An algorithm for standardization of tumor Infiltrating lymphocyte evaluation in head and neck cancers.

PURPOSE: The prognostic and predictive significance of pathologist-read tumor infiltrating lymphocytes (TILs) in head and neck cancers have been demonstrated through multiple studies over the years. TILs have not been broadly adopted clinically, perhaps due to substantial inter-observer variability. In this study, we developed a machine-based algorithm for TIL evaluation in head and neck cancers and validated its prognostic value in independent cohorts. EXPERIMENTAL DESIGN: A network classifier called NN3-17 was trained to identify and calculate tumor cells, lymphocytes, fibroblasts and "other" cells on hematoxylin-eosin stained sections using the QuPath software. These measurements were used to construct three predefined TIL variables. A retrospective collection of 154 head and neck squamous cell cancer cases was used as the discovery set to identify optimal association of TIL variables and survival. Two independent cohorts of 234 cases were used for validation. RESULTS: We found that electronic TIL variables were associated with favorable prognosis in both the HPV-positive and -negative cases. After adjusting for clinicopathologic factors, Cox regression analysis demonstrated that electronic total TILs% (p = 0.025) in the HPV-positive and electronic stromal TILs% (p < 0.001) in the HPV-negative population were independent markers of disease specific outcomes (disease free survival). CONCLUSIONS: Neural network TIL variables demonstrated independent prognostic value in validation cohorts of HPV-positive and HPV-negative head and neck cancers. These objective variables can be calculated by an open-source software and could be considered for testing in a prospective setting to assess potential clinical implications.

Image analysis-based tumor infiltrating lymphocytes measurement predicts breast cancer pathologic complete response in SWOG S0800 neoadjuvant chemotherapy trial.

We assessed the predictive value of an image analysis-based tumor-infiltrating lymphocytes (TILs) score for pathologic complete response (pCR) and event-free survival in breast cancer (BC). About 113 pretreatment samples were analyzed from patients with stage IIB-IIIC HER-2-negative BC randomized to neoadjuvant chemotherapy ± bevacizumab. TILs quantification was performed on full sections using QuPath open-source software with a convolutional neural network cell classifier (CNN11). We used easTILs% as a digital metric of TILs score defined as [sum of lymphocytes area (mm2)/stromal area(mm2)] × 100. Pathologist-read stromal TILs score (sTILs%) was determined following published guidelines. Mean pretreatment easTILs% was significantly higher in cases with pCR compared to residual disease (median 36.1 vs.14.8%, p < 0.001). We observed a strong positive correlation (r = 0.606, p < 0.0001) between easTILs% and sTILs%. The area under the prediction curve (AUC) was higher for easTILs% than sTILs%, 0.709 and 0.627, respectively. Image analysis-based TILs quantification is predictive of pCR in BC and had better response discrimination than pathologist-read sTILs%.

Objective assessment of tumor infiltrating lymphocytes as a prognostic marker in melanoma using machine learning algorithms.

BACKGROUND: The prognostic value of tumor-infiltrating lymphocytes (TILs) assessed by machine learning algorithms in melanoma patients has been previously demonstrated but has not been widely adopted in the clinic. We evaluated the prognostic value of objective automated electronic TILs (eTILs) quantification to define a subset of melanoma patients with a low risk of relapse after surgical treatment. METHODS: We analyzed data for 785 patients from 5 independent cohorts from multiple institutions to validate our previous finding that automated TIL score is prognostic in clinically-localized primary melanoma patients. Using serial tissue sections of the Yale TMA-76 melanoma cohort, both immunofluorescence and Hematoxylin-and-Eosin (H&E) staining were performed to understand the molecular characteristics of each TIL phenotype and their associations with survival outcomes. FINDINGS: Five previously-described TIL variables were each significantly associated with overall survival (p<0.0001). Assessing the receiver operating characteristic (ROC) curves by comparing the clinical impact of two models suggests that etTILs (electronic total TILs) (AUC: 0.793, specificity: 0.627, sensitivity: 0.938) outperformed eTILs (AUC: 0.77, specificity: 0.51, sensitivity: 0.938). We also found that the specific molecular subtype of cells representing TILs includes predominantly cells that are CD3+ and CD8+ or CD4+ T cells. INTERPRETATION: eTIL% and etTILs scores are robust prognostic markers in patients with primary melanoma and may identify a subgroup of stage II patients at high risk of recurrence who may benefit from adjuvant therapy. We also show the molecular correlates behind these scores. Our data support the need for prospective testing of this algorithm in a clinical trial. FUNDING: This work was also supported by a sponsored research agreements from Navigate Biopharma and NextCure and by grants from the NIH including the Yale SPORE in in Skin Cancer, P50 CA121974, the Yale SPORE in Lung Cancer, P50 CA196530, NYU SPORE in Skin Cancer P50CA225450 and the Yale Cancer Center Support Grant, P30CA016359.

