameter by evaluation of hematoxylin and eosin (H&E)-stained tumor sections. TILs have been shown to provide prognostic and potentially predictive value, particularly in triple-negative and human epidermal growth factor receptor 2-overexpressing BC.
A standardized methodology for evaluating TILs is now needed as a prerequisite for integrating this parameter in standard histopathological practice, in a research setting as well as in clinical trials. This article reviews current data on the clinical validity and utility of TILs in BC in an effort to foster better knowledge and insight in this rapidly evolving field, and to develop a standardized methodology for visual assessment on H&E sections, acknowledging the future potential of molecular/multiplexed approaches.
The methodology provided is sufficiently detailed to offer a uniformly applied, pragmatic starting point and improve consistency and reproducibility in the measurement of TILs for future studies.
To infer the prognostic value of simultaneous androgen receptor (
) and
profiling in liquid biopsies from patients with metastatic castration-resistant prostate cancer (mCRPC) starting a new line of
...signaling inhibitors (ARSi).
Between March 2014 and April 2017, we recruited patients with mCRPC (
= 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
splice variants (ARV). Low-pass whole-genome and targeted gene-body sequencing of
and
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.
Overall, no single
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.
inactivation harbored independent prognostic value HR 1.88; 95% confidence interval (CI), 1.18-3.00;
= 0.008, and outperformed ARV expression and detection of genomic
alterations. Using Cox coefficient analysis of clinical parameters and
status, we identified three prognostic groups with differing PFS estimates (median, 14.7 vs. 7.51 vs. 2.62 months;
< 0.0001), which was validated in an independent mCRPC cohort (
= 202) starting first-line ARSi (median, 14.3 vs. 6.39 vs. 2.23 months;
< 0.0001).
In an all-comer cohort, tumor burden estimates and
outperform any
perturbation to infer prognosis.See related commentary by Rebello et al., p. 1699.
Molecular characterisation of single circulating tumour cells (CTCs) holds considerable promise for predictive biomarker assessment and to explore CTC heterogeneity. We evaluate a new method, the ...DEPArray system, that allows the dielectrophoretic manipulation and isolation of single and 100% purified groups of CTCs from pre-enriched blood samples and explore the feasibility of their molecular characterisation.
Samples containing known numbers of two cell populations were used to assess cell loss during sample loading. Cultured breast cancer cells were isolated from spiked blood samples using CellSearch CTC and Profile kits. Single tumour cells and groups of up to 10 tumour cells were recovered with the DEPArray system and subjected to transcriptional and mutation analysis.
On average, 40% cell loss was observed when loading samples to the DEPArray system. Expected mutations in clinically relevant markers could be obtained for 60% of single recovered tumour cells and all groups of tumour cells. Reliable gene expression profiles were obtained from single cells and groups of up to 10 cells for 2 out of 3 spiked breast cancer cell lines.
We describe a semiautomated workflow for the isolation of small groups of 1 to 10 tumour cells from whole blood samples and provide proof of principle for the feasibility of their comprehensive molecular characterisation.
Abstract Background Expression of the androgen receptor splice variant 7 (AR-V7) is associated with poor response to second-line endocrine therapy in castration-resistant prostate cancer (CRPC). ...However, a large fraction of nonresponding patients are AR-V7–negative. Objective To investigate if a comprehensive liquid biopsy–based AR profile may improve patient stratification in the context of second-line endocrine therapy. Design, setting, and participants Peripheral blood was collected from patients with CRPC ( n = 30) before initiation of a new line of systemic therapy. We performed profiling of circulating tumour DNA via low-pass whole-genome sequencing and targeted sequencing of the entire AR gene, including introns. Targeted RNA sequencing was performed on enriched circulating tumour cell fractions to assess the expression levels of seven AR splice variants (ARVs). Outcome measurements and statistical analysis Somatic AR variations, including copy-number alterations, structural variations, and point mutations, were combined with ARV expression patterns and correlated to clinicopathologic parameters. Results and limitations Collectively, any AR perturbation, including ARV, was detected in 25/30 patients. Surprisingly, intra-AR structural variation was present in 15/30 patients, of whom 14 expressed ARVs. The majority of ARV-positive patients expressed multiple ARVs, with AR-V3 the most abundantly expressed. The presence of any ARV was associated with progression-free survival after second-line endocrine treatment (hazard ratio 4.53, 95% confidence interval 1.424–14.41; p = 0.0105). Six out of 17 poor responders were AR-V7–negative, but four carried other AR perturbations. Conclusions Comprehensive AR profiling, which is feasible using liquid biopsies, is necessary to increase our understanding of the mechanisms underpinning resistance to endocrine treatment. Patient summary Alterations in the androgen receptor are associated with endocrine treatment outcomes. This study demonstrates that it is possible to identify different types of alterations via simple blood draws. Follow-up studies are needed to determine the effect of such alterations on hormonal therapy.
Tumor-infiltrating lymphocytes (TILs) are a robust prognostic adjunct in invasive breast cancer, but their clinical role in ductal carcinoma in situ (DCIS) has not been ascertained.
We evaluated the ...prevalence and clinical relevance of TILs in a well annotated series of 1488 consecutive DCIS women with a median follow-up of 8.2years. Detailed criteria for TILs evaluation were pre-defined involving the International Immuno-Oncology Biomarker Working Group. TILs percentage was considered both as a continuous and categorical variable. Levels of TILs were examined for their associations with ipsilateral breast event (IBE), whether in situ or invasive.
Of the 1488 patients with DCIS under study, 35.1% had <1%, 58.3% 1–49% and 6.5% ≥50% peri-ductal stromal lymphocytes. The interobserver agreement in TILs evaluation, measured by the intraclass correlation coefficient (ICC) was 0.96 (95% CI 0.95–0.97). At univariable analysis, clinical factors significantly associated with TILs (P ≤0.001) were intrinsic subtype, grade, necrosis, type of surgery. Her-2 positive DCIS were more frequently associated with TILs (24% of patients with TILs ≥50%), followed by the triple negative (11%), Luminal B/Her-2 positive (9%) and Luminal A/B subtypes (1%) (P < 0.0001). We did not find any association between TILs as a continuous variable and the risk of IBEs. Likewise, when patients were stratified by TILs percentage (<1%, between 1% and 49.9%, and ≥50%), no statistically significant association was observed (10-year cumulative incidence of IBEs: 19%, 17.3%, and 18.7% respectively, P = 0.767).
TILs occur more frequently in the Her-2 positive DCIS. Although we did not find a significant association between TILs and the 10-year risk of IBE, our data suggest that immunotherapies might be considered in subsets of DCIS patients.
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