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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.

Soil pore water salinity is widely accepted to be the primary influencing factor determining coastal vegetation succession. This study investigated an ecological wetland restoration process on the Nanhui coast, Shanghai, during which seasonal changes in vegetation density, soil salinity, and coastal elevation were recorded in detail. These variables interacted with each other and showed coincident distributions. The whole restoration process was categorized into four periods according to vegetation community dynamics. Regression analysis revealed that elevation, not soil pore water salinity, was the most critical factor influencing the spatial pattern of vegetation survival, expansion, and extinction on the Nanhui coast. From a long-term perspective, the local vegetation extinction pattern may have an irreparable influence on salt marsh succession, especially in environments with intricate hydrological and sedimentary conditions such as the Nanhui coastline. Without intact vegetation communities, deficiencies in coastal ecosystem resilience may cause vegetation extinction under the influence of a strong hydrological environment. The area between vegetation communities and bare tidal flats, the “vulnerable zone”, was a critical area determining the ultimate success of coastal vegetation restoration.

Methotrexate (MTX) is broadly applied in the clinic for the treatments of cancers and autoimmune diseases. Targeted delivery of MTX is attractive to improve its efficacy and reduce off-target toxicity. However, MTX encapsulation in nanoparticle is challenging due to its high water solubility. We rationally designed a well-defined telodendrimer (TD) nanocarrier based on MTX structure to sequester it in nanoparticles. Riboflavin (Rf) and positive charges groups were precisely conjugated on TD to form multivalent hydrogen bonds, π-π stacking and electrostatic interactions with MTX. A reverse micelle approach was developed to preset MTX and TD interactions in the core of micelles, which ensures the effective MTX loading upon dispersion into aqueous solution. As results, MTX loading capacity reaches over 20% (w/w) in the optimized nanocarrier with the particle size of 20-30 nm. The nanoformulations sustain the release of MTX in a controlled manner and exhibit excellent hemocompatibility. The in vitro cellular uptake of MTX was significantly improved by the nanoformulations. The potency of MTX nanoformulations is comparable to the free MTX in cytotoxicity. A psoriasis-like skin inflammation model was induced in mouse by imiquimod (IMQ) stimulation. MTX nanoformulations improved the psoriasis targeting and exhibited a superior long-lasting efficacy in reducing skin inflammation compared with the free MTX in psoriasis treatment.

BackgroundImmune checkpoint inhibitor-based therapies have achieved impressive success in the treatment of several cancer types. Predictive immune biomarkers, including PD-L1, MSI and TMB are well established as surrogate markers for immune evasion and tumor-specific neoantigens across many tumors. Positive detection across cancer types varies, but overall ~50% of patients test negative for these primary immune markers.1 In this study, we investigated the prevalence of secondary immune biomarkers outside of PD-L1, TMB and MSI.MethodsComprehensive genomic and immune profiling, including PD-L1 IHC, TMB, MSI and gene expression of 395 immune related genes was performed on 6078 FFPE tumors representing 34 cancer types, predominantly composed of lung cancer (36.7%), colorectal cancer (11.9%) and breast cancer (8.5%). Expression levels by RNA-seq of 36 genes targeted by immunotherapies in solid tumor clinical trials, identified as secondary immune biomarkers, were ranked against a reference population. Genes with a rank value ≥75th percentile were considered high and values were associated with PD-L1 (positive ≥1%), MSI (MSI-H or MSS) and TMB (high ≥10 Mut/Mb) status. Additionally, secondary immune biomarker status was segmented by tumor type and cancer immune cycle roles.ResultsIn total, 41.0% of cases were PD-L1+, 6.4% TMB+, and 0.1% MSI-H. 12.6% of cases were positive for >2 of these markers while 39.9% were triple negative (PD-L1-/TMB-/MSS). Of the PD-L1-/TMB-/MSS cases, 89.1% were high for at least one secondary immune biomarker, with 69.3% having ≥3 markers. PD-L1-/TMB-/MSS tumor types with ≥50% prevalence of high secondary immune biomarkers included brain, prostate, kidney, sarcoma, gallbladder, breast, colorectal, and liver cancer. High expression of cancer testis antigen secondary immune biomarkers (e.g., NY-ESO-1, LAGE-1A, MAGE-A4) was most commonly observed in bladder, ovarian, sarcoma, liver, and prostate cancer (≥15%). Tumors demonstrating T-cell priming (e.g., CD40, OX40, CD137), trafficking (e.g., TGFB1, TLR9, TNF) and/or recognition (e.g., CTLA4, LAG3, TIGIT) secondary immune biomarkers were most represented by kidney, gallbladder, and sarcoma (≥40%), with melanoma, esophageal, head & neck, cervical, stomach, and lung cancer least represented (≥15%).ConclusionsOur studies show comprehensive tumor profiling that includes gene expression can detect secondary immune biomarkers targeted by investigational therapies in ~90% of PD-L1-/TMB-/MSS cases. While genomic profiling could also provide therapeutic choices for a percentage of these patients, detection of secondary immune biomarkers by RNA-seq provides additional options for patients without a clear therapeutic path as determined by PD-L1 testing and genomic profiling alone.ReferenceHuang R S P, Haberberger J, Severson E, et al. A pan-cancer analysis of PD-L1 immunohistochemistry and gene amplification, tumor mutation burden and microsatellite instability in 48,782 cases. Mod Pathol 2021;34: 252–263.

