Microbiome modulates immunotherapy response in cutaneous squamous cell carcinoma.

The gut microbiome is increasingly recognized to alter cancer risk, progression and response to treatments such as immunotherapy, especially in cutaneous melanoma. However, whether the microbiome influences immune checkpoint inhibitor (ICI) immunotherapy response to non-melanoma skin cancer has not yet been defined. As squamous cell carcinomas (SCC) are in closest proximity to the skin microbiome, we hypothesized that the skin microbiome, which regulates cutaneous immunity, might affect SCC-associated anti-PD1 immunotherapy treatment response. We used ultraviolet radiation to induce SCC in SKH1 hairless mice. We then treated the mice with broad-band antibiotics to deplete the microbiome, followed by colonisation by candidate skin and gut bacteria or persistent antibiotic treatment, all in parallel with ICI treatment. We longitudinally monitored skin and gut microbiome dynamics by 16S rRNA gene sequencing and tumour burden by periodic tumour measurements and histologic assessment. Our study revealed that antibiotics-induced abrogation of the microbiome reduced the tumour burden, suggesting a functional role of the microbiome in non-melanoma skin cancer therapy response.

Microbiome modulates immunotherapy response in cutaneous squamous cell carcinoma.

The gut microbiome is increasingly recognized to alter cancer risk, progression, and response to treatments such as immunotherapy, especially in cutaneous melanoma. However, whether the microbiome influences immune checkpoint inhibitor (ICI) immunotherapy response to non-melanoma skin cancer has not yet been defined. As squamous cell carcinomas (SCC) are in closest proximity to the skin microbiome, we hypothesized that the skin microbiome, which regulates cutaneous immunity, might affect SCC-associated anti-PD1 immunotherapy treatment response. We used ultraviolet radiation to induce SCC in SKH1 hairless mice. We then treated the mice with broad-band antibiotics to deplete the microbiome, followed by colonization by candidate skin and gut bacteria or persistent antibiotic treatment, all in parallel with ICI treatment. We longitudinally monitored skin and gut microbiome dynamics by 16S rRNA gene sequencing, and tumor burden by periodic tumor measurements and histologic assessment. Our study revealed that antibiotics-induced abrogation of the microbiome reduced tumor burden, suggesting a functional role of the microbiome in non-melanoma skin cancer therapy response.

Associations of the skin, oral and gut microbiome with aging, frailty and infection risk reservoirs in older adults.

Older adults represent a vulnerable population with elevated risk for numerous morbidities. To explore the association of the microbiome with aging and age-related susceptibilities including frailty and infectious disease risk, we conducted a longitudinal study of the skin, oral, and gut microbiota in 47 community- or skilled nursing facility-dwelling older adults vs. younger adults. We found that microbiome changes were not associated with chronological age so much as frailty: we identified prominent changes in microbiome features associated with susceptibility to pathogen colonization and disease risk, including diversity, stability, heterogeneity, and biogeographic determinism, which were moreover associated with a loss of Cutibacterium (C.) acnes in the skin microbiome. Strikingly, the skin microbiota were also the primary reservoir for antimicrobial resistance, clinically important pathobionts, and nosocomial strains, suggesting a potential role particularly for the skin microbiome in disease risk and dissemination of multidrug resistant pathogens.

Skin Microbiome Variation with Cancer Progression in Human Cutaneous Squamous Cell Carcinoma.

The skin microbiome plays a critical role in skin homeostasis and disorders. UVR is the major cause of nonmelanoma skin cancer, but other risk factors, including immune suppression, chronic inflammation, and antibiotic usage, suggest the microbiome as an additional, unexplored risk factor and potential disease biomarker. The overarching goal was to study the skin microbiome in squamous cell carcinoma (SCC) and premalignant actinic keratosis compared with that in healthy skin to identify skin cancer‒associated changes in the skin microbiome. We performed a high-resolution analysis of shotgun metagenomes of actinic keratosis and SCC in healthy skin, revealing the microbial community shifts specific to actinic keratosis and SCC. Most prominently, the relative abundance of pathobiont Staphylococcus aureus was increased at the expense of commensal Cutibacterium acnes in SCC compared with that in healthy skin, and enrichment of functional pathways in SCC reflected this shift. Notably, C. acnes associated with lesional versus healthy skin differed at the strain level, suggesting the specific functional changes associated with its depletion in SCC. Our study revealed a transitional microbial dysbiosis from healthy skin to actinic keratosis to SCC, supporting further investigation of the skin microbiome for use as a biomarker and providing hypotheses for studies investigating how these microbes might influence skin cancer progression.

