Pretreatment Serum Concentrations of 25-Hydroxyvitamin D and Breast Cancer Prognostic Characteristics: A Case-Control and a Case-Series Study

Results from epidemiologic studies on the relationship between vitamin D and breast cancer risk are inconclusive. It is possible that vitamin D may be effective in reducing risk only of specific subtypes due to disease heterogeneity.In case-control and case-series analyses, we examined serum concentrations of 25-hydroxyvitamin D (25OHD) in relation to breast cancer prognostic characteristics, including histologic grade, estrogen receptor (ER), and molecular subtypes defined by ER, progesterone receptor (PR) and HER2, among 579 women with incident breast cancer and 574 controls matched on age and time of blood draw enrolled in the Roswell Park Cancer Institute from 2003 to 2008. We found that breast cancer cases had significantly lower 25OHD concentrations than controls (adjusted mean, 22.8 versus 26.2 ng/mL, p<0.001). Among premenopausal women, 25OHD concentrations were lower in those with high- versus low-grade tumors, and ER negative versus ER positive tumors (p≤0.03). Levels were lowest among women with triple-negative cancer (17.5 ng/mL), significantly different from those with luminal A cancer (24.5 ng/mL, p = 0.002). In case-control analyses, premenopausal women with 25OHD concentrations above the median had significantly lower odds of having triple-negative cancer (OR = 0.21, 95% CI = 0.08-0.53) than those with levels below the median; and every 10 ng/mL increase in serum 25OHD concentrations was associated with a 64% lower odds of having triple-negative cancer (OR = 0.36, 95% CI = 0.22-0.56). The differential associations by tumor subtypes among premenopausal women were confirmed in case-series analyses.In our analyses, higher serum levels of 25OHD were associated with reduced risk of breast cancer, with associations strongest for high grade, ER negative or triple negative cancers in premenopausal women. With further confirmation in large prospective studies, these findings could warrant vitamin D supplementation for reducing breast cancer risk, particularly those with poor prognostic characteristics among premenopausal women

Assessment of variation in immunosuppressive pathway genes reveals TGFBR2 to be associated with risk of clear cell ovarian cancer.

BACKGROUND: Regulatory T (Treg) cells, a subset of CD4+ T lymphocytes, are mediators of immunosuppression in cancer, and, thus, variants in genes encoding Treg cell immune molecules could be associated with ovarian cancer. METHODS: In a population of 15,596 epithelial ovarian cancer (EOC) cases and 23,236 controls, we measured genetic associations of 1,351 SNPs in Treg cell pathway genes with odds of ovarian cancer and tested pathway and gene-level associations, overall and by histotype, for the 25 genes, using the admixture likelihood (AML) method. The most significant single SNP associations were tested for correlation with expression levels in 44 ovarian cancer patients. RESULTS: The most significant global associations for all genes in the pathway were seen in endometrioid ( p = 0.082) and clear cell ( p = 0.083), with the most significant gene level association seen with TGFBR2 ( p = 0.001) and clear cell EOC. Gene associations with histotypes at p < 0.05 included: IL12 ( p = 0.005 and p = 0.008, serous and high-grade serous, respectively), IL8RA ( p = 0.035, endometrioid and mucinous), LGALS1 ( p = 0.03, mucinous), STAT5B ( p = 0.022, clear cell), TGFBR1 ( p = 0.021 endometrioid) and TGFBR2 ( p = 0.017 and p = 0.025, endometrioid and mucinous, respectively). CONCLUSIONS: Common inherited gene variation in Treg cell pathways shows some evidence of germline genetic contribution to odds of EOC that varies by histologic subtype and may be associated with mRNA expression of immune-complex receptor in EOC patients

Predictors of Immunosuppressive Regulatory T Lymphocytes in Healthy Women

Immunosuppressive regulatory T (Treg) cells play an important role in antitumor immunity, self-tolerance, transplantation tolerance, and attenuation of allergic response. Higher proportion of Treg cells has been observed in peripheral blood of cancer cases compared to controls. Little is known about potential epidemiological predictors of Treg cell levels in healthy individuals. We conducted a cross-sectional study including 75 healthy women, between 20 and 80 years of age, who participated in the Data Bank and BioRepository (DBBR) program at Roswell Park Cancer Institute (RPCI), Buffalo, NY, USA. Peripheral blood levels of CD4+CD25+FOXP3+ Treg cells were measured using flow cytometric analysis. A range of risk factors was evaluated using Wilcoxon Rank-Sum test, Kruskal-Wallis test, and linear regression. Age, smoking, medications for treatment of osteoporosis, postmenopausal status, body mass index (BMI), and hormone replacement therapy (HRT) were found to be significant positive predictors of Treg cell levels in peripheral blood (P≤0.05). Higher education, exercise, age at first birth, oral contraceptives, and use of Ibuprofen were found be significant (P<0.05) negative predictors of Treg levels. Thus, various epidemiological risk factors might explain interindividual variation in immune response to pathological conditions, including cancer

Next generation sequencing of PD-L1 for predicting response to immune checkpoint inhibitors

