Background
Immunotherapy with immune checkpoint inhibitors (ICIs) is highly effective in microsatellite instability–high (MSI‐H) metastatic colorectal cancer (mCRC); however, specific predictive ...biomarkers are lacking.
Patients and Methods
Data and samples from 85 patients with MSI‐H mCRC treated with ICIs were gathered. Tumor infiltrating lymphocytes (TILs) and tumor mutational burden (TMB) were analyzed in an exploratory cohort of “super” responders and “clearly” refractory patients; TILs were then evaluated in the whole cohort of patients. Primary objectives were the correlation between the number of TILs and TMB and their role as biomarkers of ICI efficacy. Main endpoints included response rate (RR), progression‐free survival (PFS), and overall survival (OS).
Results
In the exploratory cohort, an increasing number of TILs correlated to higher TMB (Pearson's test, p = .0429). In the whole cohort, median number of TILs was 3.6 in responders compared with 1.8 in nonresponders (Mann‐Whitney test, p = .0448). RR was 70.6% in patients with high number of TILs (TILs‐H) compared with 42.9% in patients with low number of TILs (odds ratio = 3.20, p = .0291). Survival outcomes differed significantly in favor of TILs‐H (PFS: hazard ratio HR = 0.42, p = .0278; OS: HR = 0.41, p = .0463).
Conclusion
A significant correlation between higher TMB and increased number of TILs was shown. A significantly higher activity and better PFS and OS with ICI in MSI‐H mCRC were reported in cases with high number of TILs, thus supporting further studies of TIL count as predictive biomarker of ICI efficacy.
Implications for Practice
Microsatellite instability is the result of mismatch repair protein deficiency, caused by germline mutations or somatic modifications in mismatch repair genes. In metastatic colorectal cancer (mCRC), immunotherapy (with immune checkpoint inhibitors ICIs) demonstrated remarkable clinical benefit in microsatellite instability–high (MSI‐H) patients. ICI primary resistance has been observed in approximately 25% of patients with MSI‐H mCRC, underlining the need for predictive biomarkers. In this study, tumor mutational burden (TMB) and tumor infiltrating lymphocyte (TIL) analyses were performed in an exploratory cohort of patients with MSI‐H mCRC treated with ICIs, demonstrating a significant correlation between higher TMB and increased number of TILs. Results also demonstrated a significant correlation between high number of TILs and clinical responses and survival benefit in a large data set of patients with MSI‐H mCRC treated with ICI. TMB and TILs could represent predictive biomarkers of ICI efficacy in MSI‐H mCRC and should be incorporated in future trials testing checkpoint inhibitors in colorectal cancer.
This article explores the role of tumor mutational burden and number of tumor infiltrating lymphocytes in determining response and outcome of patients with microsatellite high metastatic colorectal cancer treated with immune checkpoint inhibitors.
Perturbations in the transcriptional programs specifying epidermal differentiation cause diverse skin pathologies ranging from impaired barrier function to inflammatory skin disease. However, the ...global scope and organization of this complex cellular program remain undefined. Here we report single-cell RNA sequencing profiles of 92,889 human epidermal cells from 9 normal and 3 inflamed skin samples. Transcriptomics-derived keratinocyte subpopulations reflect classic epidermal strata but also sharply compartmentalize epithelial functions such as cell-cell communication, inflammation, and WNT pathway modulation. In keratinocytes, ∼12% of assessed transcript expression varies in coordinate patterns, revealing undescribed gene expression programs governing epidermal homeostasis. We also identify molecular fingerprints of inflammatory skin states, including S100 activation in the interfollicular epidermis of normal scalp, enrichment of a CD1C+CD301A+ myeloid dendritic cell population in psoriatic epidermis, and IL1βhiCCL3hiCD14+ monocyte-derived macrophages enriched in foreskin. This compendium of RNA profiles provides a critical step toward elucidating epidermal diseases of development, differentiation, and inflammation.
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•Stereotyped keratinocyte subpopulations modularly comprise human epidermis•Scalp keratinocytes exhibit an inherent inflammatory transcriptional program•Myeloid dendritic cells predominate APCs in psoriatic epidermis•Macrophages represent major APCs in foreskin epidermis
Cheng et al. report single-cell RNA sequencing of normal and inflamed human epidermis, revealing a discrete set of specialized keratinocytes that exhibit a distinct composition at different anatomic sites. Myeloid dendritic cells and macrophages also vary sharply with epidermal anatomic site and inflammation, indicating dynamic programming of antigen-presenting cells.