Cultivation of common bacterial species and strains from human skin, oral, and gut microbiota.

BACKGROUND: Genomics-driven discoveries of microbial species have provided extraordinary insights into the biodiversity of human microbiota. In addition, a significant portion of genetic variation between microbiota exists at the subspecies, or strain, level. High-resolution genomics to investigate species- and strain-level diversity and mechanistic studies, however, rely on the availability of individual microbes from a complex microbial consortia. High-throughput approaches are needed to acquire and identify the significant species- and strain-level diversity present in the oral, skin, and gut microbiome. Here, we describe and validate a streamlined workflow for cultivating dominant bacterial species and strains from the skin, oral, and gut microbiota, informed by metagenomic sequencing, mass spectrometry, and strain profiling. RESULTS: Of total genera discovered by either metagenomic sequencing or culturomics, our cultivation pipeline recovered between 18.1-44.4% of total genera identified. These represented a high proportion of the community composition reconstructed with metagenomic sequencing, ranging from 66.2-95.8% of the relative abundance of the overall community. Fourier-Transform Infrared spectroscopy (FT-IR) was effective in differentiating genetically distinct strains compared with whole-genome sequencing, but was less effective as a proxy for genetic distance. CONCLUSIONS: Use of a streamlined set of conditions selected for cultivation of skin, oral, and gut microbiota facilitates recovery of dominant microbes and their strain variants from a relatively large sample set. FT-IR spectroscopy allows rapid differentiation of strain variants, but these differences are limited in recapitulating genetic distance. Our data highlights the strength of our cultivation and characterization pipeline, which is in throughput, comparisons with high-resolution genomic data, and rapid identification of strain variation.

Meta-analysis of fecal metagenomes reveals global microbial signatures that are specific for colorectal cancer.

Association studies have linked microbiome alterations with many human diseases. However, they have not always reported consistent results, thereby necessitating cross-study comparisons. Here, a meta-analysis of eight geographically and technically diverse fecal shotgun metagenomic studies of colorectal cancer (CRC, n = 768), which was controlled for several confounders, identified a core set of 29 species significantly enriched in CRC metagenomes (false discovery rate (FDR) < 1 × 10-5). CRC signatures derived from single studies maintained their accuracy in other studies. By training on multiple studies, we improved detection accuracy and disease specificity for CRC. Functional analysis of CRC metagenomes revealed enriched protein and mucin catabolism genes and depleted carbohydrate degradation genes. Moreover, we inferred elevated production of secondary bile acids from CRC metagenomes, suggesting a metabolic link between cancer-associated gut microbes and a fat- and meat-rich diet. Through extensive validations, this meta-analysis firmly establishes globally generalizable, predictive taxonomic and functional microbiome CRC signatures as a basis for future diagnostics.

Extensive transmission of microbes along the gastrointestinal tract.

The gastrointestinal tract is abundantly colonized by microbes, yet the translocation of oral species to the intestine is considered a rare aberrant event, and a hallmark of disease. By studying salivary and fecal microbial strain populations of 310 species in 470 individuals from five countries, we found that transmission to, and subsequent colonization of, the large intestine by oral microbes is common and extensive among healthy individuals. We found evidence for a vast majority of oral species to be transferable, with increased levels of transmission in colorectal cancer and rheumatoid arthritis patients and, more generally, for species described as opportunistic pathogens. This establishes the oral cavity as an endogenous reservoir for gut microbial strains, and oral-fecal transmission as an important process that shapes the gastrointestinal microbiome in health and disease.

Tuning of human MAIT cell activation by commensal bacteria species and MR1-dependent T-cell presentation.

Human mucosal-associated invariant T (MAIT) cell receptors (TCRs) recognize bacterial riboflavin pathway metabolites through the MHC class 1-related molecule MR1. However, it is unclear whether MAIT cells discriminate between many species of the human microbiota. To address this, we developed an in vitro functional assay through human T cells engineered for MAIT-TCRs (eMAIT-TCRs) stimulated by MR1-expressing antigen-presenting cells (APCs). We then screened 47 microbiota-associated bacterial species from different phyla for their eMAIT-TCR stimulatory capacities. Only bacterial species that encoded the riboflavin pathway were stimulatory for MAIT-TCRs. Most species that were high stimulators belonged to Bacteroidetes and Proteobacteria phyla, whereas low/non-stimulator species were primarily Actinobacteria or Firmicutes. Activation of MAIT cells by high- vs low-stimulating bacteria also correlated with the level of riboflavin they secreted or after bacterial infection of macrophages. Remarkably, we found that human T-cell subsets can also present riboflavin metabolites to MAIT cells in a MR1-restricted fashion. This T-T cell-mediated signaling also induced IFNγ, TNF and granzyme B from MAIT cells, albeit at lower level than professional APC. These findings suggest that MAIT cells can discriminate and categorize complex human microbiota through computation of TCR signals depending on antigen load and presenting cells, and fine-tune their functional responses.