BACKGROUND Non-small cell lung cancer (NSCLC) accounts for ~50% of brain metastases. We present the genomic and immune biomarker landscape for a cohort with metastatic NSCLC to the brain. METHODS We analyzed brain metastases FFPE tissue (n=137; ages 40-85y (mean 65y), 52% female, 48% male) vs. primary sites (n=5533; ages 24-100+y (mean 71y), 51% female, 49% male) for advanced or metastatic NSCLC patients with comprehensive genomic and immune biomarker profiling, including PD-L1 IHC, tumor mutational burden (TMB), Tumor Immunogenic Signature (TIGS), Cell Proliferation (CP), and Cancer Testis Antigen Burden (CTAB). RESULTS Genomic alteration (GA) frequency for NSCLC brain metastasis versus primary sites were similar among the most frequently mutated genes except for KRAS which was significantly higher among brain metastases (39.9% vs 25.5%, p< 0.0005). No significant differences were observed for TP53 (50.7% vs 50.2%), STK11 (11.5% vs 10.9%), CDKN2A (10.1% vs 7%), or EGFR (8.0% vs 11.5%). PD-L1 expression for all cases by IHC was not significantly different (mean TPS 29.9% vs 26.3%); however, brain metastases were more likely to be PD-L1 negative (TPS< 1%) (46.3% vs 33.3%, p< 0.005). Conversely, TMB was higher in brain metastases versus primary sites (12.9 vs 10.2 muts/MB, p< 8x10-10), with more TMB-high cases (10 muts/Mb) (57.0% vs 33.5%, p< 3x10-8). Similarly, brain metastases had a higher proportion of CTAB-high cases (68.6% vs 57.6%, p=0.01). The immune response biomarker score, TIGS, was more often weak in brain metastases (52.6% vs 34.0%, p< 9x10-6). CONCLUSION In NSCLC, metastatic brain lesions have a larger antigen burden, with increased TMB and CTAB, likely due to the immune privileged nature of the brain, which is reflected in the lower TIGS scores and PD-L1 positivity. Despite lower PD-L1 positivity, NSCLC brain metastases with negative PD-L1 IHC may potentially benefit from immunotherapy strategies given the high TMB and CTAB.

Objectives Aberrant expression of the mesenchymal epithelial transition factor (MET) gene has been observed in several malignancies, and drugs targeting the MET gene have been implicated in clinical trials with promising results. Hence, MET is a potentially targetable oncogenic driver. We explored the frequency of MET gene high copy number in melanomas and carcinomas. Methods The study group included 135 patients. Tissue microarrays were constructed with 19 melanomas and 116 carcinomas diagnosed from 2010 to 2012. We screened MET gene copy number by fluorescence in situ hybridization analysis using probes for MET gene and CEP7 as control. Results We found MET gene amplification in 2 (11%) of 19 melanoma cases, whereas 5 (26%) of 19 melanoma cases showed polysomy. For carcinomas, there was no MET gene amplification identified. However, 8 (7%) of 116 cases showed polysomy. Conclusions In our study, MET gene amplification was identified in 11% of melanomas and is relatively concordant with few reported studies. However, about 26% of the additional melanoma cases showed MET gene polysomy, which has not been reported as per our knowledge. If these results are validated with further orthogonal studies, more of the melanoma cases could potentially benefit from targeted therapy with MET tyrosine kinase inhibitors.

Timely and accurate identification of molecular alterations in solid tumors is essential for proper management of patients with advanced cancers. This has created a need for rapid, scalable comprehensive genomic profiling (CGP) systems that detect an increasing number of therapeutically-relevant variant types and molecular signatures. In this study, we assessed the analytical performance of the TruSight Oncology 500 High-Throughput assay for detection of somatic alterations from formalin-fixed paraffin-embedded tissue specimens. In parallel, we developed supporting software and automated sample preparation systems designed to process up to 70 clinical samples in a single NovaSeq 6000TM sequencing run with a turnaround time of 99% overall accuracy and precision. Our results demonstrate that the high-throughput CGP assay is a reliable method for accurate detection of molecular alterations in support of precision therapeutics in oncology. The supporting systems and scalable workflow allow for efficient interpretation and prompt reporting of hundreds of patient cancer genomes per week with excellent analytical performance.

Purpose of Study: Cancer testis antigens (CTA) are highly immunogenic genes with the ability to cause cancer-specific immune responses when expressed. Their tumor cell-specific expression makes them a key target of natural T cell response, cancer vaccines, immune checkpoint blockade (ICB), and cell-based immunotherapies in a wide range of tumor types. In this study, we assess the pan-cancer distribution and ICB survival association of CTA burden (CTAB) in real-world solid tumors. Procedure: Three tumor sample cohorts were studied: 1) a pan-cancer discovery cohort to develop a low- and high-CTAB cutoff (n=5450, 39 tumor types), 2) a TCGA cohort (n=19923, 32 tumor types) used to validate the classifier based on CTAB distribution and serve as a non-ICB-treated population, and 3) an ICB-treated retrospective cohort to validate the classification on overall survival (OS) (n=242, 3 tumor types). The expression levels of 17 CTA were measured using targeted RNA-Seq of FFPE tumor samples and then ranked against a pan-cancer reference population. CTAB was calculated for each sample, cohort and tumor type as the sum of the 17 CTA gene expression ranks. The discovery cohort median CTAB of 171 was used to classify all three cohorts into high- and low-CTAB groups. OS analysis was performed on the TCGA and ICB-treated cohorts using a CoxPH regression model to determine the Hazard Ratio (HR). Results: The three cohorts demonstrated overlapping single-peak, left-skewed CTAB distribution curves centered at CTAB values between 170 (discovery cohort) and 256 (retrospective cohort). When grouping by tumor types and ordering by median CTAB, the CTAB distributions for tumor types within all three cohorts were comparable. CoxPH regression analysis revealed an association between the CTAB threshold classifier and OS in both the ICB-treated retrospective and non-ICB TCGA cohorts. However, the direction of this association differed between the two cohorts, with high-CTAB samples having better survival (HR=0.936, p=0.076) in the ICB-treated retrospective cohort and worse survival (HR: 1.007, p=0.084) in the non-ICB-treated cohort. Conclusion: Our studies show that the CTAB distribution was maintained across the discovery and TCGA cohorts and a wide range of tumor types, supporting that the CTAB classifier is valid and histology agnostic. Additionally, when evaluating the ICB and non-ICB-treated cohorts, CTAB demonstrated the ability to predict OS, pointing to the utility of ICB in supporting CTA-specific natural immune response. However, further studies are necessary to verify these mechanisms of response to ICB as well as cancer vaccines and cell-based immunotherapies. Additional validation is needed to establish the predictive utility of CTAB alone and in combination with other immune oncology biomarkers for resistance or response. Citation Format: R.J. Seager, Erik Van Roey, Shuang Gao, Blake Burgher, Paul DePietro, Mary Nesline, Roger Klein, Shengle Zhang, Jeffrey M. Conroy, Sarabjot Pabla. Cancer testis antigen burden: Pan-cancer distribution and survival implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5137.

