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Introduction: Matching patients to an effective targeted therapy or immunotherapy is a challenge for advanced and metastatic non-small cell lung cancer (NSCLC), especially when relying on assays that test one marker at a time. Unlike traditional single marker tests, comprehensive genomic profiling (CGP) can simultaneously assess NSCLC tumors for hundreds of genomic biomarkers and markers for immunotherapy response, leading to quicker and more precise matches to therapeutics. Methods: In this study, we performed CGP on 7,606 patients with advanced or metastatic NSCLC using the Illumina TruSight Oncology 500 (TSO 500) CGP assay to show its coverage and utility in detecting known and novel features of NSCLC. Results: Testing revealed distinct genomic profiles of lung adenocarcinoma and squamous cell carcinomas and detected variants with a current targeted therapy or clinical trial in >72% of patient tumors. Known associations between genomic alterations and immunotherapy markers were observed including significantly lower TMB levels in tumors with therapy-associated alterations and significantly higher PD-L1 levels in tumors with ALK , MET , BRAF , or ROS1 driver mutations. Co-occurrence analysis followed by network analysis with gene module detection revealed known and novel co-occurrences between genomic alterations. Further, certain modules of genes with co-occurring genomic alterations had dose-dependent relationships with histology and increasing or decreasing levels of PD-L1 and TMB, suggesting a complex relationship between PD-L1, TMB, and genomic alterations in these gene modules. Discussion: This study is the largest clinical study to date utilizing the TSO 500. It provides an opportunity to further characterize the landscape of NSCLC using this newer technology and show its clinical utility in detecting known and novel facets of NSCLC to inform treatment decision-making. [ABSTRACT FROM AUTHOR]

Clinical management of non-small cell lung cancer (NSCLC) requires accurate identification of tumor-specific genetic alterations to inform treatment options. Historically, providers have relied on single-gene testing (SGT) for actionable variants due to a perception of cost-effectiveness and/or efficient turnaround time compared to next-generation sequencing (NGS). However, not all actionable variants may be evaluated through SGT modalities, and an SGT approach can exhaust valuable tissue needed for more comprehensive analyses. In contrast, comprehensive genomic profiling (CGP) tests employ NGS to sequence megabases of DNA and RNA to evaluate all relevant molecular alterations, providing a broader genetic profile to identify actionable alterations that SGT may not accurately or efficiently assess. Here, we briefly describe four cases from a large reference laboratory in which actionable alterations were identified by CGP but not SGT. The discussion highlights the utility and advantages of using CGP to provide complete and timely treatment options and clinical trial opportunities for patients with NSCLC. [ABSTRACT FROM AUTHOR]

Disparities in cancer diagnosis, treatment, and outcomes based on self-identified race and ethnicity (SIRE) are well documented, yet these variables have historically been excluded from clinical research. Without SIRE, genetic ancestry can be inferred using single-nucleotide polymorphisms (SNPs) detected from tumor DNA using comprehensive genomic profiling (CGP). However, factors inherent to CGP of tumor DNA increase the difficulty of identifying ancestry-informative SNPs, and current workflows for inferring genetic ancestry from CGP need improvements in key areas of the ancestry inference process. This study used genomic data from 4274 diverse reference subjects and CGP data from 491 patients with solid tumors and SIRE to develop and validate a workflow to obtain accurate genetically inferred ancestry (GIA) from CGP sequencing results. We use consensus-based classification to derive confident ancestral inferences from an expanded reference dataset covering eight world populations (African, Admixed American, Central Asian/Siberian, European, East Asian, Middle Eastern, Oceania, South Asian). Our GIA calls were highly concordant with SIRE (95%) and aligned well with reference populations of inferred ancestries. Further, our workflow could expand on SIRE by (i) detecting the ancestry of patients that usually lack appropriate racial categories, (ii) determining what patients have mixed ancestry, and (iii) resolving ancestries of patients in heterogeneous racial categories and who had missing SIRE. Accurate GIA provides needed information to enable ancestry-aware biomarker research, ensure the inclusion of underrepresented groups in clinical research, and increase the diverse representation of patient populations eligible for precision medicine therapies and trials. [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibitors have changed the treatment landscape for various malignancies; however, their benefit is limited to a subset of patients. The immune machinery includes both mediators of suppression/immune evasion, such as PD-1, PD-L1, CTLA-4, and LAG-3, all of which can be inhibited by specific antibodies, and immune-stimulatory molecules, such as T-cell co-stimulatory receptors that belong to the tumor necrosis factor receptor superfamily (TNFRSF), including OX40 receptor (CD134; TNFRSF4), 4-1BB (CD137; TNFRSF9), and glucocorticoid-induced TNFR-related (GITR) protein (CD357; TNFRSF18). In particular, OX40 and its binding ligand OX40L (CD134L; TNFSF4; CD252) are critical for immunoregulation. When OX40 on activated T cells binds OX40L on antigen-presenting cells, T-cell activation and immune stimulation are initiated via enhanced T-cell survival, proliferation and cytotoxicity, memory T-cell formation, and abrogation of regulatory T cell (Treg) immunosuppressive functions. OX40 agonists are in clinical trials both as monotherapy and in combination with other immunotherapy agents, in particular specific checkpoint inhibitors, for cancer treatment. To date, however, only a minority of patients respond. Transcriptomic profiling reveals that OX40 and OX40L expression vary between and within tumor types, and that only ~ 17% of cancer patients have high OX40 and low OX40L, one of the expression patterns that might be theoretically amenable to OX40 agonist enhancement. Taken together, the data suggest that the OX40/OX40L machinery is a critical part of the immune stimulatory system and that understanding endogenous expression patterns of these molecules and co-existing checkpoints merits further investigation in the context of a precision immunotherapy strategy for cancer therapy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Introduction: Younger patients with non-small cell lung cancer (NSCLC) (<50 years) represent a significant patient population with distinct clinicopathological features and enriched targetable genomic alterations compared to older patients. However, previous studies of younger NSCLC suffer from inconsistent findings, few studies have incorporated sex into their analyses, and studies targeting age-related differences in the tumor immune microenvironment are lacking. Methods: We performed a retrospective analysis of 8,230 patients with NSCLC, comparing genomic alterations and immunogenic markers of younger and older patients while also considering differences between male and female patients. We defined older patients as those ≥65 years and used a 5-year sliding threshold from <45 to <65 years to define various groups of younger patients. Additionally, in an independent cohort of patients with NSCLC, we use our observations to inform testing of the combinatorial effect of age and sex on survival of patients given immunotherapy with or without chemotherapy. Results: We observed distinct genomic and immune microenvironment profiles for tumors of younger patients compared to tumors of older patients. Younger patient tumors were enriched in clinically relevant genomic alterations and had gene expression patterns indicative of reduced immune system activation, which was most evident when analyzing male patients. Further, we found younger male patients treated with immunotherapy alone had significantly worse survival compared to male patients ≥65 years, while the addition of chemotherapy reduced this disparity. Contrarily, we found younger female patients had significantly better survival compared to female patients ≥65 years when treated with immunotherapy plus chemotherapy, while treatment with immunotherapy alone resulted in similar outcomes. Discussion: These results show the value of comprehensive genomic and immune profiling (CGIP) for informing clinical treatment of younger patients with NSCLC and provides support for broader coverage of CGIP for younger patients with advanced NSCLC. [ABSTRACT FROM AUTHOR]

