Uncovering the microbiota in renal cell carcinoma tissue using 16S rRNA gene sequencing.

INTRODUCTION: Increasing evidence indicates an important role of microbiota in cancer development and progression, while little is known about the correlation between microbiota and renal cell carcinoma (RCC). Thus, we performed this study to profile the intratumoral microbiota possibly associated with RCC. MATERIALS AND METHODS: Paired RCC and adjacent normal tissue samples were collected from 24 patients with RCC. V3-V4 variable region of microbial 16S rRNA gene was sequenced using Illumina MiSeq. Sequencing reads were processed using QIIME. Differentially abundant bacterial taxa between groups were identified by LEfSe, and their potential functions were inferred by PICRUSt. RESULTS: Decreased species diversity was presented in RCC tissues (Simpson index, P = 0.0340), and the composition of the bacterial community in RCC tissues was significantly distinct from that in normal tissues (unweighted UniFrac distance, P = 0.026; weighted UniFrac distance, P = 0.017). Compared with normal tissues, 25 taxa increased and 47 reduced taxa were identified in RCC tissues. Among these taxa, the class Chloroplast (AUC = 0.91, P < 0.0001) and the order Streptophyta (AUC = 0.89, P < 0.0001) showed high indication accuracy to discriminate RCC tissues from normal tissues. Furthermore, nine altered pathways were identified in RCC tissues to reveal the potential microbial function. CONCLUSIONS: Our results have uncovered the presence of distinct microbiota in RCC and adjacent normal tissues and provided a better understanding of the possible role of the intratumoral microbiota in RCC. Further studies are required to confirm our results and determine the real correlation between microbiota and RCC.

Randomized trial assessing impact of probiotic supplementation on gut microbiome and clinical outcome from targeted therapy in metastatic renal cell carcinoma.

Studies suggest a link between the gut microbiome and metastatic renal cell carcinoma (mRCC) outcomes, including evidence that mRCC patients possess a lower abundance of Bifidobacterium spp. compared to healthy adults. We sought to assess if a Bifidobacterium-containing yogurt product could modulate the gut microbiome and clinical outcome from vascular endothelial growth factor-tyrosine kinase inhibitors (VEGF-TKIs). mRCC patients initiating VEGF-TKIs, regardless of the line of therapy, were randomized to probiotic-supplemented (two 4 oz. servings of the probiotic yogurt product daily) or probiotic-restricted arms. Stool samples were collected prior to therapy and at weeks 2, 3, 4, and 12. Microbiome composition was assessed using whole-metagenome sequencing. A total of 20 patients were randomized. Bifidobacterium animalis, the active ingredient of the probiotic supplement, reached detectable levels in all patients in the probiotic-supplemented arm versus two patients in the probiotic-restricted arm. Clinical benefit rate was similar in probiotic-supplemented versus probiotic-restricted arms (70% vs. 80%, p = 0.606). Linear discriminant analysis (LDA) effect size analysis of MetaPhIAn2 abundance data predicted 25 enriched species demonstrating an LDA score >3 in either clinical benefit or no clinical benefit. In patients with clinical benefit (vs. no clinical benefit), Barnesiella intestinihominis and Akkermansia muciniphila were significantly more abundant (p = 7.4 × 10-6 and p = 5.6 × 10-3 , respectively). This is the first prospective randomized study demonstrating modulation of the gut microbiome with a probiotic in mRCC. Probiotic supplementation successfully increased the Bifidobacterium spp. levels. Analysis of longitudinal stool specimens identified an association between B. intestinihominis, A. muciniphila, and clinical benefit with therapy. Trial Registration: NCT02944617.

Stool Microbiome Profiling of Patients with Metastatic Renal Cell Carcinoma Receiving Anti-PD-1 Immune Checkpoint Inhibitors.

Preclinical models and early clinical data suggest an interplay between the gut microbiome and response to immunotherapy in solid tumors including metastatic renal cell carcinoma (mRCC). We sought to characterize the stool microbiome of mRCC patients receiving a checkpoint inhibitor (CPI) and to assess treatment-related changes in microbiome composition over the course of CPI therapy. Stool was collected from 31 patients before initiation of nivolumab (77%) or nivolumab plus ipilimumab (23%) therapy, of whom 58% experienced clinical benefit. Greater microbial diversity was associated with clinical benefit from CPI therapy (p = 0.001), and multiple species were associated with clinical benefit or lack thereof. Temporal profiling of the microbiome indicated that the relative abundance of Akkermansia muciniphila increased in patients deriving clinical benefit from CPIs. This study substantiates results from previous CPI-related microbiome profiling studies in mRCC. Temporal changes in microbiome composition suggest potential utility in modulating the microbiome for more successful CPI outcomes. PATIENT SUMMARY: We compared the composition and diversity of the gut microbiome in patients receiving immunotherapy for renal cell carcinoma. We found that higher microbial diversity is associated with better treatment outcomes. Treatment response is characterized by changes in microbial species over the course of treatment.

Gut Bacteria Composition Drives Primary Resistance to Cancer Immunotherapy in Renal Cell Carcinoma Patients.