PURPOSE: The immunohistochemistry companion diagnostic test for pembrolizumab (IHC 22C3 pharmDx) lacks sensitivity, challenging immunotherapy selection for NSCLC patients with lower levels of expression. Unlike IHC 22C3, which restricts assessment of PD-L1 expression to viable tumor cells as a tumor proportion score (% TPS), mRNA next generation sequencing (RNA-seq) measures PD-L1 expression in the tumor microenvironment for both tumor and inflammatory background cells. RNA-seq previously demonstrated concordance with IHC and may be a robust alternative testing method for multiple tumor types. Here, we sought to optimize PD-L1 RNA-seq cutoff values in NSCLC to improve clinical sensitivity. PROCEDURE: NSCLC patients included in the study (n=3,465) were tested for PD-L1 expression by IHC 22C3 and clinically validated RNA-seq, measured as % rank (0-100) relative to a reference population based on normalized reads per million (nRPM). Patients were divided into an RNA-seq cut-off discovery cohort (n=3,168), and a test cohort pembrolizumab treated patients. Principal components analysis (PCA) was used to classify patients based on test results and explore cut-off values in the discovery cohort. Kaplan Meier curves and a Cox proportional hazards regression models assessed overall survival (OS) hazard ratios (HR) for RNA-seq versus standard of care IHC cut-offs in the test cohort. RESULTS: Unsupervised PCA clustering identified three distinct PD-L1 groups separated by combinations of significant over- and under-representation of RNA-seq and IHC result measures from prior testing. The groups were labeled as “low” (rank ≤40), “moderate” (rank 41-73), and “high” (rank ≥74), based on the median RNA-seq rank for each group (+/- 1SD for low and high). Both the low and moderate groups were overrepresented by patients in the PD-L1 IHC low and negative groups. The moderate group was overrepresented by patients with moderately high PD-L1 RNA-seq ranks (median=70), while the low group was overrepresented by patients that were not PD-L1 high by RNA-seq. The high group was overrepresented by patients high for PD-L1 by both IHC and RNA-seq. OS HRs were better for RNA-seq high versus moderate (HR=0.05, CI 0.00-0.63, p=.02), and RNA-seq high versus low (HR=0.16, CI 0.03-0.86, p=.03) groups compared to standard of care IHC 22C3 high versus low groups, (HR=0.21, CI 0.04-1.07, p=.06). Findings were non-significant for the RNA-seq moderate versus low groups, likely due to the limited and disproportionately high number of patients with poor performance status in these groups. CONCLUSIONS: PD-L1 expression by RNA-seq demonstrated improved clinical sensitivity in predicting OS versus standard of care PD-LI IHC in a pembrolizumab treated NSCLC patient cohort. Additional studies are needed to further define cut-offs in the context of performance status, and better understand immune escape mechanisms in the moderate group. Citation Format: Mary K. Nesline, Sarabjot Pabla, Yong Hee Lee, Paul DePietro, Amy Early, Roger Klein, Shengle Zhang, Jeffrey Conroy. PD-L1 expression by RNA-sequencing and survival from pembrolizumab in non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1259.

2623 Background: Immune checkpoint inhibitors (ICIs) have emerged as effective treatments in non-small cell lung cancer (NSCLC). While the clinical utility of single agent ICI or in combination with chemotherapy has been well established, there remains an unmet need for the development of biomarkers that can better predict response. To address this need, we developed and applied a combination genomic and immune biomarker strategy to ICI-treated NSCLC patients which identified distinct patient subgroups with differential benefit among single agent or combination ICI treatment strategies. Methods: A discovery cohort (DC) of 5450 tumors across 37 histologies were evaluated by comprehensive genomic and immune profiling of the tumor immune microenvironment. Individual and combination biomarker assessment included PD-L1 IHC, TMB, tumor inflammation (TIGS), cell proliferation (CP) and cancer testis antigen burden (CTAB). From this cohort, combinations of molecular and immune biomarkers were identified and applied to a retrospective cohort (RC) of 225 metastatic NSCLC patients treated with pembrolizumab + chemo or pembrolizumab alone to correlate with response. Comparison of objective response rates (ORR) was performed using Chi-square test. Kaplan-Meir analysis was performed to test for differences in overall survival (OS) and 1-year OS. Results: Unsupervised analysis of the DC revealed four distinct biomarker combination groups that describe underlying tumor immunobiology: tumor dominant (CTAB, TMB, CP High), proliferative (CP High), inflamed (TIGS High), and checkpoint (PDL1, TIGS and TMB High). Application of these biomarker groups to the RC demonstrated significant differences in response to ICI regimens between groups (p = 0.04). Patients in the proliferative group (35.1%, 79/225; median PD-L1 = 20% TPS) treated with single agent pembrolizumab showed a significantly higher ORR (59%; 16/27) compared to pembrolizumab + chemo (27%; 14/52; p = 0.005), significantly improved 1-yr OS (p = 0.03), and trend towards better OS (p = 0.14). Importantly, patients in the inflamed group (16%, 36/225; median PD-L1 = 1% TPS), suggested that pembrolizumab + chemo (ORR 26.1%; 6/23) was not associated with ORR compared to pembrolizumab (ORR 31%; 4/13, p = 0.76), or OS (p = 0.37) and 1-yr OS (p = 0.57). Conclusions: Comprehensive genomic and immune profiling may identify PD-L1 low NSCLC patients who benefit from single agent pembrolizumab. PD-L1 low NSCLC patients with a proliferative phenotype may benefit from single agent pembrolizumab, whereas PD-L1 low cases with an inflamed phenotype may benefit from both single agent and combination pembrolizumab. Although further clinical validation of these predictive biomarker combinations is required, this data-driven approach demonstrates the potential to provide treatment decision support when selecting an ICI therapeutic strategy in lung cancer.

