Immunologic Factors Associated with Differential Response to Neoadjuvant Chemoimmunotherapy in Triple-Negative Breast Cancer.

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.

Transcriptomic analysis of GITR and GITR ligand reveals cancer immune heterogeneity with implications for GITR targeting.

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.

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

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.

OX40/OX40 ligand and its role in precision immune oncology.

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.

The Impact of Prior Single-Gene Testing on Comprehensive Genomic Profiling Results for Patients with Non-Small Cell Lung Cancer.

INTRODUCTION: Tissue-based broad molecular profiling of guideline-recommended biomarkers is advised for the therapeutic management of patients with non-small cell lung cancer (NSCLC). However, practice variation can affect whether all indicated biomarkers are tested. We aimed to evaluate the impact of common single-gene testing (SGT) on subsequent comprehensive genomic profiling (CGP) test outcomes and results in NSCLC. METHODS: Oncologists who ordered SGT for guideline-recommended biomarkers in NSCLC patients were prospectively contacted (May-December 2022) and offered CGP (DNA and RNA sequencing), either following receipt of negative SGT findings, or instead of SGT for each patient. We describe SGT patterns and compare CGP completion rates, turnaround time, and recommended biomarker detection for NSCLC patients with and without prior negative SGT results. RESULTS: Oncologists in > 80 community practices ordered CGP for 561 NSCLC patients; 135 patients (27%) first had negative results from 30 different SGT combinations; 84% included ALK, EGFR and PD-L1, while only 3% of orders included all available SGTs for guideline-recommended genes. Among patients with negative SGT results, CGP was attempted using the same tissue specimen 90% of the time. There were also significantly more CGP order cancellations due to tissue insufficiency (17% vs. 7%), DNA sequencing failures (13% vs. 8%), and turnaround time > 14 days (62% vs. 29%) than among patients who only had CGP. Forty-six percent of patients with negative prior SGT had positive CGP results for recommended biomarkers, including targetable genomic variants in genes beyond ALK and EGFR, such as ERBB2, KRAS (non-G12C), MET (exon 14 skipping), NTRK2/3, and RET . CONCLUSION: For patients with NSCLC, initial use of SGT increases subsequent CGP test cancellations, turnaround time, and the likelihood of incomplete molecular profiling for guideline-recommended biomarkers due to tissue insufficiency.

Patients with non-small cell lung cancer (NSCLC) should have their tumor tissue tested for all recommended biomarkers that can help identify their best treatment options. Traditional tests look at gene biomarkers one by one (single-gene testing), and doctors can order some or all these tests individually or in a group. However, some recommended biomarkers cannot be tested by traditional single-gene tests at all. Newer technology (next-generation sequencing) covers all current recommended treatment biomarkers in one test (comprehensive genomic profiling), but this testing is more expensive and can take more time. Our study shows that NSCLC patients do not get all recommended treatment biomarkers tested when a single-gene testing approach is taken. Single-gene testing also used up some patients' tumor tissue entirely, such that further testing by comprehensive genomic profiling could not be done at all (17% vs. 7% for patients with no prior single-gene tests), resulted in more sequencing failures (13% vs. 8%), and had turnaround time for results greater than 14 days for more patients (62% vs. 29%). When comprehensive genomic profiling was completed, 46% of patients with negative results from prior single-gene testing had positive results for recommended treatment biomarkers that were not included in the initial single-gene tests. To ensure that NSCLC patients receive testing for all recommended biomarkers, comprehensive genomic profiling must be performed first.

LAG-3 transcriptomic expression correlates linearly with other checkpoints, but not with clinical outcomes.

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 R2 = 0.21 (P < 0.001)), PD-1 (R2 = 0.24 (P < 0.001)) and CTLA-4 (R2 = 0.19 (P < 0.001)); when examined for Spearman correlation, significance did not change. LAG-3 expression (dichotomized at ≥ 75th (high) versus < 75th (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.

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

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.

High CTLA-4 transcriptomic expression correlates with high expression of other checkpoints and with immunotherapy outcome.

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.

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.