Tumor immune microenvironment of self-identified African American and non-African American triple negative breast cancer.

Differences in the tumor immune microenvironment may result in differences in prognosis and response to treatment in cancer patients. We hypothesized that differences in the tumor immune microenvironment may exist between African American (AA) and NonAA patients, due to ancestry-related or socioeconomic factors, that may partially explain differences in clinical outcomes. We analyzed clinically matched triple-negative breast cancer (TNBC) tissues from self-identified AA and NonAA patients and found that stromal TILs, PD-L1 IHC-positivity, mRNA expression of immune-related pathways, and immunotherapy response predictive signatures were significantly higher in AA samples (p < 0.05; Fisher's Exact Test, Mann-Whitney Test, Permutation Test). Cancer biology and metabolism pathways, TAM-M2, and Immune Exclusion were significantly higher in NonAA samples (p < 0.05; Permutation Test, Mann-Whitney Test). There were no differences in somatic tumor mutation burden. Overall, there is greater immune infiltration and inflammation in AA TNBC and these differences may impact response to immune checkpoint inhibitors and other therapeutic agents that modulate the immune microenvironment.

Systematically higher Ki67 scores on core biopsy samples compared to corresponding resection specimen in breast cancer: a multi-operator and multi-institutional study.

Ki67 has potential clinical importance in breast cancer but has yet to see broad acceptance due to inter-laboratory variability. Here we tested an open source and calibrated automated digital image analysis (DIA) platform to: (i) investigate the comparability of Ki67 measurement across corresponding core biopsy and resection specimen cases, and (ii) assess section to section differences in Ki67 scoring. Two sets of 60 previously stained slides containing 30 core-cut biopsy and 30 corresponding resection specimens from 30 estrogen receptor-positive breast cancer patients were sent to 17 participating labs for automated assessment of average Ki67 expression. The blocks were centrally cut and immunohistochemically (IHC) stained for Ki67 (MIB-1 antibody). The QuPath platform was used to evaluate tumoral Ki67 expression. Calibration of the DIA method was performed as in published studies. A guideline for building an automated Ki67 scoring algorithm was sent to participating labs. Very high correlation and no systematic error (p = 0.08) was found between consecutive Ki67 IHC sections. Ki67 scores were higher for core biopsy slides compared to paired whole sections from resections (p ≤ 0.001; median difference: 5.31%). The systematic discrepancy between core biopsy and corresponding whole sections was likely due to pre-analytical factors (tissue handling, fixation). Therefore, Ki67 IHC should be tested on core biopsy samples to best reflect the biological status of the tumor.

Programmed Death-Ligand 1 and Programmed Death-Ligand 2 mRNAs Measured Using Closed-System Quantitative Real-Time Polymerase Chain Reaction Are Associated With Outcome and High Negative Predictive Value in Immunotherapy-Treated NSCLC.

