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Background: 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. Methods and Findings: 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 lowgrade 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 caseseries analyses. [ABSTRACT FROM AUTHOR]

Background: Global DNA hypomethylation may result in chromosomal instability and oncogene activation, and as a surrogate of systemic methylation activity, may be associated with breast cancer risk. Methods: Samples and data were obtained from women with incident early-stage breast cancer (I–IIIa) and women who were cancer free, frequency matched on age and race. In preliminary analyses, genomic methylation of leukocyte DNA was determined by measuring 5-methyldeoxycytosine (5-mdC), as well as methylation analysis of the LINE-1-repetitive DNA element. Further analyses used only 5-mdC levels. Logistic regression models were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for risk of breast cancer in relation to amounts of methylation. Results: In a subset of samples tested (n = 37), 5-mdC level was not correlated with LINE-1 methylation. 5-mdC level in leukocyte DNA was significantly lower in breast cancer cases than healthy controls (P = 0.001), but no significant case–control differences were observed with LINE-1 methylation (P = 0.176). In the entire data set, we noted significant differences in 5-mdC levels in leukocytes between cases (n = 176) and controls (n = 173); P value < 0.001. Compared with women in the highest 5-mdC tertile (T3), women in the second (T2; OR = 1.49, 95% CI = 0.84–2.65) and lowest tertile (T1; OR = 2.86, 95% CI = 1.65–4.94) had higher risk of breast cancer (P for trend ≤0.001). Among controls only and cases and controls combined, only alcohol intake was found to be inversely associated with methylation levels. Conclusion: These findings suggest that leukocyte DNA hypomethylation is independently associated with development of breast cancer. [ABSTRACT FROM PUBLISHER]

Background: The understanding of molecular characteristics of HER2-low breast cancer is evolving since the establishment of trastuzumab deruxtecan. Here, we explore the differences in expression patterns of immune-related genes in the tumor immune microenvironment (TME) and survival between HER2-low and HER2-zero breast cancers., Methods: Comprehensive genomic and immune profiling, including RNA-seq gene expression assessment of 395 immune genes, was performed on FFPE samples from 129 patients with advanced HER2-negative (immunohistochemistry (IHC) 0, 1+ or 2+ with negative ERBB2 amplification by in-situ hybridization) breast cancer. Both estrogen receptor (ER) and HER2 statuses were obtained from available pathology reports. mRNA expressions of immune biomarkers, except for PD-L1 IHC and TMB, were derived from RNA-seq. Statistical comparisons were performed using the Kruskal-Wallis or Wilcoxon Rank-Sum test or the two-sample test for equality of proportions with continuity correction (p≤0.05 for significance). Survival differences were calculated using Kaplan-Meier analysis (p≤0.05 for significance)., Results: There were no significant differences in mRNA expressions of immune-related genes between HER2-low and HER2-zero breast cancers. However, HER2-low breast cancers were associated with a higher proportion of ER-positivity. When ER was analyzed along with HER2, we observed a significantly higher tumor immunogenic signature (TIGS) expression in HER2-zero/ER-negative tumors than in HER2-low/ER-positive tumors (p=0.0088). Similarly, lower expression of PD-L1 and T cell immunoglobulin and ITIM domain (TIGIT) mRNA was observed in HER2-low/ER-positive tumors when compared to HER2-zero/ER-negative tumors (p=0.014 and 0.012, respectively). Patients with HER2-low tumors had a longer median OS than those with HER2-zero tumors (94 months vs 42 months, p=0.0044)., Conclusion: Patients with HER2-low breast cancer have longer survivals yet display no differences in immune-related gene expression when compared to those with HER2-zero cancers. The differences in survival can be attributed to the higher rate of ER-positivity seen in HER2-low breast cancers, compared to HER2-zero tumors., Competing Interests: HCK, RJS, SP, MFS, EVR, SG, KCS, RAP, MFG, MC, MKN, SBH, KAA, SZ, JMC, TJJ, ME, BC, EAS, and SR are employees of Labcorp Oncology, a business unit of Labcorp. SG is an employee at Roswell Park Comprehensive Cancer Center. ME and BJC are employees of Labcorp. RJS, EVR, SG, SP, and JMC are listed as authors on a pending patent US2022/049867 entitled Methods and Systems for Analyzing and Utilizing Cancer Testis Antigen Burden. JC and SP report a pending patent US20220136070A1 Methods and Systems for Characterizing Tumor Response to Immunotherapy Using an Immunogenic Profile pending to OmniSeq, Inc. SP reports patents US11427873B2 Methods and systems for assessing proliferative potential and resistance to immune checkpoint blockade and US11515008B2 Methods and systems of prioritizing treatments, vaccination, testing and/or activities while protecting the privacy of individuals issued to Omniseq. TJJ, EAS, MKN, KCS are shareholder of Labcorp. TJJ is a shareholder in Fortrea. The authors report no other conflicts of interest in this work., (© 2024 Ko et al.)