e21167 Background: CGIP analyzes FFPE tumor tissue by DNA/RNA sequencing for SNVs, indels, copy gain/loss, fusions, splice variants, MSI and TMB, along with PD-L1 IHC. A purported advantage of CGIP in NSCLC is the ability to identify targeted and immunotherapy biomarkers to inform clinical management. However, the extent to which CGIP supports treatment decisions and benefits NSCLC patients in various treatment settings is limited. Methods: A retrospective analysis of OmniSeq CGIP results (June 2017-March 2019) and real-world clinical data (through March 2020) for NSCLC patients (n = 300) was performed to evaluate treatment strategies at Roswell Park Comprehensive Cancer Center. Patient targeted and immunotherapies following CGIP were classified as “matched” to biomarker results (established or potentially clinically significant) at the indication level (single or multi-marker results, histology, treatment line) based on AMP/ASCO/CAP guidance for strength of biomarker clinical evidence. We estimated overall survival (OS) from CGIP report date for patients who first received either matched therapy or chemotherapy (and no subsequent matched therapy), and assessed the predictive value of matched therapy for OS in the first or subsequent line setting, adjusting for clinicopathologic covariates. Results: Most CGIP tested patients were female (55%), stage IIIB/IV (89%), ECOG < 2 (83%), non-squamous (86%), treatment naïve (62%), ever smokers (88%). 74% (228) of patients were treated post-CGIP, with 71% receiving at least one matched therapy. Matched therapies received in the frontline setting were supported by the highest (Tier 1A) category of evidence more often than subsequent line therapies (97% vs. 68%). 90% of patients with oncogenic driver mutations received targeted agents (17% of total) and 57% received matched immunotherapy. In the frontline setting, compared to chemotherapy, OS was highest for patients who first received matched targeted therapy (median = 23.4 mo; HR 0.26; p = .004; 95% CI 0.13-0.68) vs matched immunotherapy (median = 17.9 mo; HR 0.38; p = .001; 95% CI 0.21-0.69). Subsequent line, OS was also highest for patients who first received matched targeted therapy (median not est., mean = 27.5 mo; HR 0.20; p = .063; 95% CI 0.04-1.09) vs matched immunotherapy (median = 17.4 mo; HR 0.20; 95% CI 0.04-1.09), however, these differences were non-significant. Conclusions: CGIP supports evidence-based clinical decision making for NSCLC in the first and subsequent line settings and leads to improved survival for patients who receive matched targeted or immunotherapy compared to chemotherapy. Better predictive markers are needed to identify NSCLC patients who are more likely to respond to immunotherapies. Heterogeneity of patient biomarker profiles and treatment strategies over time in real world practice are a challenge to assessing CGIP efficacy.

Theory of self-organization, i.e., scale-dependent feedback (SDF), has been widely used to explain mechanisms of spatial patterns in different ecosystems. Studies have demonstrated that self-organization is one of the mechanisms through which ecosystem resilience is maintained. However, the application of SDF in real ecological restoration practices is a challenge due to the lack of a controlled experimental validation. In the present study, multiple scales of vegetation patches were constructed along an elevation gradient in the saltmarsh ecosystem on Nanhui coasts and were investigated to verify if there was an effect of SDF. Results of the density-variation curves analyses revealed that most constructed self-organized patches could survive and an optimal curve was found of which the density-dependent feedback was proven through fitting with the asymptotic regression model. The large vegetation patches exhibited considerable increases in density when compared to the small vegetation patches, which occurred in challenging environments, i.e., on the verges of elevation thresholds, and with a tendency to shrink. Analyses using one-way ANOVA revealed that there was an optimal patch scale and elevation in the study area, i.e., 1 m × 1 m scale and 3.2 m, respectively. Optimal scale and elevation provide a comprehensively explanations of SDF, although with the positive effects gradually decreased along the distance away from the optimal condition. The present study provides novel insights on applying the theory of SDF in facilitating the restoration process of coastal saltmarshes.