INTRODUCTION: Immune checkpoint inhibitors (ICIs) have become standard of care in lung cancer management, but only a relatively small percentage of patients treated respond. Current predictive biomarkers, including immunohistochemical detection of programmed death-ligand 1 (PD-L1), are insufficient for determining who will respond or, more importantly in the adjuvant setting, who will not respond to ICI therapy. Here, we investigate an alternative method of assessment of PD-L1 to predict nonresponse. METHODS: This study uses a research use only quantitative real-time reverse transcription polymerase chain reaction assay on the GeneXpert system, to test for the association between four target immune genes, CD274 (PD-L1), PDCD1LG2 (programmed death-ligand 2 [PD-L2]), CD8A, and IRF1, and response to ICI therapy. Tissues were collected from 122 patients with advanced NSCLC before ICI therapy in a retrospective cohort, macrodissected, and analyzed using the GeneXpert. RESULTS: Both high PD-L1 and PD-L2 mRNA expression levels were associated with improved long-term benefit at 24 months (p = 0.047 for both PD-L1 and PD-L2) and overall survival (PD-L1, p = 0.048; PD-L2, p = 0.049). Both PD-L1 and PD-L2 mRNA levels were higher in patients with KRAS mutations. Most importantly, low PD-L1 mRNA level had a high negative predictive value of 0.92 for absence of long-term benefit. CONCLUSIONS: With further validation of this assay in low-stage patients, an assessment of PD-L1 mRNA rather than protein, could be a method to determine which low-stage patients that should not be treated with ICIs in the adjuvant setting. This approach may also be a useful objective method for selecting patients for treatment in the advanced setting.

Quantitative measurement of HER2 expression to subclassify ERBB2 unamplified breast cancer.

The efficacy of the antibody drug conjugate (ADC) Trastuzumab deruxtecan (T-DXd) in HER2 low breast cancer patients suggests that the historical/conventional assays for HER2 may need revision for optimal patient care. Specifically, the conventional assay is designed to distinguish amplified HER2 from unamplified cases but is not sensitive enough to stratify the lower ranges of HER2 expression. Here we determine the optimal dynamic range for unamplified HER2 detection in breast cancer and then redesign an assay to increase the resolution of the assay to stratify HER2 expression in unamplified cases. We used the AQUA™ method of quantitative immunofluorescence to test a range of antibody concentrations to maximize the sensitivity within the lower range of HER2 expression. Then, using a cell line microarray with HER2 protein measured by mass spectrometry we determined the amount of HER2 protein in units of attomols/mm2. Then by calculation of the limits of detection, quantification, and linearity of this assay we determined that low HER2 range expression in unamplified cell lines is between 2 and 20 attomol/mm2. Finally, application of this assay to a serial collection of 364 breast cancer cases from Yale shows 67% of the population has HER2 expression above the limit of quantification and below the levels seen in HER2 amplified breast cancer. In the future, this assay could be used to determine the levels of HER2 required for response to T-DXd or similar HER2 conjugated ADCs.

Predictive Markers of Response to Neoadjuvant Durvalumab with Nab-Paclitaxel and Dose-Dense Doxorubicin/Cyclophosphamide in Basal-Like Triple-Negative Breast Cancer.

PURPOSE: We examined gene expression, germline variant, and somatic mutation features associated with pathologic response to neoadjuvant durvalumab plus chemotherapy in basal-like triple-negative breast cancer (bTNBC). EXPERIMENTAL DESIGN: Germline and somatic whole-exome DNA and RNA sequencing, programmed death ligand 1 (PD-L1) IHC, and stromal tumor-infiltrating lymphocyte scoring were performed on 57 patients. We validated our results using 162 patients from the GeparNuevo randomized trial. RESULTS: Gene set enrichment analysis showed that pathways involved in immunity (adaptive, humoral, innate), JAK-STAT signaling, cancer drivers, cell cycle, apoptosis, and DNA repair were enriched in cases with pathologic complete response (pCR), whereas epithelial-mesenchymal transition, extracellular matrix, and TGFß pathways were enriched in cases with residual disease (RD). Immune-rich bTNBC with RD was enriched in CCL-3, -4, -5, -8, -23, CXCL-1, -3, -6, -10, and IL1, -23, -27, -34, and had higher expression of macrophage markers compared with immune-rich cancers with pCR that were enriched in IFNγ, IL2, -12, -21, chemokines CXCL-9, -13, CXCR5, and activated T- and B-cell markers (GZMB, CD79A). In the validation cohort, an immune-rich five-gene signature showed higher expression in pCR cases in the durvalumab arm (P = 0.040) but not in the placebo arm (P = 0.923) or in immune-poor cancers. Independent of immune markers, tumor mutation burden was higher, and PI3K, DNA damage repair, MAPK, and WNT/ß-catenin signaling pathways were enriched in germline and somatic mutations in cases with pCR. CONCLUSIONS: The TGFß pathway is associated with immune-poor phenotype and RD in bTNBC. Among immune-rich bTNBC RD, macrophage/neutrophil chemoattractants dominate the cytokine milieu, and IFNγ and activated B cells and T cells dominate immune-rich cancers with pCR.