Background PD-L1 immunohistochemistry (IHC) has been traditionally used for predicting clinical responses to immune checkpoint inhibitors (ICIs). However, there are at least 4 different assays and antibodies used for PD-L1 IHC, each developed with a different ICI. We set to test if next generation RNA sequencing (RNA-seq) is a robust method to determine PD-L1 mRNA expression levels and furthermore, efficacy of predicting response to ICIs as compared to routinely used, standardized IHC procedures. Methods A total of 209 cancer patients treated on-label by FDA-approved ICIs, with evaluable responses were assessed for PD-L1 expression by RNA-seq and IHC, based on tumor proportion score (TPS) and immune cell staining (ICS). A subset of serially diluted cases was evaluated for RNA-seq assay performance across a broad range of PD-L1 expression levels. Results Assessment of PD-L1 mRNA levels by RNA-seq demonstrated robust linearity across high and low expression ranges. PD-L1 mRNA levels assessed by RNA-seq and IHC (TPS and ICS) were highly correlated (p < 2e-16). Sub-analyses showed sustained correlation when IHC results were classified as high or low by clinically accepted cut-offs (p < 0.01), and results did not differ by tumor type or anti-PD-L1 antibody used. Overall, a combined positive PD-L1 result (≥1% IHC TPS and high PD-L1 expression by RNA-Seq) was associated with a 2-to-5-fold higher overall response rate (ORR) compared to a double negative result. Standard assessments of sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) showed that a PD-L1 positive assessment for melanoma samples by RNA-seq had the lowest sensitivity (25%) but the highest PPV (72.7%). Among the three tumor types analyzed in this study, the only non-overlapping confidence interval for predicting response was for “RNA-seq low vs high” in melanoma. Conclusions Measurement of PD-L1 mRNA expression by RNA-seq is comparable to PD-L1 expression by IHC both analytically and clinically in predicting ICI response. RNA-seq has the added advantages of being amenable to standardization and avoidance of interpretation bias. PD-L1 by RNA-seq needs to be validated in future prospective ICI clinical studies across multiple histologies

Predicting response to checkpoint inhibitors in melanoma beyond PD-L1 and mutational burden

Background: Immune checkpoint inhibitors (ICIs) have changed the clinical management of melanoma. However, not all patients respond, and current biomarkers including PD-L1 and mutational burden show incomplete predictive performance. The clinical validity and utility of complex biomarkers have not been studied in melanoma. Methods: Cutaneous metastatic melanoma patients at eight institutions were evaluated for PD-L1 expression, CD8+ T-cell infiltration pattern, mutational burden, and 394 immune transcript expression. PD-L1 IHC and mutational burden were assessed for association with overall survival (OS) in 94 patients treated prior to ICI approval by the FDA (historical-controls), and in 137 patients treated with ICIs. Unsupervised analysis revealed distinct immune clusters with separate response rates. This comprehensive immune profiling data were then integrated to generate a continuous Response Score (RS) based upon response criteria (RECIST v.1.1). RS was developed using a single institution training cohort (n = 48) and subsequently tested in a separate eight institution validation cohort (n = 29) to mimic a real-world clinical scenario. Results: PD-L1 positivity ≥1% correlated with response and OS in ICI-treated patients, but demonstrated limited predictive performance. High mutational burden was associated with response in ICI-treated patients, but not with OS. Comprehensive immune profiling using RS demonstrated higher sensitivity (72.2%) compared to PD-L1 IHC (34.25%) and tumor mutational burden (32.5%), but with similar specificity. Conclusions: In this study, the response score derived from comprehensive immune profiling in a limited melanoma cohort showed improved predictive performance as compared to PD-L1 IHC and tumor mutational burden

High indoleamine 2,3-dioxygenase transcript levels predict better outcome after front-line cancer immunotherapy

Summary: Indoleamine 2,3-dioxygenase 1 (IDO1), which catabolizes tryptophan, is a potential target to unlock the immunosuppressive tumor microenvironment. Correlations between IDO1 and immune checkpoint inhibitor (ICI) efficacy remain unclear. Herein, we investigated IDO1 transcript expression across cancers and clinical outcome correlations. High IDO1 transcripts were more frequent in uterine (54.2%) and ovarian cancer (37.2%) but varied between and within malignancies. High IDO1 RNA expression was associated with high expression of PD-L1 (immune checkpoint ligand), CXCL10 (an effector T cell recruitment chemokine), and STAT1 (a component of the JAK-STAT pathway) (all multivariable p < 0.05). PIK3CA and CTCF alterations were more frequent in the high IDO1 group. High IDO1 expression was an independent predictor of progression-free survival (adjusted HR = 0.44, 95% CI 0.20–0.99, p = 0.049) and overall survival (adjusted HR = 0.31, 95% CI 0.11–0.87, p = 0.026) after front-line ICIs. IDO1 expression warrants further exploration as a predictive biomarker for immunotherapy. Moreover, co-expressed immunoregulatory molecules merit exploration for co-targeting

Evaluation of tumor microenvironment and biomarkers of immune checkpoint inhibitor response in metastatic renal cell carcinoma