PD-L1 Expression by RNA-Sequencing in Non-Small Cell Lung Cancer: Concordance with Immunohistochemistry and Associations with Pembrolizumab Treatment Outcomes.

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.

Challenges and prospects of CSF1R targeting for advanced malignancies.

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.

Cancer-Immunity Marker RNA Expression Levels across Gynecologic Cancers: Implications for Immunotherapy.

Our objective was to characterize cancer-immunity marker expression in gynecologic cancers and compare immune landscapes between gynecologic tumor subtypes and with nongynecologic solid tumors. RNA expression levels of 51 cancer-immunity markers were analyzed in patients with gynecologic cancers versus nongynecologic cancers, and normalized to a reference population of 735 control cancers, ranked from 0 to 100, and categorized as low (0-24), moderate (25-74), or high (75-100) percentile rank. Of the 72 patients studied, 43 (60%) had ovarian, 24 (33%) uterine, and 5 (7%) cervical cancer. No two immune profiles were identical according to expression rank (0-100) or rank level (low, moderate, or high). Patients with cervical cancer had significantly higher expression level ranks of immune activating, proinflammatory, tumor-infiltrating lymphocyte markers, and checkpoints than patients with uterine or ovarian cancer (P < 0.001 for all comparisons). However, there were no significant differences in immune marker expression between uterine and ovarian cancers. Tumors with PD-L1 tumor proportional score (TPS) ≥1% versus 0% had significantly higher expression levels of proinflammatory markers (58 vs. 49%, P = 0.0004). Compared to patients with nongynecologic cancers, more patients with gynecologic cancers express high levels of IDO-1 (44 vs. 13%, P < 0.001), LAG3 (35 vs. 21%, P = 0.008), and IL10 (31 vs. 15%, P = 0.002.) Patients with gynecologic cancers have complex and heterogeneous immune landscapes that are distinct from patient to patient and from other solid tumors. High levels of IDO1 and LAG3 suggest that clinical trials with IDO1 inhibitors or LAG3 inhibitors, respectively, may be warranted in gynecologic cancers.

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

BACKGROUND: Immunotherapy 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. METHODS: Two 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. RESULTS: The 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. CONCLUSION: A 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.

Indoleamine 2,3-dioxygenase (IDO) inhibitors and cancer immunotherapy.

Strategies for unlocking immunosuppression in the tumor microenvironment have been investigated to overcome resistance to first-generation immune checkpoint blockade with anti- programmed cell death protein 1 (PD-1)/ programmed death-ligand 1 (PD-L1) and anti-cytotoxic T-lymphocyte associated protein 4 (CTLA-4) agents. Indoleamine 2,3-dioxygenase (IDO) 1, an enzyme catabolizing tryptophan to kynurenine, creates an immunosuppressive environment in preclinical studies. Early phase clinical trials investigating inhibition of IDO1, especially together with checkpoint blockade, provided promising results. Unfortunately, the phase 3 trial of the IDO1 inhibitor epacadostat combined with the PD-1 inhibitor pembrolizumab did not show clinical benefit when compared with pembrolizumab monotherapy in patients with advanced malignant melanoma, which dampened enthusiasm for IDO inhibitors. Even so, several molecules, such as the aryl hydrocarbon receptor and tryptophan 2,3-dioxygenase, were reported as additional potential targets for the modulation of the tryptophan pathway, which might enhance clinical effectiveness. Furthermore, the combination of IDO pathway blockade with agents inhibiting other signals, such as those generated by PIK3CA mutations that may accompany IDO1 upregulation, may be a novel way to enhance activity. Importantly, IDO1 expression level varies by tumor type and among patients with the same tumor type, suggesting that patient selection based on expression levels of IDO1 may be warranted in clinical trials.

Integration of tumor inflammation, cell proliferation, and traditional biomarkers improves prediction of immunotherapy resistance and response.