TIM-3, an inhibitory checkpoint receptor, may invoke anti-PD-1/anti-PD-L1 immune checkpoint inhibitor (ICI) resistance. The predictive impact of TIM-3 RNA expression in various advanced solid tumors among patients treated with ICIs is yet to be determined, and their prognostic significance also remains unexplored. We investigated TIM-3 transcriptomic expression and clinical outcomes. We examined TIM-3 RNA expression data through the OmniSeq database. TIM-3 transcriptomic patterns were calibrated against a reference population (735 tumors), adjusted to internal housekeeping genes, and calculated as percentiles. Overall, 514 patients (31 cancer types; 489 patients with advanced/metastatic disease and clinical annotation) were assessed. Ninety tumors (17.5% of 514) had high (≥75 th percentile RNA rank) TIM-3 expression. Pancreatic cancer had the greatest proportion of TIM-3 high expressors (36% of 55 patients). Still, there was variability within cancer types with, for instance, 12.7% of pancreatic cancers harboring low TIM-3 (<25 th percentile) levels. High TIM-3 expression independently and significantly correlated with high PD-L2 RNA expression (odds ratio (OR) 9.63, 95% confidence interval (CI) 4.91-19.4, P<0.001) and high VISTA RNA expression (OR 2.71, 95% CI 1.43-5.13, P=0.002), all in multivariate analysis. High TIM-3 RNA did not correlate with overall survival (OS) from time of metastatic disease in the 272 patients who never received ICIs, suggesting that it is not a prognostic factor. However, high TIM-3 expression predicted longer median OS (but not progression-free survival) in 217 ICI-treated patients (P=0.0033; median OS, 2.84 versus 1.21 years (high versus not-high TIM-3)), albeit not retained in multivariable analysis. In summary, TIM-3 RNA expression was variable between and within malignancies, and high levels associated with high PD-L2 and VISTA checkpoints and with pancreatic cancer. Individual tumor immunomic assessment and co-targeting co-expressed checkpoints merits exploration in prospective trials as part of a precision immunotherapy strategy., Competing Interests: Dr. Kurzrock has received research funding from Boehringer Ingelheim, Debiopharm, Foundation Medicine, Genentech, Grifols, Guardant, Incyte, Konica Minolta, Medimmune, Merck Serono, Omniseq, Pfizer, Sequenom, Takeda, and TopAlliance and from the NCI; as well as consultant and/or speaker fees and/or advisory board for Actuate Therapeutics, AstraZeneca, Bicara Therapeutics, Inc., Biological Dynamics, Caris, Datar Cancer Genetics, Daiichi, EISAI, EOM Pharmaceuticals, Iylon, Merck, NeoGenomics, Neomed, Pfizer, Precirix, Prosperdtx, Regeneron, Roche, TD2/Volastra, Turning Point Therapeutics, X-Biotech; has an equity interest in CureMatch Inc. and IDbyDNA; serves on the Board of CureMatch and CureMetrix, and is a co-founder of CureMatch. Jason K Sicklick receives consultant fees from Deciphera, Aadi and Grand Rounds; serves as a consultant for CureMatch, received speakers’ fees from Deciphera, La-Hoffman Roche, Foundation Medicine, Merck, QED and Daiichi Sankyo; and owns stock in Personalis and CureMatch. Shumei Kato serves as a consultant for Medpace, Foundation Medicine, NeoGenomics and CureMatch. He receives speaker’s fee from Chugai, Roche/Genentech and Bayer, and advisory board for Pfizer. He has research funding from ACT Genomics, Sysmex, Konica Minolta, OmniSeq, Personalis and Function Oncology. Mary K Nesline, Sarabjot Pabla, Jeffrey M Conroy and Paul DePietro all have full-time employment at Labcorp., (AJCR Copyright © 2024.)