Background: KEYNOTE-522 resulted in FDA approval of the immune checkpoint inhibitor pembrolizumab in combination with neoadjuvant chemotherapy for patients with early-stage, high-risk, triple-negative breast cancer (TNBC). Unfortunately, pembrolizumab is associated with several immune-related adverse events (irAEs). We aimed to identify potential tumor microenvironment (TME) biomarkers which could predict patients who may attain pathological complete response (pCR) with chemotherapy alone and be spared the use of anti-PD-1 immunotherapy. Methods: Comprehensive immune profiling, including RNA-seq gene expression assessment of 395 immune genes, was performed on matched FFPE tumor samples from 22 stage I-III TNBC patients (14 patients treated with neoadjuvant chemotherapy alone (NAC) and 8 treated with neoadjuvant chemotherapy combined with pembrolizumab (NAC+I)). Results: Differential gene expression analysis revealed that in the NAC group, IL12B and IL13 were both significantly associated with pCR. In the NAC+I group, LCK and TP63 were significantly associated with pCR. Patients in both treatment groups exhibiting pCR tended to have greater tumor inflammation than non-pCR patients. In the NAC+I group, patients with pCR tended to have greater cell proliferation and higher PD-L1 expression, while in the NAC group, patients with pCR tended to have lower cancer testis antigen expression. Additionally, the NAC+I group trended toward a lower relative dose intensity averaged across all chemotherapy drugs, suggesting that more dose reductions or treatment delays occurred in the NAC+I group than the NAC group. Conclusions: A comprehensive understanding of immunologic factors could potentially predict pCR to chemotherapy alone, enabling the avoidance of the unnecessary treatment of these patients with checkpoint inhibitors. [ABSTRACT FROM AUTHOR]

Background: CTLA-4 impedes the immune system's antitumor response. There are two Food and Drug Administration-approved anti-CTLA-4 agents – ipilimumab and tremelimumab – both used together with anti-PD-1/PD-L1 agents. Objective: To assess the prognostic implications and immunologic correlates of high CTLA-4 in tumors of patients on immunotherapy and those on non-immunotherapy treatments. Design/methods: We evaluated RNA expression levels in a clinical-grade laboratory and clinical correlates of CTLA-4 and other immune checkpoints in 514 tumors, including 489 patients with advanced/metastatic cancers and full outcome annotation. A reference population (735 tumors; 35 histologies) was used to normalize and rank transcript abundance (0–100 percentile) to internal housekeeping gene profiles. Results: The most common tumor types were colorectal (140/514, 27%), pancreatic (55/514, 11%), breast (49/514, 10%), and ovarian cancers (43/514, 8%). Overall, 87 of 514 tumors (16.9%) had high CTLA-4 transcript expression (⩾75th percentile rank). Cancers with the largest proportion of high CTLA-4 transcripts were cervical cancer (80% of patients), small intestine cancer (33.3%), and melanoma (33.3%). High CTLA-4 RNA independently/significantly correlated with high PD-1, PD- L2, and LAG3 RNA levels (and with high PD-L1 in univariate analysis). High CTLA-4 RNA expression was not correlated with survival from the time of metastatic disease [ N = 272 patients who never received immune checkpoint inhibitors (ICIs)]. However, in 217 patients treated with ICIs (mostly anti-PD-1/anti-PD- L1), progression-free survival (PFS) and overall survival (OS) were significantly longer among patients with high versus non-high CTLA-4 expression [hazard ratio, 95% confidence interval: 0.6 (0.4–0.9) p = 0.008; and 0.5 (0.3–0.8) p = 0.002, respectively]; results were unchanged when 18 patients who received anti-CTLA-4 were omitted. Patients whose tumors had high CTLA-4 and high PD-L1 did best; those with high PD-L1 but non-high CTLA-4 and/or other expression patterns had poorer outcomes for PFS (p = 0.004) and OS (p = 0.009) after immunotherapy. Conclusion: High CTLA-4, especially when combined with high PD-L1 transcript expression, was a significant positive predictive biomarker for better outcomes (PFS and OS) in patients on immunotherapy. Plain language summary: High CTLA-4 expression and immunotherapy outcome High CTLA-4 expression was not a prognostic factor for survival in patients not receiving ICIs but was a significant positive predictive biomarker for better outcome (PFS and OS) in patients on immunotherapy, perhaps because it correlated with expression of other checkpoints such as PD-1 and PD-L2. [ABSTRACT FROM AUTHOR]