Impact of a randomized weight loss trial on breast tissue markers in breast cancer survivors.

Few trials have examined the effect of lifestyle behavioral interventions on tissue markers in patients with cancer. The purpose of this study was to examine the feasibility and impact of a 6-month weight loss intervention on breast tissue and serum biomarkers in women with breast cancer. Fifty-one women who completed breast cancer treatment and had a BMI ≥ 25.0 kg/m2 were randomized to a weight loss intervention or usual care. Breast tissue biopsies, fasting blood draw and body composition were collected at baseline and 6 months, with between-group changes examined using analysis of covariance method. Baseline and post-intervention biopsies were conducted in 49 and 42 women, respectively, with pre- and post-epithelial tissue available from 25 tissue samples. Average 6-month weight loss was 6.7% for the weight loss group and 2.0% increase for the usual care group (p < 0.0001). At baseline, body fat and serum insulin levels were inversely associated with breast tissue insulin receptor levels and CD68 (p < 0.05). At 6 months, favorable changes were observed in serum leptin and adiponectin levels and tissue CD163 among women randomized to weight loss vs. adverse change in women randomized to usual care (p < 0.05). Breast tissue biopsies are feasible to collect in a clinical research setting among breast cancer survivors, with weight loss favorably impacting metabolic and inflammatory markers associated with breast cancer.

Expression of the transcription factor PU.1 induces the generation of microglia-like cells in human cortical organoids.

Microglia play a role in the emergence and preservation of a healthy brain microenvironment. Dysfunction of microglia has been associated with neurodevelopmental and neurodegenerative disorders. Investigating the function of human microglia in health and disease has been challenging due to the limited models of the human brain available. Here, we develop a method to generate functional microglia in human cortical organoids (hCOs) from human embryonic stem cells (hESCs). We apply this system to study the role of microglia during inflammation induced by amyloid-ß (Aß). The overexpression of the myeloid-specific transcription factor PU.1 generates microglia-like cells in hCOs, producing mhCOs (microglia-containing hCOs), that we engraft in the mouse brain. Single-cell transcriptomics reveals that mhCOs acquire a microglia cell cluster with an intact complement and chemokine system. Functionally, microglia in mhCOs protect parenchyma from cellular and molecular damage caused by Aß. Furthermore, in mhCOs, we observed reduced expression of Aß-induced expression of genes associated with apoptosis, ferroptosis, and Alzheimer's disease (AD) stage III. Finally, we assess the function of AD-associated genes highly expressed in microglia in response to Aß using pooled CRISPRi coupled with single-cell RNA sequencing in mhCOs. In summary, we provide a protocol to generate mhCOs that can be used in fundamental and translational studies as a model to investigate the role of microglia in neurodevelopmental and neurodegenerative disorders.

Interplay between copy number alterations and immune profiles in the early breast cancer Scandinavian Breast Group 2004-1 randomized phase II trial: results from a feasibility study.