BackgroundWhen expressed in cancer cells, cancer testis antigens (CTAs) are highly immunogenic and have the capacity to elicit cancer-specific immune responses in diverse malignancies. With their expression limited to tumor cells, CTAs have become a prime target of natural T cell response, immune cell-based therapy, and cancer vaccines. In this study, we investigated CTA burden in real-world clinical tumors spanning multiple histologies, revealing a novel prognostic gene expression-based biomarker.MethodsTargeted RNA-seq was performed on 5450 FFPE tumors representing 39 histologic types, predominantly composed of lung cancer (40.4%) followed by colorectal cancer (10.6%) and breast cancer (8.6%). Using an amplicon-based NGS approach, expression levels of 17 CTA genes were ranked against a reference population. Cancer Testis Antigen Burden (CTAB) was calculated as the sum of the gene expression rank for each CTA gene. The median CTAB of ≥171 was used as cutoff for CTAB High versus Low classification. We estimated Pearson’s correlation for all CTA genes to discover co-expression patterns between CTAs and histologies. Overall survival (OS) analysis was performed using CoxPh regression model whereas response analysis was performed using logistic regression model with p-values reported.ResultsWithin the tumor samples, CTAB values ranged from 0–1700, with kidney cancer demonstrating overall lowest mean CTAB (110) and melanoma the highest (550). NSCLC had an average CTAB of 283. In an immune checkpoint blockade treated retrospective cohort of 110 NSCLC patients, High CTAB showed better OS compared to Low CTA (HR: 0.55, p=0.07). Additionally, when combined with tumor inflammation and cell proliferation biomarkers, highly inflamed but poorly proliferative tumors with High CTAB had improved OS (HR: 0.27, p=0.05). No significant association with response was detectedConclusionsOur studies show that co-expression of multiple CTA genes occurs in many tumor types and can be reliably detected using a targeted RNA-seq approach. Utilization of this co-expression pattern to calculate CTAB reveals tumor-type associated signatures, which in a small NSCLC cohort is associated with the overall survival. The findings suggest that these immunogenic antigens expose the tumor cells to natural or immunotherapy augmented cell-based immune response, and that CTAB is a potential predictive marker for therapeutic response to checkpoint inhibitors. Further studies are needed to establish the predictive value in other tumor types, as well as the role of CTAB in immune cell therapies and vaccinations.

BackgroundCancer of unknown primary (CUP) is a rare tumor type accounting for 2% of solid cancers. In the subset of CUP cases where tumor of origin is posited and treated as such, no clear clinical benefit has been demonstrated. Furthermore, CUP patients treated by empiric platinum-based regimens have low response and survival rates of approximately 20%.1 2 Support of tissue-agnostic marker-directed immunotherapy is growing because it targets the immune system rather than the tumor, with some efficacy evidence emerging for CUP.3 Identifying new targets for immunotherapeutic opportunities in this heterogeneous and difficult to treat patient group is a critical unmet need.MethodsComprehensive genomic and immune marker profiling by NGS4 was performed on FFPE tissue for CUP tumors (n=298) as indicated by physicians’ test orders from >100 clinical practice sites. Histology was verified by a molecular pathologist as part of pre-analytic test quality control, with cases representing tumors with adenocarcinoma (58%), carcinoma (26%), squamous (10%), and neuroendocrine (6%) histologic features. RNA-expression levels of immune genes that are current targets in non-CUP immunotherapy clinical trials (n=36) were ranked against a reference population (≥75th percentile=high), and described by histologic type, along with PD-L1 IHC (22C3) expression, tumor mutational burden (TMB) and genomic variants.Results90% of all CUP tumors had at least 1 highly expressed immune gene target in active immunotherapy trials, with the most frequent being TGFB1 (47%) and CCL2 (39%). 55% of CUP tumors were PD-L1 IHC 22C3 positive (>=1% TPS), and 21% had high TMB (>=10 mut/Mb) in CUP tumors with neuroendocrine (32%), carcinoma (30%), squamous cell (21%), and adenocarcinoma (17%) histologic features. Overall, 26% of CUP patient tumors, mostly adenocarcinomas (28%) and carcinomas (27%), harbored genomic variants (n=77) with FDA approved targeted therapies in other tumor types. The most frequently immunogenic CUP tumors were carcinomas, showing high RNA-seq expression of 26/36 genes in at least 20% of patients, most represented by CD20, CD27, TLR8, and PD-L1. High expression of CD40, CSF1R, TIM3, and VISTA was most common in adenocarcinomas. Squamous cell carcinomas were relatively immunogenic, with frequent high expression of 17/36 immune genes, uniquely including MAGEA4. Neuroendocrine tumors were the least immunogenic, with frequent high expression in only 4/36 genes, including ADORA2A (42%) and MAGEA1 (37%).ConclusionsCUP tumors diversely express both standard marker and novel immunotherapeutic targets based on histology and may benefit from selective access to clinical trials for these therapies.ReferencesNational Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) Occult Primary (Cancer of Unknown Primary [CUP]), Version 2.2021. Fort Washington, Pennsylvania: National Comprehensive Cancer Network; 2021. https://www.nccn.org/professionals/physician_gls/pdf/occult.pdf.Laprovitera N, Riefolo M, Ambrosini E, Klec C, Pichler M, Ferracin M. Cancer of unknown primary: Challenges and progress in clinical management. Cancers (Basel) 2021;13(3):1–30. doi:10.3390/cancers130304513.Naing A, Meric-Bernstam F, Stephen B, et al. Phase 2 study of pembrolizumab in patients with advanced rare cancers. J Immunother Cancer 2020;8(1):e000347. doi:10.1136/jitc-2019-0003474.Conroy JM, Pabla S, Glenn ST, et al. Analytical validation of a next-generation sequencing assay to monitor immune responses in solid tumors. J Mol Diagnostics 2018;20(1):95–109. doi:10.1016/j.jmoldx.2017.10.001