BackgroundImmunotherapy combinations including ipilimumab and nivolumab are now the standard of care for untreated metastatic renal cell carcinoma (mRCC). Biomarkers of response are lacking to predict patients who will have a favorable or unfavorable response to immunotherapy. This study aimed to use the OmniSeq transcriptome-based platform to develop biomarkers of response to immunotherapy.MethodsTwo cohorts of patients were retrospectively collected. These included an investigational cohort of patients with mRCC treated with immune checkpoint inhibitor therapy from five institutions, and a subsequent validation cohort of patients with mRCC treated with combination ipilimumab and nivolumab from two institutions (Duke Cancer Institute and Cleveland Clinic Taussig Cancer Center). Tissue-based RNA sequencing was performed using the OmniSeq Immune Report Card on banked specimens to identify gene signatures and immune checkpoints associated with differential clinical outcomes. A 5-gene expression panel was developed based on the investigational cohort and was subsequently evaluated in the validation cohort. Clinical outcomes including progression-free survival (PFS) and overall survival (OS) were extracted by retrospective chart review. Objective response rate (ORR) was assessed by Response Evaluation Criteria in Solid Tumors (RECIST) V.1.1.ResultsThe initial investigation cohort identified 86 patients with mRCC who received nivolumab (80%, 69/86), ipilimumab/nivolumab (14%, 12/86), or pembrolizumab (6%, 5/86). A gene expression score was created using the top five genes found in responders versus non-responders (FOXP3, CCR4, KLRK1, ITK, TIGIT). The ORR in patients with high gene expression (GEhigh) on the 5-gene panel was 29% (14/48), compared with low gene expression (GElow) 3% (1/38, χ2 p=0.001). The validation cohort was comprised of 62 patients who received ipilimumab/nivolumab. There was no difference between GEhigh and GElow in terms of ORR (44% vs 38.5%), PFS (HR 1.5, 95% CI 0.58 to 3.89), or OS (HR 0.96, 95% CI 0.51 to 1.83). Similarly, no differences in ORR, PFS or OS were observed when patients were stratified by tumor mutational burden (high=top 20%), PD-L1 (programmed death-ligand 1) expression by immunohistochemistry or RNA expression, or CTLA-4 (cytotoxic T-lymphocytes-associated protein 4) RNA expression. The International Metastatic RCC Database Consortium (IMDC) risk score was prognostic for OS but not PFS.ConclusionA 5-gene panel that was associated with improved ORR in a predominantly nivolumab monotherapy population of patients with mRCC was not predictive for radiographic response, PFS, or OS among patients with mRCC treated with ipilimumab and nivolumab

Cancer testis antigen burden (CTAB): a novel biomarker of tumor-associated antigens in lung cancer

Abstract Background Cancer-testis antigens (CTAs) are tumor antigens that are normally expressed in the testes but are aberrantly expressed in several cancers. CTA overexpression drives the metastasis and progression of lung cancer, and is associated with poor prognosis. To improve lung cancer diagnosis, prognostic prediction, and drug discovery, robust CTA identification and quantitation is needed. In this study, we examined and quantified the co-expression of CTAs in lung cancer to derive cancer testis antigen burden (CTAB), a novel biomarker of immunotherapy response. Methods Formalin fixed paraffin embedded (FFPE) tumor samples in discovery cohort (n = 5250) and immunotherapy and combination therapy treated non-small cell lung cancer (NSCLC) retrospective (n = 250) cohorts were tested by comprehensive genomic and immune profiling (CGIP), including tumor mutational burden (TMB) and the mRNA expression of 17 CTAs. PD-L1 expression was evaluated by IHC. CTA expression was summed to derive the CTAB score. The median CTAB score for the discovery cohort of 170 was applied to the retrospective cohort as cutoff for CTAB “high” and “low”. Biomarker and gene expression correlation was measured by Spearman correlation. Kaplan–Meier survival analyses were used to detect overall survival (OS) differences, and objective response rate (ORR) based on RECIST criteria was compared using Fisher’s exact test. Results The CTAs were highly co-expressed (p < 0.05) in the discovery cohort. There was no correlation between CTAB and PD-L1 expression (R = 0.011, p = 0.45) but some correlation with TMB (R = 0.11, p = 9.2 × 10–14). Kaplan–Meier survival analysis of the immunotherapy-treated NSCLC cohort revealed better OS for the pembrolizumab monotherapy treated patients with high CTAB (p = 0.027). The combination group demonstrated improved OS compared to pembrolizumab monotherapy group (p = 0.04). The pembrolizumab monotherapy patients with high CTAB had a greater ORR than the combination therapy group (p = 0.02). Conclusions CTA co-expression can be reliably measured using CGIP in solid tumors. As a biomarker, CTAB appears to be independent from PD-L1 expression, suggesting that CTAB represents aspects of tumor immunogenicity not measured by current standard of care testing. Improved OS and ORR for high CTAB NSCLC patients treated with pembrolizumab monotherapy suggests a unique underlying aspect of immune response to these tumor antigens that needs further investigation