Emerging data indicate that genomic alterations can shape immune cell composition in early breast cancer. However, there is a need for complementary imaging and sequencing methods for the quantitative assessment of combined somatic copy number alteration (SCNA) and immune profiling in pathological samples. Here, we tested the feasibility of three approaches-CUTseq, for high-throughput low-input SCNA profiling, multiplexed fluorescent immunohistochemistry (mfIHC) and digital-image analysis (DIA) for quantitative immuno-profiling- in archival formalin-fixed paraffin-embedded (FFPE) tissue samples from patients enrolled in the randomized SBG-2004-1 phase II trial. CUTseq was able to reproducibly identify amplification and deletion events with a resolution of 100 kb using only 6 ng of DNA extracted from FFPE tissue and pooling together 77 samples into the same sequencing library. In the same samples, mfIHC revealed that CD4 + T-cells and CD68 + macrophages were the most abundant immune cells and they mostly expressed PD-L1 and PD-1. Combined analysis showed that the SCNA burden was inversely associated with lymphocytic infiltration. Our results set the basis for further applications of CUTseq, mfIHC and DIA to larger cohorts of early breast cancer patients.

An Open-Source, Automated Tumor-Infiltrating Lymphocyte Algorithm for Prognosis in Triple-Negative Breast Cancer.

PURPOSE: Although tumor-infiltrating lymphocytes (TIL) assessment has been acknowledged to have both prognostic and predictive importance in triple-negative breast cancer (TNBC), it is subject to inter and intraobserver variability that has prevented widespread adoption. Here we constructed a machine-learning based breast cancer TIL scoring approach and validated its prognostic potential in multiple TNBC cohorts. EXPERIMENTAL DESIGN: Using the QuPath open-source software, we built a neural-network classifier for tumor cells, lymphocytes, fibroblasts, and "other" cells on hematoxylin-eosin (H&E)-stained sections. We analyzed the classifier-derived TIL measurements with five unique constructed TIL variables. A retrospective collection of 171 TNBC cases was used as the discovery set to identify the optimal association of machine-read TIL variables with patient outcome. For validation, we evaluated a retrospective collection of 749 TNBC patients comprised of four independent validation subsets. RESULTS: We found that all five machine TIL variables had significant prognostic association with outcomes (P ≤ 0.01 for all comparisons) but showed cell-specific variation in validation sets. Cox regression analysis demonstrated that all five TIL variables were independently associated with improved overall survival after adjusting for clinicopathologic factors including stage, age, and histologic grade (P ≤ 0.0003 for all analyses). CONCLUSIONS: Neural net-driven cell classifier-defined TIL variables were robust and independent prognostic factors in several independent validation cohorts of TNBC patients. These objective, open-source TIL variables are freely available to download and can now be considered for testing in a prospective setting to assess clinical utility.See related commentary by Symmans, p. 5446.

TET2 Protects Against Vascular Smooth Muscle Cell Apoptosis and Intimal Thickening in Transplant Vasculopathy.

BACKGROUND: Coronary allograft vasculopathy (CAV) is a devastating sequela of heart transplant in which arterial intimal thickening limits coronary blood flow. There are currently no targeted therapies to prevent or reduce this pathology that leads to transplant failure. Vascular smooth muscle cell (VSMC) phenotypic plasticity is critical in CAV neointima formation. TET2 (TET methylcytosine dioxygenase 2) is an important epigenetic regulator of VSMC phenotype, but the role of TET2 in the progression of CAV is unknown. METHODS: We assessed TET2 expression and activity in human CAV and renal transplant samples. We also used the sex-mismatched murine aortic graft model of graft arteriopathy (GA) in wild-type and inducible smooth muscle-specific Tet2 knockout mice; and in vitro studies in murine and human VSMCs using knockdown, overexpression, and transcriptomic approaches to assess the role of TET2 in VSMC responses to IFNγ (interferon γ), a cytokine elaborated by T cells that drives CAV progression. RESULTS: In the present study, we found that TET2 expression and activity are negatively regulated in human CAV and renal transplant samples and in the murine aortic graft model of GA. IFNγ was sufficient to repress TET2 and induce an activated VSMC phenotype in vitro. TET2 depletion mimicked the effects of IFNγ, and TET2 overexpression rescued IFNγ-induced dedifferentiation. VSMC-specific TET2 depletion in aortic grafts, and in the femoral wire restenosis model, resulted in increased VSMC apoptosis and medial thinning. In GA, this apoptosis was tightly correlated with proliferation. In vitro, TET2-deficient VSMCs undergo apoptosis more readily in response to IFNγ and expressed a signature of increased susceptibility to extrinsic apoptotic signaling. Enhancing TET2 enzymatic activity with high-dose ascorbic acid rescued the effect of GA-induced VSMC apoptosis and intimal thickening in a TET2-dependent manner. CONCLUSIONS: TET2 is repressed in CAV and GA, likely mediated by IFNγ. TET2 serves to protect VSMCs from apoptosis in the context of transplant vasculopathy or IFNγ stimulation. Promoting TET2 activity in vivo with systemic ascorbic acid reduces VSMC apoptosis and intimal thickening. These data suggest that promoting TET2 activity in CAV may be an effective strategy for limiting CAV progression.