Sebaceous carcinomas (SeC) are cutaneous malignancies that, in rare cases, metastasize and prove fatal. Here we report whole-exome sequencing on 32 SeC, revealing distinct mutational classes that ...explain both cancer ontogeny and clinical course. A UV-damage signature predominates in 10/32 samples, while nine show microsatellite instability (MSI) profiles. UV-damage SeC exhibited poorly differentiated, infiltrative histopathology compared to MSI signature SeC (p = 0.003), features previously associated with dissemination. Moreover, UV-damage SeC transcriptomes and anatomic distribution closely resemble those of cutaneous squamous cell carcinomas (SCC), implicating sun-exposed keratinocytes as a cell of origin. Like SCC, this UV-damage subclass harbors a high somatic mutation burden with >50 mutations per Mb, predicting immunotherapeutic response. In contrast, ocular SeC acquires far fewer mutations without a dominant signature, but show frequent truncations in the ZNF750 epidermal differentiation regulator. Our data exemplify how different mutational processes convergently drive histopathologically related but clinically distinct cancers.
BackgroundHepatitis B virus (HBV) infection is associated with approximately 25% of liver cancer cases in the US and 56% worldwide. Low rates of HBV vaccination in people 50 years or older combined ...with the fact that HBV infection is highest amongst the 40–49 year old demographic (i.e. individuals born prior to 1986) is a cause for concern as the median age for liver cancer diagnosis is 66 years of age. Despite the impact of immunotherapies in liver cancer over the past decade, durable responses remain elusive. Our approach to significantly improving outcomes is to activate and mobilize tissue-resident and systemic cancer-specific T effector memory cells (TEM), especially for the treatment of cancer in organs with low T cell count such as the liver.MethodsWe synthesized a novel capsid-modified adenovirus, SynBAd, for use as a therapeutic cancer vaccine targeting systemic immunity without causing commonly-reported adenovirus-mediated hepatotoxicity. We further engineered SynBAd to express a polytope of HBV antigens to activate the elimination of HBV+ tumor cells. SynBAd, administered intravenously (IV) for systemic and liver-specific T cell activation, was evaluated in a tumor-protection mouse model of liver cancer.ResultsIV administration of SynBAd expressing a polytope comprised of HBV epitopes significantly extended the lives of mice with a liver-localized tumor bearing the same epitopes in contrast to control animals. Moreover, recipients of SynBAd expressing an irrelevant antigen did not demonstrate comparable levels of protection. SynBAd further induced antigen specific CD8 T cell immunity in mice and a population of effector memory T cells at levels equivalent with those induced by human Adenovirus serotype 5 (Ad5). The virus predominantly localized to the liver and spleen and induced antigen-specific T cells though more robustly in the latter than Ad5. Although SynBAd is a potent immunogen, antibodies generated in mice immunized with Ad5 only weakly neutralized SynBAd infection in vitro, suggesting that preexisting Ad5 antibodies will not dramatically influence SynBAd efficacy.ConclusionsThe model used is representative of colon metastases to the liver as well as HBV-driven hepatocellular carcinoma. Localization of the vaccine after administration, activation of antigen specific T cells, mobilization of effector memory T cells, and efficacy in the liver cancer model suggest that this platform promotes a systemic tumor controlling immune response with wide-ranging clinical potential beyond liver cancer.
BackgroundCombination immune therapy has demonstrated curative potential in multi-agent pre-clinical studies and early clinical studies. We have created a single-agent combination immune therapy ...platform, dubbed virus-like DNA (VLD), to activate multiple anti-cancer immune signals leveraging the robust T cell responses elicited by viral infection. VLD was designed to avoid neutralizing antibody responses, immunotherapy-mediated toxicities, and extensive manufacturing cost and time. VLD comprises of a) antigen, composed of a polytope selected based on genomic variants to activate CD4+ and CD8+ T cells, b) a proprietary protein that activates three independent T helper 1 and cytotoxic T cell signaling pathways, and c) a proprietary dual CTLA-4/PD-1 engaging agent. We hypothesized that a rationally-designed combination of different classes of immune modulating factors will elicit tumor antigen-specific effector T cell responses and eradicate established tumors.MethodsMice were implanted with tumor cells prior to treatment with VLD. VLD expressing immunomodulators and predicted T cell epitopes was administered intravenously to tumor-bearing mice. Tumor measurements and animal health were monitored for the ensuing 2 months, upon which, surviving animals were rechallenged with tumor cells.ResultsIntravenous administration of VLD to animals with established tumors completely eradicated or significantly delayed tumors in recipient animals. Combining VLD with anti-PD-L1 far outperformed anti-PD-L1 alone which was not significantly better than untreated controls. Furthermore, rechallenge of tumor-free animals with tumor cells demonstrated complete protection 60 days after initial challenge, suggesting VLD-mediated formation of immunological memory. VLD’s combination of immunomodulators uniquely activated splenic effector memory and antigen-reactive CD8+ T cells.ConclusionsThese data suggest that VLD provides a scalable, modular, rapid, robust, and cost-effective means of inducing a durable anti-tumor host response by harnessing critical components of vaccines and gene therapies into one immunogene product to eradicate cancer by activating antigen-reactive T cells.