BACKGROUND: The development of immune checkpoint blockade (ICB) has revolutionized the clinical outcome of renal cell carcinoma (RCC). Nevertheless, improvement of durability and prediction of responses remain unmet medical needs. While it has been recognized that antibiotics (ATBs) decrease the clinical activity of ICB across various malignancies, little is known about the direct impact of distinct intestinal nonpathogenic bacteria (commensals) on therapeutic outcomes of ICB in RCC. OBJECTIVE: To evaluate the predictive value of stool bacteria composition for ICB efficacy in a cohort of advanced RCC patients. DESIGN, SETTING, AND PARTICIPANTS: We prospectively collected fecal samples from 69 advanced RCC patients treated with nivolumab and enrolled in the GETUG-AFU 26 NIVOREN microbiota translational substudy phase 2 trial (NCT03013335) at Gustave Roussy. We recorded patient characteristics including ATB use, prior systemic therapies, and response criteria. We analyzed 2994 samples of feces from healthy volunteers (HVs). In parallel, preclinical studies performed in RCC-bearing mice that received fecal transplant (FMT) from RCC patients resistant to ICB (NR-FMT) allowed us to draw a cause-effect relationship between gut bacteria composition and clinical outcomes for ICB. The influence of tyrosine kinase inhibitors (TKIs) taken before starting nivolumab on the microbiota composition has also been assessed. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Metagenomic data (MG) from whole genome sequencing (WGS) were analyzed by multivariate and pairwise comparisons/fold ratio to identify bacterial fingerprints related to ATB or prior TKI exposure and patients' therapeutic response (overall response and progression-free survival), and compared with the data from cancer-free donors. RESULTS AND LIMITATIONS: Recent ATB use (n = 11; 16%) reduced objective response rates (from 28% to 9%, p < 0.03) and markedly affected the composition of the microbiota, facilitating the dominance of distinct species such as Clostridium hathewayi, which were also preferentially over-represented in stools from RCC patients compared with HVs. Importantly, TKIs taken prior to nivolumab had implications in shifting the microbiota composition. To establish a cause-effect relationship between gut bacteria composition and ICB efficacy, NR-FMT mice were successfully compensated with either FMT from responding RCC patients or beneficial commensals identified by WGS-MG (Akkermansia muciniphila and Bacteroides salyersiae). CONCLUSIONS: The composition of the microbiota is influenced by TKIs and ATBs, and impacts the success of immunotherapy. Future studies will help sharpen the role of these specific bacteria and their potential as new biomarkers. PATIENT SUMMARY: We used quantitative shotgun DNA sequencing of fecal microbes as well as preclinical models of fecal or bacterial transfer to study the association between stool composition and (pre)clinical outcome to immune checkpoint blockade. Novel insights into the pathophysiological relevance of intestinal dysbiosis in the prognosis of kidney cancer may lead to innovative therapeutic solutions, such as supplementation with probiotics to prevent primary resistance to therapy.

The Microbiome in Benign Renal Tissue and in Renal Cell Carcinoma.

INTRODUCTION: The renal bacterial colonization has not been explored so far. OBJECTIVE: The aim of this study was to describe the renal microbiome and to determine differences of the renal microbiome in healthy and tumor-bearing parenchyma. METHODS: Ten biopsies from patients undergoing laparoscopic nephrectomy for renal carcinoma with no history of urinary tract infections within the last 6 months were included in this study. The identification of all microorganisms was done using 16S DNA sequencing. The beta diversity analysis was performed by Bray-Curtis dissimilarity. RESULTS: In all kidney samples, a plethora of microorganisms was found, with significant differences between benign and malignant renal tissue (p < 0.0001). CONCLUSIONS: There is evidence that healthy kidney tissue as well as renal cell cancer tissue have a specific microbiome, thus opening new perspectives in renal physiology and tumor pathogenesis.

Yeast extract inhibits the proliferation of renal cell carcinoma cells via regulation of iron metabolism.

The microbiome has recently attracted research interest in a variety of subjects, including cancer. In the present study, it was determined that reinforced clostridium media (RCM) for microbiome culture, exerts antitumor effects on renal cell carcinoma cells when compared to the microbiome 'X'. The antitumor effects of RCM were investigated for all ingredients of RCM, and the results revealed that yeast extract could be a candidate for the ingredient driving this phenomenon. Further experiments including MTT assay, cell counting, cell death analysis, cell cycle analysis and western blotting were conducted with yeast extract on renal cell carcinoma cells (Caki­1 and Caki­2) and normal human proximal tubular cells (HK­2). As a result, yeast extract exhibited dose­dependent antitumor effects on Caki­1 and Caki­2, but only slight effects on HK­2. In addition, yeast extract only exhibited slight effects on necrosis, autophagy, or apoptosis of Caki­1 and Caki­2. Yeast extract produced cell cycle arrest with an increased G0/G1 fraction and a decreased S fraction, and this was considered to be related to the decreased cyclin D1. Although yeast extract treatment increased anti­oxidant activities, the antitumor effects of yeast extract were also related to iron metabolism, based on the decreased transferrin receptor and increased ferritin. In addition, decreased GPX4 may be related to iron­dependent cell death, particularly in Caki­2. These results revealed that yeast extract may inhibit proliferation of renal cell carcinoma cells by regulating iron metabolism. Since an increased iron requirement is a classic phenomenon of cancer cells, yeast extract may be a candidate for adjuvant treatment of renal cell carcinoma.