Objective. Secretory carcinoma is a recently described entity with characteristic immunoprofile and ETV6 (12p13) rearrangement. Before its initial description, it was generally diagnosed as acinic cell carcinoma (ACCi). We evaluated immunoprofile and ETV6 rearrangement in cytological and surgical cases of previously diagnosed ACCi, in an attempt to identify any misclassified SC. Methods. Fifteen cytology and surgical cases of ACCi diagnosed over a 13-year period were retrieved and subjected to immunohistochemistry for S-100, mammaglobin, GATA-3 and DOG-1 as well as FISH for ETV6 (12p13). Results. Of the 8 cytology cases, only 1 was positive for S100, GATA-3, and mammaglobin, and negative for DOG-1. It also demonstrated ETV6 rearrangement and was reclassified as SC. The same immunoprofile was present in 2 of the 13 surgical cases. ETV6 rearrangement characterized by 3′ interstitial deletion was detected in one of these cases and was reclassified as SC. Immunohistochemistry and ETV6 rearrangement were useful in identifying 2 (13.3%) cases misclassified as ACCi. Conclusions. Characteristic immunoprofile and ETV6 gene rearrangement may prove useful in identifying cases of SC. The presence of ETV6 3′ interstitial deletion in one of our cases suggests that there may be additional ETV6 related genetic alterations contributing to the pathogenesis of SC.

We have developed two linear-dendritic telodendrimers (TDs) with rational design using amphiphilic riboflavin (Rf) as building blocks for efficient doxorubicin (DOX) delivery. Micellar TD nanoparticles (NPs) are composed of a hydrophilic polyethylene glycol (PEG) shell and a Rf-containing affinitive core for DOX encapsulation. Strong DOX-Rf interactions and amphiphilic Rf structure render these nanocarriers with an ultra-high DOX loading capacity (>1/1, DOX/TD, w/w), ∼100% loading efficiency, the sustained drug release and the optimal particle sizes (20–40 nm) for efficient tumor-targeted drug delivery. These nanoformulations significantly prolonged DOX circulation time in the blood without the accelerated clearance observed after multiple injections. DOX-TDs target several types of tumors efficiently in vivo , e.g. Raji lymphoma, MDA-MB-231 breast cancer, and SKOV-3 ovarian cancer. In vivo maximum tolerated dose (MTD) of DOX was increased by 2–2.5 folds for the nanoformulations in mice relative to those of free DOX and Doxil ® . These nanoformulations significantly inhibited tumor growth and prolonged survival of mice bearing SKOV-3 ovarian cancer xenografts. In summary, Rf-containing nanoformulations with high DOX loading capacity, improved stability and efficient tumor targeting lead to superior antitumor efficacy, which merit the further development for clinical application.

Dystroglycanopathies are a group of congenital muscular dystrophies (CMD) often caused by mutations in genes encoding glycosyltransferases that lead to hypoglycosylation of α-dystroglycan (α-DG) and reduce its extracellular matrix-binding activity. Overexpressing LARGE (formerly known as like-glycosyltransferase) generates an extracellular matrix-binding carbohydrate epitope in cells with CMD-causing mutations in not only LARGE but also other glycosyltransferases, including POMT1, POMGnT1, and fukutin, creating the possibilities of a one-for-all gene therapy. To determine the feasibility of LARGE gene therapy, a serotype 9 adeno-associated viral vector for overexpressing LARGE (AAV9-LARGE) was injected intracardially into newborns of two mouse models of CMD: the natural LARGE mutant Large(myd) mice and protein O-mannose N-acetylglucosaminyltransferase 1 (POMGnT1) knockout mice. AAV9-LARGE virus treatment yielded partial restoration of α-DG glycosylation and ligand-binding activity. The muscular dystrophy phenotype in skeletal muscles was ameliorated as revealed by significantly reduced fibrosis, necrosis, and numbers of centrally located nuclei with improved motor function. These results indicate that LARGE overexpression in vivo by AAV9-mediated gene therapy is effective at restoring functional glycosylation of α-DG and rescuing the muscular dystrophy phenotype in deficiency of not only LARGE but also POMGnT1, providing evidence that in vivo LARGE gene therapy may be broadly useful in dystroglycanopathies.

Objective. Secretory carcinoma is a recently described entity with characteristic immunoprofile and ETV6 (12p13) rearrangement. Before its initial description, it was generally diagnosed as acinic cell carcinoma (ACCi). We evaluated immunoprofile and ETV6 rearrangement in cytological and surgical cases of previously diagnosed ACCi, in an attempt to identify any misclassified SC. Methods. Fifteen cytology and surgical cases of ACCi diagnosed over a 13-year period were retrieved and subjected to immunohistochemistry for S-100, mammaglobin, GATA-3 and DOG-1 as well as FISH for ETV6 (12p13). Results. Of the 8 cytology cases, only 1 was positive for S100, GATA-3, and mammaglobin, and negative for DOG-1. It also demonstrated ETV6 rearrangement and was reclassified as SC. The same immunoprofile was present in 2 of the 13 surgical cases. ETV6 rearrangement characterized by 3′ interstitial deletion was detected in one of these cases and was reclassified as SC. Immunohistochemistry and ETV6 rearrangement were useful in identifying 2 (13.3%) cases misclassified as ACCi. Conclusions. Characteristic immunoprofile and ETV6 gene rearrangement may prove useful in identifying cases of SC. The presence of ETV6 3′ interstitial deletion in one of our cases suggests that there may be additional ETV6 related genetic alterations contributing to the pathogenesis of SC.