Growing evidence indicates a role for the gut microbiota in modulating anti-tumor treatment efficacy in human cancer. Here we study mucosa-associated invariant T (MAIT) cells to look for evidence of ...bacterial antigen recognition in human colon, lung, and kidney carcinomas. Using mass cytometry and single-cell mRNA sequencing, we identify a tumor-infiltrating MAIT cell subset expressing CD4 and Foxp3 and observe high expression of CD39 on MAIT cells from colorectal cancer (CRC) only, which we show in vitro to be expressed specifically after TCR stimulation. We further reveal that these cells are phenotypically and functionally exhausted. Sequencing data show high bacterial infiltration in CRC tumors and highlight an enriched species, Fusobacteria nucleatum, with capability to activate MAIT cells in a TCR-dependent way. Our results provide evidence of a MAIT cell response to microbial antigens in CRC and could pave the way for manipulating MAIT cells or the microbiome for cancer therapy.
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Comprehensive profiling of tumor-infiltrating MAIT cells in colorectal cancer (CRC)Identification of a tumor-infiltrating MAIT cell subset expressing CD4 and Foxp3TCR-driven CD39 expression is enriched in tumor-infiltrating MAIT cellsCRC-related Fusobacterium nucleatum can activate MAIT cells in a TCR-dependent way
By investigating human colon, lung, and kidney carcinomas, Li et al. propose MAIT cells as part of the missing link between the gut microbiota and tumor biology. This work will open new avenues in cancer therapy by manipulating MAIT cells or MAIT-related bacterial ligands in the context of microbiota-influenced cancers.
Abstract
We engineered a novel adenovirus with a modified capsid for use as an integrated anticancer gene therapy and to circumvent pre-existing adenovirus immunity. The modified adenovirus, SynBAd, ...when administered intravenously, induced antigen-specific CD8 T cell immunity in the liver of mice (C57BL/6) and a splenic population of effector memory T cells at levels equivalent with those induced by human Adenovirus serotype 5 (Ad5). Although the virus elicited equivalent liver and splenic T cell responses, SynBAd did not elevate liver enzymes as commonly seen with Ad5. SynBAd bound factor X with similar affinity to Ad5, though it localized preferentially to the spleen relative to Ad5, suggesting greater systemic immune stimulation. CD8 T cells in the liver and spleen increased with SynBAd administration, indicating that the virus acts as a potent mobilizer of T cells. Although SynBAd is a potent immunogen, antibodies generated in mice immunized with Ad5 only weakly neutralized SynBAd infection in vitro, suggesting that preexisting Ad5 antibodies will not dramatically influence SynBAd efficacy. Localization of the adenovirus after administration, activation of antigen specific T cells, and mobilization of effector memory T cells, suggest that this approach will promote an antitumor immune response that could be used in multiple tumor indications.
Transcriptome profiling can provide information of great value in clinical decision-making, yet RNA from readily available formalin-fixed paraffin-embedded (FFPE) tissue is often too degraded for ...quality sequencing. To assess the clinical utility of FFPE-derived RNA, we performed ribo-deplete RNA extractions on > 3200 FFPE slide samples; 25 of these had direct FFPE vs. fresh frozen (FF) replicates, 57 were sequenced in 2 different labs, 87 underwent multiple library analyses, and 16 had direct microdissected vs. macrodissected replicates. Poly-A versus ribo-depletion RNA extraction methods were compared using transcriptomes of TCGA cohort and 3116 FFPE samples. Compared to FF, FFPE transcripts coding for nuclear/cytoplasmic proteins involved in DNA packaging, replication, and protein synthesis were detected at lower rates and zinc finger family transcripts were of poorer quality. The greatest difference in extraction methods was in histone transcripts which typically lack poly-A tails. Encouragingly, the overall sequencing success rate was 81%. Exome coverage was highly concordant in direct FFPE and FF replicates, with 98% agreement in coding exon coverage and a median correlation of whole transcriptome profiles of 0.95. We provide strong rationale for clinical use of FFPE-derived RNA based on the robustness, reproducibility, and consistency of whole transcriptome profiling.