A new tool for technical standardization of the Ki67 immunohistochemical assay.

Ki67, a nuclear proliferation-related protein, is heavily used in anatomic pathology but has not become a companion diagnostic or a standard-of-care biomarker due to analytic variability in both assay protocols and interpretation. The International Ki67 Working Group in breast cancer has published and has ongoing efforts in the standardization of the interpretation of Ki67, but they have not yet assessed technical issues of assay production representing multiple sources of variation, including antibody clones, antibody formats, staining platforms, and operators. The goal of this work is to address these issues with a new standardization tool. We have developed a cell line microarray system in which mixes of human Karpas 299 or Jurkat cells (Ki67+) with Sf9 (Spodoptera frugiperda) (Ki67-) cells are present in incremental standardized ratios. To validate the tool, six different antibodies, including both ready-to-use and concentrate formats from six vendors, were used to measure Ki67 proliferation indices using IHC protocols for manual (bench-top) and automated platforms. The assays were performed by three different laboratories at Yale and analyzed using two image analysis software packages, including QuPath and Visiopharm. Results showed statistically significant differences in Ki67 reactivity between each antibody clone. However, subsets of Ki67 assays using three clones performed in three different labs show no significant differences. This work shows the need for analytic standardization of the Ki67 assay and provides a new tool to do so. We show here how a cell line standardization system can be used to normalize the staining variability in proliferation indices between different antibody clones in a triple negative breast cancer cohort. We believe that this cell line standardization array has the potential to improve reproducibility among Ki67 assays and laboratories, which is critical for establishing Ki67 as a standard-of-care assay.

Neoadjuvant durvalumab plus weekly nab-paclitaxel and dose-dense doxorubicin/cyclophosphamide in triple-negative breast cancer.

The goal of this Phase I/II trial is to assess the safety and efficacy of administering durvalumab concurrent with weekly nab-paclitaxel and dose-dense doxorubicin/cyclophosphamide (ddAC) neoadjuvant therapy for stages I-III triple-negative breast cancer. The primary endpoint is pathologic complete response (pCR:ypT0/is, ypN0). The response was correlated with PDL1 expression and stromal tumor-infiltrating lymphocytes (sTILs). Two dose levels of durvalumab (3 and 10 mg/kg) were assessed. PD-L1 was assessed using the SP263 antibody; ≥1% immune and tumor cell staining was considered positive; sTILs were calculated as the area occupied by mononuclear inflammatory cells over the total intratumoral stromal area. 59 patients were evaluable for toxicity and 55 for efficacy in the Phase II study (10 mg/kg dose). No dose-limiting toxicities were observed in Phase I. In Phase II, pCR rate was 44% (95% CI: 30-57%); 18 patients (31%) experienced grade 3/4 treatment-related adverse events (AE), most frequently neutropenia (n = 4) and anemia (n = 4). Immune-related grade 3/4 AEs included Guillain-Barre syndrome (n = 1), colitis (n = 2), and hyperglycemia (n = 2). Of the 50 evaluable patients for PD-L1, 31 (62%) were PD-L1 positive. pCR rates were 55% (95% CI: 0.38-0.71) and 32% (95% CI: 0.12-0.56) in the PD-L1 positive and negative groups (p = 0.15), respectively. sTIL counts were available on 52 patients and were significantly higher in the pCR group (p = 0.0167). Concomitant administration of durvalumab with sequential weekly nab-paclitaxel and ddAC neoadjuvant chemotherapy resulted in a pCR rate of 44%; pCR rates were higher in sTIL-high cancers.