[Microbial Biomarkers for Early Cancer Detection]. / Mikrobielle Biomarker zur Krebsfrüherkennung.

The human microbiome - the vast amount of microbes that colonize our body - play an important role in maintaining our health. Changes in microbiome composition have been linked to multiple diseases including cancer. Although mechanisms and causalities of these associations still have to be uncovered, microbiome alterations across various stages of disease can be utilized for novel diagnostic and prognostic tests. Research on biomarkers extracted from the gut microbiome has in particular focused on colorectal cancer, where clinical use is already on the horizon. For example, multiple microbial taxonomic markers such as Fusobacterium nucleatum and other oral pathogens have been identified in human feces with potential for non-invasive diagnostics and prognostics. The article summarizes the recent developments, but also limitations and challenges for the development of microbiome-based biomarkers for cancer early detection.

Colorectal Cancer and the Human Gut Microbiome: Reproducibility with Whole-Genome Shotgun Sequencing.

Accumulating evidence indicates that the gut microbiota affects colorectal cancer development, but previous studies have varied in population, technical methods, and associations with cancer. Understanding these variations is needed for comparisons and for potential pooling across studies. Therefore, we performed whole-genome shotgun sequencing on fecal samples from 52 pre-treatment colorectal cancer cases and 52 matched controls from Washington, DC. We compared findings from a previously published 16S rRNA study to the metagenomics-derived taxonomy within the same population. In addition, metagenome-predicted genes, modules, and pathways in the Washington, DC cases and controls were compared to cases and controls recruited in France whose specimens were processed using the same platform. Associations between the presence of fecal Fusobacteria, Fusobacterium, and Porphyromonas with colorectal cancer detected by 16S rRNA were reproduced by metagenomics, whereas higher relative abundance of Clostridia in cancer cases based on 16S rRNA was merely borderline based on metagenomics. This demonstrated that within the same sample set, most, but not all taxonomic associations were seen with both methods. Considering significant cancer associations with the relative abundance of genes, modules, and pathways in a recently published French metagenomics dataset, statistically significant associations in the Washington, DC population were detected for four out of 10 genes, three out of nine modules, and seven out of 17 pathways. In total, colorectal cancer status in the Washington, DC study was associated with 39% of the metagenome-predicted genes, modules, and pathways identified in the French study. More within and between population comparisons are needed to identify sources of variation and disease associations that can be reproduced despite these variations. Future studies should have larger sample sizes or pool data across studies to have sufficient power to detect associations that are reproducible and significant after correction for multiple testing.

Durable coexistence of donor and recipient strains after fecal microbiota transplantation.

Fecal microbiota transplantation (FMT) has shown efficacy in treating recurrent Clostridium difficile infection and is increasingly being applied to other gastrointestinal disorders, yet the fate of native and introduced microbial strains remains largely unknown. To quantify the extent of donor microbiota colonization, we monitored strain populations in fecal samples from a recent FMT study on metabolic syndrome patients using single-nucleotide variants in metagenomes. We found extensive coexistence of donor and recipient strains, persisting 3 months after treatment. Colonization success was greater for conspecific strains than for new species, the latter falling within fluctuation levels observed in healthy individuals over a similar time frame. Furthermore, same-donor recipients displayed varying degrees of microbiota transfer, indicating individual patterns of microbiome resistance and donor-recipient compatibilities.

Temporal and technical variability of human gut metagenomes.

BACKGROUND: Metagenomics has become a prominent approach for exploring the role of the gut microbiota in human health. However, the temporal variability of the healthy gut microbiome has not yet been studied in depth using metagenomics and little is known about the effects of different sampling and preservation approaches. We performed metagenomic analysis on fecal samples from seven subjects collected over a period of up to two years to investigate temporal variability and assess preservation-induced variation, specifically, fresh frozen compared to RNALater. We also monitored short-term disturbances caused by antibiotic treatment and bowel cleansing in one subject. RESULTS: We find that the human gut microbiome is temporally stable and highly personalized at both taxonomic and functional levels. Over multiple time points, samples from the same subject clustered together, even in the context of a large dataset of 888 European and American fecal metagenomes. One exception was observed in an antibiotic intervention case where, more than one year after the treatment, samples did not resemble the pre-treatment state. Clustering was not affected by the preservation method. No species differed significantly in abundance, and only 0.36% of gene families were differentially abundant between preservation methods. CONCLUSIONS: Technical variability is small compared to the temporal variability of an unperturbed gut microbiome, which in turn is much smaller than the observed between-subject variability. Thus, short-term preservation of fecal samples in RNALater is an appropriate and cost-effective alternative to freezing of fecal samples for metagenomic studies.