BACKGROUND: Contemporary to the rapidly evolving landscape of cancer immunotherapy is the equally changing understanding of immune tumor microenvironments (TMEs) which is crucial to the success of these therapies. Their reliance on a robust host immune response necessitates clinical grade measurements of immune TMEs at diagnosis. In this study, we describe a stable tumor immunogenic profile describing immune TMEs in multiple tumor types with ability to predict clinical benefit from immune checkpoint inhibitors (ICIs). METHODS: A tumor immunogenic signature (TIGS) was derived from targeted RNA-sequencing (RNA-seq) and gene expression analysis of 1323 clinical solid tumor cases spanning 35 histologies using unsupervised analysis. TIGS correlation with ICI response and survival was assessed in a retrospective cohort of NSCLC, melanoma and RCC tumor blocks, alone and combined with TMB, PD-L1 IHC and cell proliferation biomarkers. RESULTS: Unsupervised clustering of RNA-seq profiles uncovered a 161 gene signature where T cell and B cell activation, IFNg, chemokine, cytokine and interleukin pathways are over-represented. Mean expression of these genes produced three distinct TIGS score categories: strong (n = 384/1323; 29.02%), moderate (n = 354/1323; 26.76%), and weak (n = 585/1323; 44.22%). Strong TIGS tumors presented an improved ICI response rate of 37% (30/81); with highest response rate advantage occurring in NSCLC (ORR = 36.6%; 16/44; p = 0.051). Similarly, overall survival for strong TIGS tumors trended upward (median = 25 months; p = 0.19). Integrating the TIGS score categories with neoplastic influence quantified via cell proliferation showed highly proliferative and strong TIGS tumors correlate with significantly higher ICI ORR than poorly proliferative and weak TIGS tumors [14.28%; p = 0.0006]. Importantly, we noted that strong TIGS and highly [median = not achieved; p = 0.025] or moderately [median = 16.2 months; p = 0.025] proliferative tumors had significantly better survival compared to weak TIGS, highly proliferative tumors [median = 7.03 months]. Importantly, TIGS discriminates subpopulations of potential ICI responders that were considered negative for response by TMB and PD-L1. CONCLUSIONS: TIGS is a comprehensive and informative measurement of immune TME that effectively characterizes host immune response to ICIs in multiple tumors. The results indicate that when combined with PD-L1, TMB and cell proliferation, TIGS provides greater context of both immune and neoplastic influences on the TME for implementation into clinical practice.

Immune profiling and immunotherapeutic targets in pancreatic cancer.

BACKGROUND: Immunotherapeutic approaches for pancreatic ductal adenocarcinoma (PDAC) are less successful as compared to many other tumor types. In this study, comprehensive immune profiling was performed in order to identify novel, potentially actionable targets for immunotherapy. METHODS: Formalin-fixed paraffin embedded (FFPE) specimens from 68 patients were evaluated for expression of 395 immune-related markers (RNA-seq), mutational burden by complete exon sequencing of 409 genes, PD-L1 expression by immunohistochemistry (IHC), pattern of tumor infiltrating lymphocytes (TILs) infiltration by CD8 IHC, and PD-L1/L2 copy number by fluorescent in situ hybridization (FISH). RESULTS: The seven classes of actionable genes capturing myeloid immunosuppression, metabolic immunosuppression, alternative checkpoint blockade, CTLA-4 immune checkpoint, immune infiltrate, and programmed cell death 1 (PD-1) axis immune checkpoint, discerned 5 unique clinically relevant immunosuppression expression profiles (from most to least common): (I) combined myeloid and metabolic immunosuppression [affecting 25 of 68 patients (36.8%)], (II) multiple immunosuppressive mechanisms (29.4%), (III) PD-L1 positive (20.6%), (IV) highly inflamed PD-L1 negative (10.3%); and (V) immune desert (2.9%). The Wilcoxon rank-sum test was used to compare the PDAC cohort with a comparison cohort (n=1,416 patients) for the mean expressions of the 409 genes evaluated. Multiple genes including TIM3, VISTA, CCL2, CCR2, TGFB1, CD73, and CD39 had significantly higher mean expression versus the comparison cohort, while three genes (LAG3, GITR, CD38) had significantly lower mean expression. CONCLUSIONS: This study demonstrates that a clinically relevant unique profile of immune markers can be identified in PDAC and be used as a roadmap for personalized immunotherapeutic decision-making strategies.