Ki67 reproducibility using digital image analysis: an inter-platform and inter-operator study.

Ki67 expression has been a valuable prognostic variable in breast cancer, but has not seen broad adoption due to lack of standardization between institutions. Automation could represent a solution. Here we investigate the reproducibility of Ki67 measurement between three image analysis platforms with supervised classifiers performed by the same operator, by multiple operators, and finally we compare their accuracy in prognostic potential. Two breast cancer patient cohorts were used for this study. The standardization was done with the 30 cases of ER+ breast cancer that were used in phase 3 of International Ki67 in Breast Cancer Working Group initiatives where blocks were centrally cut and stained for Ki67. The outcome cohort was from 149 breast cancer cases from the Yale Pathology archives. A tissue microarray was built from representative tissue blocks with median follow-up of 120 months. The Mib-1 antibody (Dako) was used to detect Ki67 (dilution 1:100). HALO (IndicaLab), QuantCenter (3DHistech), and QuPath (open source software) digital image analysis (DIA) platforms were used to evaluate Ki67 expression. Intraclass correlation coefficient (ICC) was used to measure reproducibility. Between-DIA platform reproducibility was excellent (ICC: 0.933, CI: 0.879-0.966). Excellent reproducibility was found between all DIA platforms and the reference standard Ki67 values of Spectrum Webscope (QuPath-Spectrum Webscope ICC: 0.970, CI: 0.936-0.986; HALO-Spectrum Webscope ICC: 0.968, CI: 0.933-0.985; QuantCenter-Spectrum Webscope ICC: 0.964, CI: 0.919-0.983). All platforms showed excellent intra-DIA reproducibility (QuPath ICC: 0.992, CI: 0.986-0.996; HALO ICC: 0.972, CI: 0.924-0.988; QuantCenter ICC: 0.978, CI: 0.932-0.991). Comparing each DIA against outcome, the hazard ratios were similar. The inter-operator reproducibility was particularly high (ICC: 0.962-0.995). Our results showed outstanding reproducibility both within and between-DIA platforms, including one freely available DIA platform (QuPath). We also found the platforms essentially indistinguishable with respect to prediction of breast cancer patient outcome. Results justify multi-institutional DIA studies to assess clinical utility.

An international multicenter study to evaluate reproducibility of automated scoring for assessment of Ki67 in breast cancer.

The nuclear proliferation biomarker Ki67 has potential prognostic, predictive, and monitoring roles in breast cancer. Unacceptable between-laboratory variability has limited its clinical value. The International Ki67 in Breast Cancer Working Group investigated whether Ki67 immunohistochemistry can be analytically validated and standardized across laboratories using automated machine-based scoring. Sets of pre-stained core-cut biopsy sections of 30 breast tumors were circulated to 14 laboratories for scanning and automated assessment of the average and maximum percentage of tumor cells positive for Ki67. Seven unique scanners and 10 software platforms were involved in this study. Pre-specified analyses included evaluation of reproducibility between all laboratories (primary) as well as among those using scanners from a single vendor (secondary). The primary reproducibility metric was intraclass correlation coefficient between laboratories, with success considered to be intraclass correlation coefficient >0.80. Intraclass correlation coefficient for automated average scores across 16 operators was 0.83 (95% credible interval: 0.73-0.91) and intraclass correlation coefficient for maximum scores across 10 operators was 0.63 (95% credible interval: 0.44-0.80). For the laboratories using scanners from a single vendor (8 score sets), intraclass correlation coefficient for average automated scores was 0.89 (95% credible interval: 0.81-0.96), which was similar to the intraclass correlation coefficient of 0.87 (95% credible interval: 0.81-0.93) achieved using these same slides in a prior visual-reading reproducibility study. Automated machine assessment of average Ki67 has the potential to achieve between-laboratory reproducibility similar to that for a rigorously standardized pathologist-based visual assessment of Ki67. The observed intraclass correlation coefficient was worse for maximum compared to average scoring methods, suggesting that maximum score methods may be suboptimal for consistent measurement of proliferation. Automated average scoring methods show promise for assessment of Ki67 scoring, but requires further standardization and subsequent clinical validation.