Simple Summary: We compared RNA next-generation sequencing (RNA-seq) to standard immunohistochemistry (IHC) for PD-L1 expression measurement and associations with pembrolizumab immunotherapy outcomes in NSCLC patient tumors. RNA-seq and IHC PD-L1 score interpretation agreed for 80% of patients, and an RNA-seq "high" cutoff that accurately separated IHC high versus low or negative expression was identified. However, RNA-seq could not discern PD-L1 IHC from negative expression due to the limited sensitivity of IHC as a reference test. High PD-L1 expression by RNA-seq alone and in combination with IHC high or low status was associated with better pembrolizumab outcomes in NSCLC patients than IHC alone. Programmed cell death ligand (PD-L1) expression by immunohistochemistry (IHC) lacks sensitivity for pembrolizumab immunotherapy selection in non-small cell lung cancer (NSCLC), particularly for tumors with low expression. We retrospectively evaluated transcriptomic PD-L1 by mRNA next-generation sequencing (RNA-seq). In an unselected NSCLC patient cohort (n = 3168) tested during standard care (2017–2021), PD-L1 IHC and RNA-seq demonstrated moderate concordance, with 80% agreement overall. Most discordant cases were either low or negative for PD-L1 expression by IHC but high by RNA-seq. RNA-seq accurately discriminated PD-L1 IHC high from low tumors by receiver operator curve (ROC) analysis but could not distinguish PD-L1 IHC low from negative tumors. In a separate pembrolizumab monotherapy cohort (n = 102), NSCLC tumors classified as PD-L1 high versus not high by RNA-seq had significantly improved response, progression-free survival, and overall survival as an individual measure and in combination with IHC high or low status. PD-L1 IHC status (high or low) trended toward but had no significant associations with improved outcomes. Conventional PD-L1 IHC testing has inherent limitations, making it an imperfect reference standard for evaluating novel testing technologies. RNA-seq offers an objective PD-L1 measure that could represent a complementary method to IHC to improve NSCLC patient selection for immunotherapy. [ABSTRACT FROM AUTHOR]

Biomarkers predicting immunotherapy response in metastatic renal cell cancer (mRCC) are lacking. PD-L1 immunohistochemistry is a complementary diagnostic for immune checkpoint inhibitors (ICIs) in mRCC, but has shown minimal clinical utility and is not used in routine clinical practice. Tumor specimens from 56 patients with mRCC who received nivolumab were evaluated for PD-L1, cell proliferation (targeted RNA-seq), and outcome. For 56 patients treated with nivolumab as a standard of care, there were 2 complete responses and 8 partial responses for a response rate of 17.9%. Dividing cell proliferation into tertiles, derived from the mean expression of 10 proliferation-associated genes in a reference set of tumors, poorly proliferative tumors (62.5%) were more common than moderately (30.4%) or highly proliferative (8.9%) counterparts. Moderately proliferative tumors were enriched for PD-L1 positive (41.2%), compared to poorly proliferative counterparts (11.4%). Objective response for moderately proliferative (29.4%) tumors was higher than that of poorly (11.4%) proliferative counterparts, but not statistically significant (p =.11). When cell proliferation and negative PD-L1 tumor proportion scores were combined statistically significant results were achieved (p =.048), showing that patients with poorly proliferative and PD-L1 negative tumors have a very low response rate (6.5%) compared to moderately proliferative PD-L1 negative tumors (30%). Cell proliferation has value in predicting response to nivolumab in clear cell mRCC patients, especially when combined with PD-L1 expression. Further studies which include the addition of progression-free survival (PFS) along with sufficiently powered subgroups are required to further support these findings. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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]