Proliferative potential and response to nivolumab in clear cell renal cell carcinoma patients.

Background: 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. Methods: Tumor specimens from 56 patients with mRCC who received nivolumab were evaluated for PD-L1, cell proliferation (targeted RNA-seq), and outcome. Results: 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%). Conclusions: 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.

Economic Burden of Checkpoint Inhibitor Immunotherapy for the Treatment of Non-Small Cell Lung Cancer in US Clinical Practice.

PURPOSE: The efficacy of checkpoint inhibitor (CPI) immunotherapy in patients with non-small cell lung cancer (NSCLC) is limited by a lack of strongly predictive response markers, subjecting patients to potential underutilization of alternative effective treatments, increased risk for futile care, and unnecessary costs. Here, we characterize the extent to which basic molecular tumor-marker testing has been performed for NSCLC therapy selection in the United States, and compare medical resource utilization and costs in CPI-treated patients versus CPI-eligible patients treated with other therapies. METHODS: We identified a cohort of CPI-treated patients with NSCLC and a propensity score-matched cohort of CPI-eligible patients with NSCLC treated with non-CPI therapies (3095 patients in each group), using US administrative claims data covering the pre- and postinitial FDA-approval period for nivolumab, pembrolizumab, and atezolizumab (October 2012 to September 2017). We describe the utilization of recommended baseline molecular testing for CPI selection (pre-index date for CPI or other anticancer therapy), including programmed death ligand 1 (PD-L1) immunohistochemistry, ALK rearrangement and EGFR mutation testing, and pre- and postindex treatment patterns. All-cause medical resource utilization and semiannual total reimbursement (costs) were compared between CPI-treated and non-CPI-treated patients. FINDINGS: At baseline, in the propensity score-matched CPI- and non-CPI-treated patient cohorts, mean PD-L1 immunohistochemistry test utilization for CPI selection was moderate (0.6 vs 0.7 per patient, respectively). However, we observed much lower mean utilization of testing for EGFR mutations (0.1 vs 0.1 per patient) and ALK rearrangements (0.1 vs 0.2 per patient). Postindex, the use of both chemotherapy and ALK- and EGFR-targeted therapies were decreased in both cohorts. The CPI-treated group had significantly higher mean medical resource utilization in nearly all categories in the postindex period, and total per-patient semiannual costs, than did the CPI-eligible patients who received other therapies (141,537 vs 75,429 US dollars [USD]; P < 0.0001), driven by CPI drug reimbursement. Median (interquartile range) time on CPI was longest with pembrolizumab (113 [106-127] days), followed by nivolumab (105 [97-106] days) and atezolizumab (64 [50-85] days). Despite being associated with the lowest drug cost and the shortest treatment duration, atezolizumab was associated with the highest mean total per-patient semiannual costs (160,540 USD) compared with pembrolizumab (153,003 USD) and nivolumab (138,542 USD). IMPLICATIONS: The advent of CPI treatment for NSCLC has added substantial care-related costs for patients and payers, concurrent with underutilization of minimum recommended molecular testing for therapy selection. Broad uptake of panel-based comprehensive targeted-therapy and immunotherapy profiling can promote optimal treatment selection and sequencing, reduce the likelihood of futile treatment, and further improve patient outcomes.

Development and analytical validation of a next-generation sequencing based microsatellite instability (MSI) assay.

BACKGROUND: We have developed and analytically validated a next-generation sequencing (NGS) assay to classify microsatellite instability (MSI) in formalin-fixed paraffin-embedded (FFPE) tumor specimens. METHODOLOGY: The assay relies on DNA-seq evaluation of insertion/deletion (indel) variability at 29 highly informative genomic loci to estimate MSI status without the requirement for matched-normal tissue. The assay has a clinically relevant five-day turnaround time and can be conducted on as little as 20 ng genomic DNA with a batch size of up to forty samples in a single run. RESULTS: The MSI detection method was developed on a training set (n = 94) consisting of 22 MSI-H, 24 MSS, and 47 matched normal samples and tested on an independent test set of 24 MSI-H and 24 MSS specimens. Assay performance with respect to accuracy, reproducibility, precision as well as control sample performance was estimated across a wide range of FFPE samples of multiple histologies to address pre-analytical variability (percent tumor nuclei), and analytical variability (batch size, run, day, operator). Analytical precision studies demonstrated that the assay is highly reproducible and accurate as compared to established gold standard PCR methodology and has been validated through NYS CLEP. SIGNIFICANCE: This assay provides clinicians with robust and reproducible NGS-based MSI testing without the need of matched normal tissue to inform clinical decision making for patients with solid tumors.