The characteristic immunoprofile for the diagnosis of synovial sarcoma, a neoplasm of unclear tissue origin, is expression of transducer-like enhancer of split 1 (TLE-1), CD99, partial expression of cytokeratin, and epithelial membrane antigen by immunohistochemistry (IHC). Diagnostic dilemma or misdiagnosis can occur due to overlap in IHC and morphology with carcinomas, and particularly poorly differentiated and metastatic tumors. The frequency of TLE-1 and CD99 expression in carcinomas by IHC has not been previously assessed. We evaluated TLE-1 and CD99 expression in various carcinomas and evaluated the expression of the SS18 (SYT) gene rearrangement (a characteristic biomarker for synovial sarcoma) in tumors with TLE-1 and/or CD99 expression. Immunostains of TLE-1 and CD99 were performed in 100 various carcinomas. Seven of the 98 cases (7%) of carcinomas showed TLE-1 expression, including 1 each of prostate adenocarcinoma (ADCA), esophageal ADCA, basal cell carcinoma, adrenocortical carcinoma, endometrial ADCA, ovarian serous carcinoma, and small cell carcinoma. Twenty-one of the 100 cases (21%) of carcinomas demonstrated CD99 expression, including 6 prostate ADCA, 3 esophageal ADCA, 5 squamous cell carcinomas, 2 hepatocellular carcinomas, 1 each for endometrial ADCA, renal cell carcinoma, urothelial cell carcinoma, neuroendocrine carcinoma, and mucoepidermoid carcinoma. An esophageal ADCA was positive for both TLE-1 and CD99. None of the carcinomas with positive TLE-1 (n=7) or CD99 (n=21) by IHC showed SS18 gene rearrangement by fluorescent in situ hybridization. TLE-1 and CD99 expression were identified in 7% and 21% of carcinomas, respectively. This is a potential pitfall in the IHC interpretation for diagnosis of synovial sarcoma. SS18 gene rearrangement by fluorescent in situ hybridization is helpful for the diagnostically challenging cases, either for confirmation or exclusion of synovial sarcoma.

For the multi rotor aircraft with high model order, a control strategy based on dynamic surface back-stepping is putted forward to solve the problem that the derivation of the virtual control input signal is complicated when conventional back-stepping approach is used. First, the mathematical model of multi rotor aircraft is established. Then, dynamic surface back-stepping that the low pass filter is introduced into the controller design to solve the problem of computational expansion is presented, and the Lyapunov stability analysis of the designed controller shows that it is stable and exponentially convergent. Finally, simulation results show that this approach is superior to the conventional back-stepping approach.

We report a soft tissue sarcoma from the thigh with morphologic features resembling Ewing sarcoma, clear cell sarcoma, and myoepithelial tumor of soft tissue. In addition, the genetic and immunohistochemical findings do not correspond to any established pattern, so the tumor does not clearly fit into any one classification. The karyotype analysis revealed a rare chromosomal rearrangement, t(6;22)(p22;q12), that previously has been reported in bone and epithelial tumors. Molecular studies confirmed the presence of an EWSR1–POU5F1 fusion creating a chimeric gene with the N-terminal transcriptional activation domain of EWSR1 and the C-terminal POU DNA binding domain of POU5F1 . This report is novel in that to our knowledge, it is the first complete molecular characterization of an EWSR1–POU5F1 fusion in a soft tissue sarcoma. Evaluation of existing data on the known EWSR1–POU5F1 tumors suggests that the fusion gene functions in a wide variety of cell types and may modify the differentiation state of cells, resulting in susceptibility to tumorigenesis.

An association between Hashimoto thyroiditis and papillary thyroid carcinoma has been postulated for decades. We undertook this study to identify potential precursors of papillary thyroid carcinoma in Hashimoto thyroiditis using a combination of morphologic, immunohistochemical, and molecular techniques. For the study, samples from 59 cases of Hashimoto thyroiditis were stained with antibodies to HBME1 and cytokeratin (CK)19. Tiny HBME1+ and CK19+ atypical cell clusters were identified and analyzed for the BRAF mutation by the colorimetric Mutector assay and allele-specific polymerase chain reaction. HBME1+ and CK19+ atypical cell clusters were identified in 12 (20%) of 59 cases. The minute size (

HER2 gene amplification by fluorescence in situ hybridization and protein expression by immunohistochemistry (IHC) have been used for prognosis and guiding treatment of invasive ductal carcinoma of the breast with trastuzumab. Accurate evaluation of HER2 status is important in the management of patients with candidacy for the HER2-targeting therapy. Despite previous studies, effects of polysomy of chromosome 17, at which HER2 is located, on HER2 protein expression remains controversial. In this study, we calculated the average copy numbers of HER2 and chromosome 17 (CEP17) per nucleus in 109 cases of invasive breast carcinoma and analyzed their correlations with the HER2/CEP17 ratio and protein expression. As expected, there were close correlations between HER2 protein expression and the HER2/CEP17 ratio (CC: 0.49, P0.001), along with the HER2 copy number per nucleus (CC: 0.48, P0.001) and between the CEP17 copy number per nucleus and the HER2 copy number per nucleus (CC: 0.45, P0.001). Correlation between the CEP17 copy number per nucleus and the HER2/CEP17 ratio was not significant (CC: 0.2, P0.05). There was a weak, but statistically insignificant, correlation between the CEP17 copy number per nucleus and HER2 protein expression by IHC (CC: 0.26, P0.05). The cases were then grouped on the basis of the amplification of the HER2 gene by fluorescence in situ hybridization. In the cases that showed no amplification, there was a significantly higher CEP17 copy number per nucleus in cases with strong HER2 protein expression (2.99) when compared with cases with weak (2.39) or absent (1.86) expression. In conclusion, a high HER2 gene copy number-associated polysomy 17 is a significant contributing factor in HER2 protein overexpression in unamplified invasive breast carcinomas. Cases without HER2 amplification, but carrying polysomy 17, should be further evaluated for HER2 protein overexpression by IHC. Those with polysomy 17-associated HER2 protein overexpression, like any other IHC+ ones, should be eligible candidates for trastuzumab therapy.

