Report on computational assessment of Tumor Infiltrating Lymphocytes from the International Immuno-Oncology Biomarker Working Group.

Assessment of tumor-infiltrating lymphocytes (TILs) is increasingly recognized as an integral part of the prognostic workflow in triple-negative (TNBC) and HER2-positive breast cancer, as well as many other solid tumors. This recognition has come about thanks to standardized visual reporting guidelines, which helped to reduce inter-reader variability. Now, there are ripe opportunities to employ computational methods that extract spatio-morphologic predictive features, enabling computer-aided diagnostics. We detail the benefits of computational TILs assessment, the readiness of TILs scoring for computational assessment, and outline considerations for overcoming key barriers to clinical translation in this arena. Specifically, we discuss: 1. ensuring computational workflows closely capture visual guidelines and standards; 2. challenges and thoughts standards for assessment of algorithms including training, preanalytical, analytical, and clinical validation; 3. perspectives on how to realize the potential of machine learning models and to overcome the perceptual and practical limits of visual scoring.

Circulating tumor DNA in early response assessment and monitoring of advanced colorectal cancer treated with a multi-kinase inhibitor.

Predictive biomarkers are eagerly awaited in advanced colorectal cancer (aCRC). Targeted sequencing performed on tumor and baseline plasma samples in 20 patients with aCRC treated with regorafenib identified 89 tumor-specific mutations of which ≥50% are also present in baseline plasma. Droplet digital PCR (ddPCR) assays were optimized to monitor circulating tumor DNA (ctDNA) levels in plasmatic samples collected throughout the treatment course and showed the importance of using the absolute value for ctDNA rather than the mutant/wild type ratio in monitoring the therapy outcome. High baseline cell free DNA (cfDNA) levels are associated with shorter overall survival (OS) (HR 7.38, P=0.001). An early increase (D14) in mutated copies/mL is associated with a significantly worse PFS (HR 6.12, P=0.008) and OS (HR 8.02, P=0.004). These data suggest a high prognostic value for early ctDNA level changes and support the use of blood-born genomic markers as a tool for treatment.

Reliability of tumor-infiltrating lymphocyte and tertiary lymphoid structure assessment in human breast cancer.

The presence of tumor-infiltrating lymphocytes (TIL), reflecting host immune activity, is frequently correlated with better clinical outcomes, particularly in HER2-positive and triple-negative breast cancer. Recent findings suggest that organization of immune infiltrates in tertiary lymphoid structures also has a beneficial effect on survival. This study investigated inter- and intra-observer variation in TIL assessment using conventional hematoxylin-eosin versus immunohistochemical staining to identify immune cells. Global, intratumoral, and stromal TIL, as well as tertiary lymphoid structures were scored independently by experienced pathologists on full-face tumor sections (n=124). The fidelity of scoring infiltrates in core biopsies compared to surgical specimens, and pathological assessment compared to quantitative digital analysis was also evaluated. The inter-observer concordance correlation coefficient was 0.80 for global, 0.72 for intratumoral, and 0.71 for stromal TIL, while the intra-observer concordance correlation coefficient was 0.90 for global, 0.77 for intratumoral, and 0.89 for stromal TIL using immunohistochemical stains. Correlations were lower with hematoxylin-eosin stains, particularly for intratumoral TIL, while global scores had the highest concordance correlation coefficients. Our study concluded that tertiary lymphoid structures are accurately and consistently scored using immunohistochemical but not hematoxylin-eosin stains. A strong association was observed between TIL in core biopsies and surgical samples (R2=0.74) but this did not extend to tertiary lymphoid structures (R2=0.26). TIL scored by pathologists and digital analysis were correlated but our analysis reveals a constant bias between these methods. These data challenge current criteria for TIL and tertiary lymphoid structure assessment in breast cancer and recommend that how pathologists evaluate immune infiltrates be reexamined for future studies.

Assessment of Ki67 in breast cancer: recommendations from the International Ki67 in Breast Cancer working group.