Oncologist uptake of comprehensive genomic profile guided targeted therapy.

We describe the extent to which comprehensive genomic profiling (CGP) results were used by oncologists to guide targeted therapy selection in a cohort of solid tumor patients tested as part of standard care at Roswell Park Comprehensive Cancer Center June 2016-June 2017, with adequate follow up through September 2018 (n = 620). Overall, 28.4% of CGP tests advised physicians about targeted therapy use supported by companion diagnostic or practice guideline evidence. Post-test targeted therapy uptake was highest for patients in active treatment at the time of order (86% versus 76% of treatment naïve patients), but also took longer to initiate (median 50 days versus 7 days for treatment naïve patients), with few patients (2.6%) receiving targeted agents prior to testing. 100% of patients with resistance variants did not receive targeted agents. Treatment naïve patients received immunotherapy as the most common alternative. When targeted therapy given off-label or in a trial was the best CGP option, (7%) of patients received it. Our data illustrate the appropriate and heterogeneous use of CGP by oncologists as a longitudinal treatment decision tool based on patient history and treatment needs, and that some patients may benefit from testing prior to initiation of other standard treatments.

Proliferative potential and resistance to immune checkpoint blockade in lung cancer patients.

BACKGROUND: Resistance to immune checkpoint inhibitors (ICIs) has been linked to local immunosuppression independent of major ICI targets (e.g., PD-1). Clinical experience with response prediction based on PD-L1 expression suggests that other factors influence sensitivity to ICIs in non-small cell lung cancer (NSCLC) patients. METHODS: Tumor specimens from 120 NSCLC patients from 10 institutions were evaluated for PD-L1 expression by immunohistochemistry, and global proliferative profile by targeted RNA-seq. RESULTS: Cell proliferation, derived from the mean expression of 10 proliferation-associated genes (namely BUB1, CCNB2, CDK1, CDKN3, FOXM1, KIAA0101, MAD2L1, MELK, MKI67, and TOP2A), was identified as a marker of response to ICIs in NSCLC. Poorly, moderately, and highly proliferative tumors were somewhat equally represented in NSCLC, with tumors with the highest PD-L1 expression being more frequently moderately proliferative as compared to lesser levels of PD-L1 expression. Proliferation status had an impact on survival in patients with both PD-L1 positive and negative tumors. There was a significant survival advantage for moderately proliferative tumors compared to their combined highly/poorly counterparts (p = 0.021). Moderately proliferative PD-L1 positive tumors had a median survival of 14.6 months that was almost twice that of PD-L1 negative highly/poorly proliferative at 7.6 months (p = 0.028). Median survival in moderately proliferative PD-L1 negative tumors at 12.6 months was comparable to that of highly/poorly proliferative PD-L1 positive tumors at 11.5 months, but in both instances less than that of moderately proliferative PD-L1 positive tumors. Similar to survival, proliferation status has impact on disease control (DC) in patients with both PD-L1 positive and negative tumors. Patients with moderately versus those with poorly or highly proliferative tumors have a superior DC rate when combined with any classification schema used to score PD-L1 as a positive result (i.e., TPS ≥ 50% or ≥ 1%), and best displayed by a DC rate for moderately proliferative tumors of no less than 40% for any classification of PD-L1 as a negative result. While there is an over representation of moderately proliferative tumors as PD-L1 expression increases this does not account for the improved survival or higher disease control rates seen in PD-L1 negative tumors. CONCLUSIONS: Cell proliferation is potentially a new biomarker of response to ICIs in NSCLC and is applicable to PD-L1 negative tumors.