Object Head trauma is a dynamic process characterized by a cascade of metabolic and molecular events. Erythropoietin (EPO) has been shown to have neuroprotective effects in animal models of traumatic brain injury (TBI). Acute in vivo mechanisms and pathological changes associated with EPO following TBI are unknown. In this study the authors compare acute metabolic and pathological changes following TBI with and without systemically administered EPO. Methods Right frontal lobe microdialysis cannulae and right parietal lobe percussion hubs were inserted into 16 Sprague–Dawley rats. After a 4- to 5-day recovery, TBI was induced via a DragonFly fluid-percussion device at 2.5–2.8 atm. Rats were randomized into 2 groups, which received 5000 U/kg EPO or normal saline intraperitoneally 30 minutes after TBI. Microdialysis samples for glucose, lactate, pyruvate, and glutamate were obtained every 25 minutes for 10 hours. Rats were killed, their brains processed for light microscopy, and sections stained with H & E. Results Erythropoietin administered 30 minutes after TBI directly affects acute brain metabolism. Brains treated with EPO maintain higher levels of glucose 4–10 hours after TBI (p < 0.01), lower levels of lactate 6–10 hours after TBI (p < 0.01), and lower levels of pyruvate 7.5–10 hours after TBI (p < 0.01) compared with saline-treated controls. Erythropoietin maintains aerobic metabolism after TBI. Systemic EPO administration reduces acute TBI-induced lesion volume (p < 0.05). Conclusions Following TBI, neuron use initially increases, with subsequent depletion of extracellular glucose, resulting in increased levels of extracellular lactate and pyruvate. This energy requirement can result in cell death due to increased metabolic demands. These data suggest that the neuroprotective effect of EPO may be partially due to improved energy metabolism in the acute phase in this rat model of TBI.

BRAF V600E is a mutation present in numerous neoplasms, including melanomas, thyroid, colorectal and ovarian carcinomas, gastrointestinal stromal tumors, and Langerhans’ cell histiocytosis. Vemurafenib, a BRAF V600E kinase inhibitor has been successfully used in the treatment of melanoma. The role of this mutation in unclassified high-grade sarcomas, malignancies with very limited treatment options, has not been widely studied. Because of the availability of a highly sensitive and specific antibody (VE1) against the BRAF V600E mutant protein, we tested 48 cases of unclassified high-grade sarcomas. Cytoplasmic expression intensity was graded as negative (0), or positive (2+ or 3+) by two pathologists and a pathology resident. Forty one out of 48 specimens remained intact in the cores after immunohistochemistry (IHC) processing. Six of the 41 cases (15%) were scored as positive. In addition, non-specific nuclear staining was detected in 12/88 cores (14%). The 6 positive cases were tested for the BRAF V600E mutation by RT-PCR, and all were negative. Based on these results, we concluded that BRAF V600E mutation is rare in unclassified high-grade sarcomas, and because of the non-specific staining, results should be interpreted with caution.

Papillary thyroid carcinoma and its variants can be difficult to distinguish from cellular adenomatous nodules. Prior studies have advocated various antibodies to aid in the differential diagnosis, but there is little agreement on their utility. We undertook this study to evaluate immunohistochemical markers in the diagnosis and differential diagnosis of papillary thyroid carcinoma. Ten cases of papillary thyroid carcinoma were initially stained for HBME1, CK19, fibronectin1, Ki-67, Calretinin, p16, SFTPB and CITED1. Additionally, two previously untested antibodies to molecules that have been found to be upregulated in papillary thyroid carcinoma (CST6 and EPS8) were also evaluated. Of these, only HBME1, CK19 and fibronectin1 showed diagnostic utility. These three markers were then further evaluated in 51 papillary thyroid carcinomas and 57 benign thyroids. HBME1 was the most sensitive and specific marker, staining 49/51 papillary thyroid carcinomas and only 4/57 benign thyroids. CK19 was equally sensitive staining all 51 papillary thyroid carcinomas, but it was nonspecific staining 39 of 57 benign thyroids. A negative result, however, was helpful in excluding papillary thyroid carcinoma. Fibronectin1 was positive in 35/51 papillary thyroid carcinomas (69%) and 4/57 (7%) benign thyroids, but its utility was hampered by high background staining. These findings suggest that the combination of HBME1 and CK19 has the greatest diagnostic utility in the differentiation of papillary thyroid carcinoma from its benign mimics.

Blood group-related antigens have been attractive targets for immunotherapy of cancer since their initial identification as cancer-related antigens. However, available information on the relative expression of most of these antigens on human malignant and normal tissues has been insufficient for selecting optimal antigens and tumors for immune attack. In this study, the distribution of the blood group-related antigens TF, Tn, sTn, Le(a), sialyl Le(a), Le(b), Le(x), sialyl Le(x), polyfucosyl Le(x) and Le(y) on 13 types of cancer and 16 normal tissues was compared. Our results show that sTn is strongly expressed on cancers of breast, colon, stomach, ovary, prostate and uterus; Tn on prostate cancer; TF on cancers of breast, colon, ovary, prostate and uterus; Le(y) on the cancers of colon, lung, pancreas and ovary; Le(a) and Le(x) on gastric cancer; and sialyl Le(a) and sialyl Le(x) on colon cancer. The complete absence of these antigens on cancers of neuroectodermal or mesodermal origin including melanoma, sarcoma, neuroblastoma and B cell lymphoma is as striking as their widespread presence on tumors of epithelial origin. Normal tissues were also tested. Tn and Le(b) were only detected on gastric and ovarian epithelia; sTn on Leydig cells of testis in addition to gastric and ovarian epithelia; Le(x) and sialyl Le(x) on polymorphonuclear leukocytes; and TF, Le(a), sialyl Le(a), Le(x), sialyl Le(x), polyfucosyl Le(x) and Le(y) on epithelia from a variety of tissues.