Uncontrolled proliferation is a hallmark of cancer. In breast cancer, immunohistochemical assessment of the proportion of cells staining for the nuclear antigen Ki67 has become the most widely used method for comparing proliferation between tumor samples. Potential uses include prognosis, prediction of relative responsiveness or resistance to chemotherapy or endocrine therapy, estimation of residual risk in patients on standard therapy and as a dynamic biomarker of treatment efficacy in samples taken before, during, and after neoadjuvant therapy, particularly neoadjuvant endocrine therapy. Increasingly, Ki67 is measured in these scenarios for clinical research, including as a primary efficacy endpoint for clinical trials, and sometimes for clinical management. At present, the enormous variation in analytical practice markedly limits the value of Ki67 in each of these contexts. On March 12, 2010, an international panel of investigators with substantial expertise in the assessment of Ki67 and in the development of biomarker guidelines was convened in London by the co-chairs of the Breast International Group and North American Breast Cancer Group Biomarker Working Party to consider evidence for potential applications. Comprehensive recommendations on preanalytical and analytical assessment, and interpretation and scoring of Ki67 were formulated based on current evidence. These recommendations are geared toward achieving a harmonized methodology, create greater between-laboratory and between-study comparability, and allow earlier valid applications of this marker in clinical practice.

Assessment of an RNA interference screen-derived mitotic and ceramide pathway metagene as a predictor of response to neoadjuvant paclitaxel for primary triple-negative breast cancer: a retrospective analysis of five clinical trials.

BACKGROUND: Addition of taxanes to preoperative chemotherapy in breast cancer increases the proportion of patients who have a pathological complete response (pCR). However, a substantial proportion of patients do not respond, and the prognosis is particularly poor for patients with oestrogen-receptor (ER)/progesterone-receptor (PR)/human epidermal growth factor receptor 2 (HER2; ERBB2)-negative (triple-negative) disease who do not achieve a pCR. Reliable identification of such patients is the first step in determining who might benefit from alternative treatment regimens in clinical trials. We previously identified genes involved in mitosis or ceramide metabolism that influenced sensitivity to paclitaxel, with an RNA interference (RNAi) screen in three cancer cell lines, including a triple-negative breast-cancer cell line. Here, we assess these genes as a predictor of pCR to paclitaxel combination chemotherapy in triple-negative breast cancer. METHODS: We derived a paclitaxel response metagene based on mitotic and ceramide genes identified by functional genomics studies. We used area under the curve (AUC) analysis and multivariate logistic regression to retrospectively assess the metagene in six cohorts of patients with triple-negative breast cancer treated with neoadjuvant chemotherapy; two cohorts treated with paclitaxel (n=27, 30) and four treated without paclitaxel (n=88, 28, 48, 39). FINDINGS: The metagene was associated with pCR in paclitaxel-treated cohorts (AUC 0.79 [95% CI 0.53-0.93], 0.72 [0.48-0.90]) but not in non-paclitaxel treated cohorts (0.53 [0.31-0.77], 0.59 [0.22-0.82], 0.53 [0.36-0.71], 0.64 [0.43-0.81]). In multivariate logistic regression, the metagene was associated with pCR (OR 19.92, 2.62-151.57; p=0.0039) with paclitaxel-containing chemotherapy. INTERPRETATION: The paclitaxel response metagene shows promise as a paclitaxel-specific predictor of pCR in patients with triple-negative breast cancer. The metagene is suitable for development into a reverse transcription-PCR assay, for which clinically relevant thresholds could be established in randomised clinical trials. These results highlight the potential for functional genomics to accelerate development of drug-specific predictive biomarkers without the need for training clinical trial cohorts. FUNDING: UK Medical Research Council; Cancer Research UK; the National Institute for Health Research (UK); the Danish Council for Independent Research-Medical Sciences (FSS); Breast Cancer Research Foundation (New York); Fondation Luxembourgeoise contre le Cancer; the Fonds National de la Recherche Scientifique; Brussels Region (IRSIB-IP, Life Sciences 2007) and Walloon Region (Biowin-Keymarker); Sally Pearson Breast Cancer Fund; and the European Commission.