Abstract
Background: We evaluate the feasibility, quality, and analytical potential of whole-transcriptome RNA-seq on over one thousand clinical FFPE tumor samples. Transcriptional profiling of RNA ...is used for clinical decision making in many tumor types. Clinical analysis of RNA is complicated by the common use of formalin-fixed paraffin-embedded (FFPE) tissue storage, which can cause low yield and RNA degradation.
Methods: RNA was extracted from FFPE material using commercially available kits using an RNAseH based ribodeplete. Multiple libraries per sample are sequenced using standard Illumina sequencing. Bowtie2, RSEM, and custom software are used for alignment, transcript quantification, fusion detection, and variant expression analysis.
Results: We observe a >85% success rate on whole transcriptome RNA-seq on our cohort of more than 1000 samples. We find reliable transcript quantification upon successful sequencing, and on a subset of samples quantify differences between FFPE and fresh-frozen material when using ribodeplete. We also compare transcriptional profiles of clinical FFPE samples to an independent set of fresh frozen, poly-A capture samples from The Cancer Genome Atlas (TCGA) and show differences between poly-A capture and ribodeplete RNA isolation methods as well as FFPE vs. FF effects. We present a robust mapping methodology for comparison of public FF poly-A dataset to our FFPE ribodeplete samples. We demonstrate that we are able to utilize this joint gene expression space to perform site of origin prediction on FFPE samples. This is especially important for clinical application in analyzing Cancer of Unknown Primary (CUP) samples as well as detecting outlier samples whose molecular features may suggest additional therapeutic avenues. Finally, we show reliable detection of fusion transcripts from FFPE RNA-seq material from whole-transcriptome analysis and expression of somatic tumor variants detected from DNA sequencing.
Conclusions: Large scale sequencing of RNA from clinical FFPE materials provides reliable transcriptomic results comparable to existing public databases of RNA, enabling research on cohorts of tumors that are FFPE-banked and unavailable as frozen tissue.
Citation Format: Yulia Newton, Justin Golovato, Mark Johnson, Shahrooz Rabizadeh, Zack Sanborn, Steve Benz, Charles J. Vaske. Analysis of whole transcriptome RNA-seq of large numbers of clinical FFPE samples abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3398.
Recessive dystrophic epidermolysis bullosa (RDEB) is a rare inherited skin and mucous membrane fragility disorder complicated by early-onset, highly malignant cutaneous squamous cell carcinomas ...(SCCs). The molecular etiology of RDEB SCC, which arises at sites of sustained tissue damage, is unknown. We performed detailed molecular analysis using whole-exome, whole-genome, and RNA sequencing of 27 RDEB SCC tumors, including multiple tumors from the same patient and multiple regions from five individual tumors. We report that driver mutations were shared with spontaneous, ultraviolet (UV) light-induced cutaneous SCC (UV SCC) and head and neck SCC (HNSCC) and did not explain the early presentation or aggressive nature of RDEB SCC. Instead, endogenous mutation processes associated with apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) deaminases dominated RDEB SCC. APOBEC mutation signatures were enhanced throughout RDEB SCC tumor evolution, relative to spontaneous UV SCC and HNSCC mutation profiles. Sixty-seven percent of RDEB SCC driver mutations was found to emerge as a result of APOBEC and other endogenous mutational processes previously associated with age, potentially explaining a >1000-fold increased incidence and the early onset of these SCCs. Human papillomavirus-negative basal and mesenchymal subtypes of HNSCC harbored enhanced APOBEC mutational signatures and transcriptomes similar to those of RDEB SCC, suggesting that APOBEC deaminases drive other subtypes of SCC. Collectively, these data establish specific mutagenic mechanisms associated with chronic tissue damage. Our findings reveal a cause for cancers arising at sites of persistent inflammation and identify potential therapeutic avenues to treat RDEB SCC.
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