Association of high CD4-positive T cell infiltration with mutations in HLA class II-regulatory genes in microsatellite-unstable colorectal cancer.

Besides being expressed on professional antigen-presenting cells, HLA class II antigens are expressed on various tumors of non-lymphoid origin, including a subset of colorectal cancers (CRC). Information about the regulation of HLA class II antigen expression is important for a better understanding of their role in the interactions between tumor and immune cells. Whether lack of HLA class II antigen expression in tumors reflects the selective immune destruction of HLA class II antigen-expressing tumor cells is unknown. To address this question, we tested whether lack of HLA class II antigen expression in CRC was associated with immune cell infiltration. We selected microsatellite-unstable (MSI-H) CRC, because they show pronounced tumor antigen-specific immune responses and, in a subset of tumors, lack of HLA class II antigen expression due to mutations inactivating HLA class II-regulatory genes. We examined HLA class II antigen expression, mutations in regulatory genes, and CD4-positive T cell infiltration in 69 MSI-H CRC lesions. Mutations in RFX5, CIITA, and RFXAP were found in 13 (28.9%), 3 (6.7%), and 1 (2.2%) out of 45 HLA class II antigen-negative tumors. CD4-positive tumor-infiltrating lymphocyte counts were significantly higher in HLA class II antigen-negative tumors harboring mutations in HLA class II-regulatory genes (107.4 T cells per 0.25 mm(2)) compared to tumors without mutations (55.5 T cells per 0.25 mm(2), p = 0.008). Our results suggest that the outgrowth of tumor cells lacking HLA class II antigen expression due to mutations of regulatory genes is favored in an environment of dense CD4-positive T cell infiltration.

Potential of fecal microbiota for early-stage detection of colorectal cancer.

Several bacterial species have been implicated in the development of colorectal carcinoma (CRC), but CRC-associated changes of fecal microbiota and their potential for cancer screening remain to be explored. Here, we used metagenomic sequencing of fecal samples to identify taxonomic markers that distinguished CRC patients from tumor-free controls in a study population of 156 participants. Accuracy of metagenomic CRC detection was similar to the standard fecal occult blood test (FOBT) and when both approaches were combined, sensitivity improved > 45% relative to the FOBT, while maintaining its specificity. Accuracy of metagenomic CRC detection did not differ significantly between early- and late-stage cancer and could be validated in independent patient and control populations (N = 335) from different countries. CRC-associated changes in the fecal microbiome at least partially reflected microbial community composition at the tumor itself, indicating that observed gene pool differences may reveal tumor-related host-microbe interactions. Indeed, we deduced a metabolic shift from fiber degradation in controls to utilization of host carbohydrates and amino acids in CRC patients, accompanied by an increase of lipopolysaccharide metabolism.

Prevalence of mismatch repair-deficient crypt foci in Lynch syndrome: a pathological study.