Transcriptomic expression profiles of immune checkpoint markers are of interest in order to decipher the mechanisms of immunotherapy response and resistance. Overall, 514 patients with various solid tumors were retrospectively analyzed in this study. The RNA expression levels of tumor checkpoint markers (ADORA2A, BTLA, CD276, CTLA4, IDO1, IDO2, LAG3, NOS2, PD-1, PD-L1, PD-L2, PVR, TIGIT, TIM3, VISTA, and VTCN) were ranked from 0-100 percentile based on a reference population. The expression of each checkpoint was correlated with cancer type, microsatellite instability (MSI), tumor mutational burden (TMB), and programmed death-ligand 1 (PD-L1) by immunohistochemistry (IHC). The cohort included 30 different tumor types, with colorectal cancer being the most common (27%). When RNA percentile rank values were categorized as "Low" (0-24), "Intermediate" (25-74), and "High" (75-100), each patient had a distinctive portfolio of the categorical expression of 16 checkpoint markers. Association between some checkpoint markers and cancer types were observed; NOS2 showed significantly higher expression in colorectal and stomach cancer (P < 0.001). Principal component analysis demonstrated no clear association between combined RNA expression patterns of 16 checkpoint markers and cancer types, TMB, MSI or PD-L1 IHC. Immune checkpoint RNA expression varies from patient to patient, both within and between tumor types, though colorectal and stomach cancer showed the highest levels of NOS2, a mediator of inflammation and immunosuppression. There were no specific combined expression patterns correlated with MSI, TMB or PD-L1 IHC. Next generation immunotherapy trials may benefit from individual analysis of patient tumors as selection criteria for specific immunomodulatory approaches., Competing Interests: Nicholas J Bevins serves as clinical laboratory director for Bertis Biosciences Inc., Birdrock Laboratory, and COVx laboratory. Nicholas J Bevins serves as Chief Medical Officer of Sequence Sciences. Sarabjot Pabla is/was an employee of OmniSeq and declares the following relationships: stock ownership from Labcorp. Mary K Nesline is/was an employee of OmniSeq and declares the following relationships: stock ownership from Labcorp. Sean T Glenn is/was an employee of OmniSeq and declares the following relationships: None. Jeffrey M Conroy is/was an employee of OmniSeq and declares the following relationships: stock ownership from Labcorp. Paul DePietro is/was an employee of OmniSeq and declares the following relationships: stock ownership from Labcorp. Jason K Sicklick receives consultant fees from Deciphera, Aadi and Grand Rounds; serves as a consultant for CureMatch, received speakers fees from Deciphera, La-Hoffman Roche, Foundation Medicine, Merck, QED and Daiichi Sankyo; and owns stock in Personalis. Shumei Kato serves as a consultant for Medpace, Foundation Medicine, NeoGenomics and CureMatch. He receives speaker’s fee from Roche/Genentech and Bayer, and advisory board for Pfizer. He has research funding from ACT Genomics, Sysmex, Konica Minolta, OmniSeq, Personalis and Function Oncology. Razelle Kurzrock has received research funding from Biological Dynamics, Boehringer Ingelheim, Debiopharm, Foundation Medicine, Genentech, Grifols, Guardant, Incyte, Konica Minolta, Medimmune, Merck Serono, Omniseq, Pfizer, Sequenom, Takeda, and TopAlliance; as well as consultant and/or speaker fees and/or advisory board for Actuate Therapeutics, AstraZeneca, Bicara Therapeutics, Biological Dynamics, Caris, Daiichi Sankyo, Inc., Datar Cancer Genetics, EISAI, EOM Pharmaceuticals, Iylon, Merck, NeoGenomics, Neomed, Pfizer, Prosperdtx, Roche, TD2/Volastra, Turning Point Therapeutics, X-Biotech; has an equity interest in CureMatch Inc., CureMetrix, and IDbyDNA; serves on the Board of CureMatch and CureMetrix, and is a co-founder of CureMatch., (AJCR Copyright © 2024.)