Elevated T cell activation score is associated with improved survival of breast cancer.

PURPOSE: Immune checkpoints cytotoxic T lymphocyte antigen 4 (CTLA4) and programmed cell death 1 receptor (PD-1) negatively regulate CD8+ T cell functions, impeding the capacity of effector T cells to kill tumors. Here, we study the prognostic significance of CTLA4, PD-1 and T cell activation status in breast cancer. METHODS: Using a publicly accessed RNA-seq dataset including 1087 breast cancer patients, we performed Kaplan-Meier survival curves and multivariate Cox regression models to evaluate the associations of CTLA4, PD-1, and weighted T cell activation score with patients' overall survival. RESULTS: Survival analyses showed that high CTLA4 but low PD-1 expression was associated with a poor overall survival, and that high T cell activation score was associated with an improved survival. The median survival was 216.6 months (95% CI 114.1-244.9) for the T activation group, 127.0 months (95% CI 112.3-212.1) for the intermediate, and 120.5 months (95% CI 93.8 to ∞) for the exhaustion (Log-rank p = 0.084). This association was verified in multivariate Cox regression analysis. The hazard ratios were 0.81 (95% CI 0.56-1.19) for the intermediate group, and 0.48 (95% CI 0.26-0.86) for the activation group, respectively, in comparison to the exhaustion group (p value for trend = 0.016). CONCLUSIONS: T cell activation score has significantly positive relationship with patients' overall survival, and may serve as a marker of personalized immunotherapy in breast cancer patients. Cocktail rather than single immune checkpoint blockade may yield more benefit for breast cancer patients.

Objective, domain-specific HER2 measurement in uterine and ovarian serous carcinomas and its clinical significance.

INTRODUCTION: HER2 overexpression/amplification is identified in up to 40% of uterine serous carcinomas (USC) and 10% of ovarian serous carcinomas (OSC). However, clinical trials using various HER2-targeted agents failed to show significant responses. FDA-approved HER2 assays target only the protein's intracellular domain (ICD) and not the extracellular domain (ECD). Previous quantitative studies in breast cancer by our group have shown that ICD of HER2 is expressed in some cases that do not express the HER2 ECD. We measured HER2 ICD and ECD in USC and OSC samples, and determined their relationship with clinico-pathologic characteristics and survival. METHODS: We measured HER2 ICD and ECD levels in 2 cohorts of USC and OSC comprising 102 and 175 patients, respectively. HER2 antibodies targeting ICD (CB11) and ECD (SP3) were validated and standardized using the AQUA® method of quantitative immunofluorescence (QIF) and a previously reported HER2 standardization tissue microarray (TMA). Objective, population-based cut-points were used to stratify patients according to HER2 ICD/ECD status. RESULTS: In USC, 8% of patients with high HER2 ICD had low ECD levels (6/75 patients). In OSC, 42% of patients with high HER2 ICD had low ECD levels (29/69 patients). HER2 ICD/ECD status in USC and OSC was not significantly associated with major clinico-pathological features or survival. CONCLUSION: Using objective, domain-specific HER2 measurement, 8% of USC and 42% of OSC patients with high HER2 ICD levels do not show uniform overexpression of the ECD. This may be related to the presence of p95 HER2, an oncogenic fragment generated by full protein cleavage or alternative initiation of translation. These observations raise the possibility that USC/OSCs expressing low ECD despite being HER2-positive by ICD measurement, may benefit from therapies directed against the intracellular domain (e.g. lapatinib or afatinib) alone or in combination with extracellular domain-directed drugs (e.g. trastuzumab, pertuzumab, T-DM1).