BACKGROUND: Lynch syndrome is an inherited tumour predisposition syndrome caused by germline mutations of DNA mismatch repair (MMR) genes. Mutation carriers have a high risk of developing colorectal cancer, but do not present with polyposis, a typical feature of other colorectal cancer syndromes such as familial adenomatous polyposis, in which polyposis reflects the high frequency of biallelic APC gene inactivation. We asked whether in Lynch syndrome biallelic inactivation of MMR genes occurred at a similar frequency to that of APC gene, and whether MMR inactivation resulted in detectable lesions within the intestinal mucosa. METHODS: Resections done for small and large bowel cancer between January, 2002, and January, 2011, were retrieved. We systematically analysed non-tumorous mucosa from carriers of a Lynch syndrome mutation (set 1: ten patients) and control patients without Lynch syndrome (set 1: nine patients) for MMR protein expression (MLH1, MSH2, and EPCAM) with immunohistochemistry. We validated the findings in an independent sample set (set 2: 30 Lynch syndrome patients, 79 controls). We did an analysis of microsatellite instability by PCR analysis to test lesions for mismatch repair deficiency. We applied a Poisson regression model to analyse the distribution of MMR-deficient crypt foci counts and a Fisher's exact test to compare the prevalence of these foci between mutation carriers and control patients. FINDINGS: 20 crypt foci with no MMR protein expression were detected in 20·1 cm(2) of non-tumorous mucosa from Lynch syndrome patients (set 1), an additional five were detected upon resectioning of two samples. In an independent validation set (set 2), two MMR-deficient crypt foci were noted in 2·2 cm(2) of mucosa. No MMR-deficient crypt foci were noted in non-tumorous mucosa from control patients without evidence for Lynch syndrome (set 1: 3·7 cm(2), set 2: 4·8 cm(2)). Microsatellite instability was detected in all seven MMR-deficient crypt foci analysed. A subset of these foci displayed unusual architectural and cytological abnormalities, although they had no polypous or adenomatous appearance. INTERPRETATION: We identified a novel type of lesion, the MMR-deficient crypt focus, as the manifestation of biallelic MMR gene inactivation in Lynch syndrome. The abundance of MMR-deficient crypt foci indicates a high frequency of biallelic MMR gene inactivation, which is in sharp contrast with the low number of clinically manifest cancers in Lynch syndrome. This discrepancy suggests that most MMR-deficient crypt foci do not progress to cancer. We propose Lynch syndrome as a unique model syndrome for studying initial steps of MMR deficiency, tumour initiation and, possibly, elimination. FUNDING: German Cancer Aid and German Research Foundation.

The molecular basis of EPCAM expression loss in Lynch syndrome-associated tumors.

Germline deletions affecting the epithelial cell adhesion molecule (EPCAM) gene lead to silencing of MSH2 and cause Lynch syndrome. We have recently reported that lack of EPCAM expression occurs in many, but not all tumors from Lynch syndrome patients with EPCAM germline deletions. The differences in EPCAM expression were not related to the localization of EPCAM germline deletions. We therefore hypothesized that the type of the second somatic hit, which leads to MSH2 inactivation during tumor development, determines EPCAM expression in the tumor cells. To test this hypothesis and to evaluate whether lack of EPCAM expression can already be detected in Lynch syndrome-associated adenomas, we analyzed four carcinomas and two adenomas from EPCAM germline deletion carriers for EPCAM protein expression and allelic deletion status of the EPCAM gene region by multiplex ligation-dependent probe amplification. In four out of six tumors we observed lack of EPCAM expression accompanied by biallelic deletions affecting the EPCAM gene. In contrast, monoallelic retention of the EPCAM gene was observed in the remaining two tumors with retained EPCAM protein expression. These results demonstrate that EPCAM expression in tumors from EPCAM deletion carriers depends on the localization of the second somatic hit that inactivates MSH2. Moreover, we report lack of EPCAM protein expression in a colorectal adenoma, suggesting that EPCAM immunohistochemistry may detect EPCAM germline deletions already at a precancerous stage.

Analysis of EPCAM protein expression in diagnostics of Lynch syndrome.

PURPOSE: Lynch syndrome is an inherited tumor predisposition syndrome caused by germline mutations of DNA mismatch repair (MMR) genes, mainly MLH1 and MSH2. Recently, germline deletions affecting the epithelial cell adhesion molecule (EPCAM) gene located upstream of MSH2 were identified as a novel mutational mechanism causing Lynch syndrome by epigenetic inactivation of the respective MSH2 allele. Immunohistochemical analysis of MMR protein expression is a hallmark of Lynch syndrome diagnostics, but it cannot distinguish between EPCAM deletion carriers and MSH2 mutation carriers. We hypothesized that EPCAM protein expression might be altered in tumors from patients with a germline EPCAM deletion. PATIENTS AND METHODS: Immunohistochemistry was used to assess EPCAM expression in Lynch syndrome-associated MSH2-negative tumors (n = 26). Multiplex ligation-dependent probe amplification (MLPA) analysis was performed to detect germline deletions of the EPCAM and MSH2 gene loci. RESULTS: In four MSH2-negative tumors, a concomitant lack of EPCAM expression was detected. MLPA analysis revealed heterozygous EPCAM deletions in all patients with EPCAM-negative tumors. In contrast, EPCAM expression was positive in all cancers from patients with germline alterations affecting MSH2 but not EPCAM. Two EPCAM deletions were detected in patients with an EPCAM-positive tumor. CONCLUSION: These results indicate that loss of EPCAM protein expression is frequent in tumors from patients with EPCAM germline deletions. EPCAM immunohistochemistry therefore represents a promising novel tool for the identification of Lynch syndrome patients with EPCAM germline deletions.