Glucocorticoid-induced tumor necrosis factor related protein (GITR) is a transmembrane protein expressed mostly on CD25 + CD4 + regulatory T-cells (Tregs) and upregulated on all T-cells upon activation. It is a T-cell co-stimulatory receptor and has demonstrated promising anti-tumor activity in pre-clinical studies. To date, however, the efficacy of GITR agonism has been discouraging in clinical trials. This study explores GITR and GITR ligand (GITR-L) ribonucleic acid (RNA) expression in solid tumors in an attempt to delineate causes for variable responses to GITR agonists. RNA expression levels of 514 patients with a variety of cancer types were normalized to internal housekeeping gene profiles and ranked as percentiles. 99/514 patients (19.3%) had high GITR expression (defined as ≥ 75th percentile). Breast and lung cancer had the highest proportion of patients with high GITR expression (39% and 35%, respectively). The expression of concomitant high GITR and low-moderate GITR-L expression (defined as <75th percentile) was present in 31% and 30% of patients with breast and lung cancer respectively. High GITR expression also showed a significant independent association with high RNA expression of other immune modulator proteins, namely, PD-L1 immunohistochemistry (IHC) ≥1 (odds ratio (OR) 2.15, P=0.008), CTLA4 (OR=2.17, P=0.05) and OX40 high RNA expression (OR=2.64, P=0.001). Overall, these results suggest that breast and lung cancer have a high proportion of patients with a GITR and GITR-L RNA expression profile that merits further investigation in GITR agonism studies. The association of high GITR expression with high CTLA4 and OX40 RNA expression, as well as positive PD-L1 IHC, provides a rationale for a combination approach targeting these specific immune modulator proteins in patients whose tumors show such co-expression., Competing Interests: Razelle Kurzrock has received research funding from Boehringer Ingelheim, Debiopharm, Foundation Medicine, Genentech, Grifols, Guardant, Incyte, Konica Minolta, Medimmune, Merck Serono, Omniseq, Pfizer, Sequenom, Takeda, and TopAlliance and from the NCI; as well as consultant and/or speaker fees and/or advisory board/consultant for Actuate Therapeutics, AstraZeneca, Bicara Therapeutics, Inc., Biological Dynamics, Caris, Datar Cancer Genetics, Daiichi, EISAI, EOM Pharmaceuticals, Iylon, LabCorp, Merck, NeoGenomics, Neomed, Pfizer, Prosperdtx, Regeneron, Roche, TD2/Volastra, Turning Point Therapeutics, X-Biotech; has an equity interest in CureMatch Inc. and IDbyDNA; serves on the Board of CureMatch and CureMetrix, and is a co-founder of CureMatch. Jason K Sicklick receives consultant fees from Deciphera, Aadi and Grand Rounds; serves as a consultant for CureMatch, received speakers fees from Deciphera, La-Hoffman Roche, Foundation Medicine, Merck, QED and Daiichi Sankyo; and owns stock in Personalis and CureMatch. Shumei Kato serves as a consultant for Medpace, Foundation Medicine, NeoGenomics and CureMatch. He receives speaker’s fee from Chugai, Roche/Genentech and Bayer, and advisory board for Pfizer. He has research funding from ACT Genomics, Sysmex, Konica Minolta, OmniSeq, Personalis and Function Oncology. Sarabjot Pabla, Jeffrey M Conroy, and Paul DePietro are employed by Omniseq. Mary K Nesline, and Heidi Ko are employed by Labcorp., (AJCR Copyright © 2024.)

Immune checkpoint inhibitors have revolutionized the treatment landscape for patients with cancer. Multi-omics, including next-generation DNA and RNA sequencing, have enabled the identification of exploitable targets and the evaluation of immune mediator expression. There is one FDA-approved LAG-3 inhibitor and multiple in clinical trials for numerous cancers. We analyzed LAG-3 transcriptomic expression among 514 patients with diverse cancers, including 489 patients with clinical annotation for their advanced malignancies. Transcriptomic LAG-3 expression was highly variable between histologies/cancer types and within the same histology/cancer type. LAG-3 RNA levels correlated linearly, albeit weakly, with high RNA levels of other checkpoints, including PD-L1 (Pearson's R 2 = 0.21 ( P < 0.001)), PD-1 (R 2 = 0.24 ( P < 0.001)) and CTLA-4 (R 2 = 0.19 ( P < 0.001)); when examined for Spearman correlation, significance did not change. LAG-3 expression (dichotomized at ≥ 75 th (high) versus < 75 th (moderate/low) RNA percentile level) was not a prognostic factor for overall survival (OS) in 272 immunotherapy-naïve patients with advanced/metastatic disease (Kaplan Meier analysis; P = 0.54). High LAG-3 levels correlated with longer OS after anti-PD-1/PD-L1-based checkpoint blockade (univariate (P = 0.003), but not multivariate analysis (hazard ratio, 95% confidence interval = 0.80 (0.46-1.40) (P = 0.44))); correlation with longer progression-free survival showed a weak univariate trend (P = 0.13). Taken together, these results suggest that high LAG-3 levels in and of themselves do not predict resistance to anti-PD-1/PD-L1 checkpoint blockade. Even so, since LAG-3 is often co-expressed with PD-1, PD-L1 and/or CTLA-4, selecting patients for combinations of checkpoint blockade based on immunomic co-expression patterns is a strategy that merits exploration., Competing Interests: Jacob J Adashek serves on the advisory board of CureMatch Inc. and serves as a consultant for datma. Shumei Kato serves as a consultant for Medpace, Foundation Medicine, NeoGenomics and CureMatch. He receives speaker’s fee from Roche and Bayer, and advisory board for Pfizer. He has research funding from ACT Genomics, Sysmex, Konica Minolta, OmniSeq and Personalis. Sarabjot Pabla, Mary K Nesline, Jeffrey M Conroy, Paul DePietro are/were employees of Labcorp Oncology. Rebecca A Previs is/was an employee of Labcorp and declares the following relationships: stock ownership from Labcorp. She has served on advisory boards for Myraid Genetics and Natera. Razelle Kurzrock has received research funding from Biological Dynamics, Boehringer Ingelheim, Debiopharm, Foundation Medicine, Genentech, Grifols, Guardant, Incyte, Konica Minolta, Medimmune, Merck Serono, Omniseq, Pfizer, Sequenom, Takeda, and TopAlliance; as well as consultant and/or speaker fees and/or advisory board for Actuate Therapeutics, AstraZeneca, Bicara Therapeutics, Biological Dynamics, Caris, Daiichi Sankyo, Inc., Datar Cancer Genetics, EISAI, EOM Pharmaceuticals, Iylon, LabCorp, Merck, NeoGenomics, Neomed, Pfizer, Precirix, Prosperdtx, Roche, TD2/Volastra, Turning Point Therapeutics, X-Biotech; has an equity interest in CureMatch Inc., CureMetrix, and IDbyDNA; serves on the Board of CureMatch and CureMetrix, and is a co-founder of CureMatch., (AJCR Copyright © 2024.)

CSF1R expression modulates tumor-associated macrophages, making CSF1R blockade an appealing immune-modulating therapeutic target. We evaluated the correlation between CSF1R tumor RNA expression and outcome (pan-cancer setting). RNA expression was ranked as a percentile (0-100) using a standardized internal reference population (735 tumors; 35 histologies). Among 514 patients, there was no difference in survival from biopsy between high and low CSF1R expressors (< 50 percentile versus ≥ 50 percentile rank). There was also no significant difference in median progression-free or overall survival (from treatment) based on CSF1R expression in 21 patients who received CSF1R inhibitors (all p values ≥ 0.08). Concurrent upregulation of ≥ 2 additional immune checkpoint markers (e.g. PD-L1, BTLA, CTLA4, LAG3, TIM3) was observed in all tumor samples with CSF1R expression ≥ 50th percentile. Pending further large prospective studies, patients with high tumor CSF1R expression may need treatment that co-targets the specific immune checkpoint pathways activated in order to impact outcome., Competing Interests: Ann Moeller, Suzanna Lee, and Hitendra Patel declare that they have no competing interests. Sarabjot Pabla, Mary K. Nesline, Jeffrey Conroy are employees of Omniseq, Inc. Jason K. Sicklick receives research funding from Amgen, Foundation Medicine is a consultant for Deciphera, Ethicon and speaker for Bayer, Deciphera, Foundation Medicine, La Hoffman-Roche, Merck, QED. Gregory P. Botta serves on the Advisory Board of Natera and as a consultant to TumorGen Inc. and CEND Therapeutics.Shumei Kato serves as a consultant for Foundation Medicine, NeoGenomics and CureMatch. He receives speaker’s fee from Roche and advisory board for Pfizer. He has research funding from ACT Genomics, Sysmex, Konica Minolta and OmniSeq.Jacob J. Adashek serves on the advisory board of CureMatch, Inc. Razelle Kurzrock has received research funding from Biological Dynamics, Boehringer Ingelheim, Debiopharm, Foundation Medicine, Genentech, Grifols, Guardant, Incyte, Konica Minolta, Medimmune, Merck Serono, Omniseq, Pfizer, Sequenom, Takeda, and TopAlliance; as well as consultant and/or speaker fees and/or advisory board for Actuate Therapeutics, AstraZeneca, Bicara Therapeutics, Biological Dynamics, Daiichi Sankyo, Inc., EISAI, EOM Pharmaceuticals, Iylon, Merck, NeoGenomics, Neomed, Pfizer, Prosperdtx, Roche, TD2/Volastra, Turning Point Therapeutics, X-Biotech; has an equity interest in CureMatch Inc., CureMetrix, and IDbyDNA; serves on the Board of CureMatch and CureMetrix, and is a co-founder of CureMatch., (AJCR Copyright © 2023.)