A repository of microbial marker genes related to human health and diseases for host phenotype prediction using microbiome data.

The microbiome research is going through an evolutionary transition from focusing on the characterization of reference microbiomes associated with different environments/hosts to the translational applications, including using microbiome for disease diagnosis, improving the effcacy of cancer treatments, and prevention of diseases (e.g., using probiotics). Microbial markers have been identified from microbiome data derived from cohorts of patients with different diseases, treatment responsiveness, etc, and often predictors based on these markers were built for predicting host phenotype given a microbiome dataset (e.g., to predict if a person has type 2 diabetes given his or her microbiome data). Unfortunately, these microbial markers and predictors are often not published so are not reusable by others. In this paper, we report the curation of a repository of microbial marker genes and predictors built from these markers for microbiome-based prediction of host phenotype, and a computational pipeline called Mi2P (from Microbiome to Phenotype) for using the repository. As an initial effort, we focus on microbial marker genes related to two diseases, type 2 diabetes and liver cirrhosis, and immunotherapy efficacy for two types of cancer, non-small-cell lung cancer (NSCLC) and renal cell carcinoma (RCC). We characterized the marker genes from metagenomic data using our recently developed subtractive assembly approach. We showed that predictors built from these microbial marker genes can provide fast and reasonably accurate prediction of host phenotype given microbiome data. As understanding and making use of microbiome data (our second genome) is becoming vital as we move forward in this age of precision health and precision medicine, we believe that such a repository will be useful for enabling translational applications of microbiome data.

Diversity of Gut Microbiota Metabolic Pathways in 10 Pairs of Chinese Infant Twins.

Early colonization of gut microbiota in human gut is a complex process. It remains unclear when gut microbiota colonization occurs and how it proceeds. In order to study gut microbiota composition in human early life, the present study recruited 10 healthy pairs of twins, including five monozygotic (MZ) and five dizygotic (DZ) twin pairs, whose age ranged from 0 to 6 years old. 20 fecal samples from these twins were processed by shotgun metagenomic sequencing, and their averaged data outputs were generated as 2G per sample. We used MEGAN5 to perform taxonomic and functional annotation of the metagenomic data, and systematically analyzed those 20 samples, including Jaccard index similarity, principle component, clustering, and correlation analyses. Our findings indicated that within our study group: 1) MZ-twins share more microbes than DZ twins or non-twin pairs, 2) gut microbiota distribution is relatively stable at metabolic pathways level, 3) age represents the strongest factor that can account for variation in gut microbiota, and 4) a clear metabolic pathway shift can be observed, which speculatively occurs around the age of 1 year old. This research will serve as a base for future studies of gut microbiota-related disease research.

Paracoccidioidomicose cutânea disseminada e pulmonar em paciente portador de neoplasia maligna visceral / Cutaneous and pulmonary paracoccidioidomycosis in a patient with a malignant visceral tumor

Paracoccidioidomicose é doença causada pelo fungo Paracoccidioides brasiliensis, caracterizada por quadro polimórfico e acometimento preferencial de pele, mucosas, pulmões, linfonodos, adrenais e sistema nervoso. De acordo com o local de inoculação e o estado imunológico do indivíduo, ocorrem as diversas formas da doença: tegumentar, linfonodular, visceral e mista. Relatamos caso de paciente com quadro de paracoccidioidomicose mista (tegumentar e pulmonar), com lesões cutâneas caracterizadas por pápulas e pústulas disseminadas e sintomas sistêmicos, possivelmente associada a imunossupressão causada por neoplasia maligna visceral.

Paracoccidioidomycosis is a systemic mycosis caused by the fungus Paracoccidioides brasiliensis that is characterized by polymorphous clinical manifestations principally affecting the skin, mucous membranes, lungs, lymph nodes, adrenal glands and the central nervous system. Depending on the site of inoculation and the individual's immunological status, the disease may take various different forms, affecting the skin, lymph nodes, viscera or a combination of these. The present report describes a patient with extensive cutaneous and pulmonary paracoccidioidomycosis, with disseminated papules and pustules, fever and pulmonary symptoms, probably related to immunosuppression caused by a renal carcinoma.

Cutaneous and pulmonary paracoccidioidomycosis in a patient with a malignant visceral tumor.

Paracoccidioidomycosis is a systemic mycosis caused by the fungus Paracoccidioides brasiliensis that is characterized by polymorphous clinical manifestations principally affecting the skin, mucous membranes, lungs, lymph nodes, adrenal glands and the central nervous system. Depending on the site of inoculation and the individual's immunological status, the disease may take various different forms, affecting the skin, lymph nodes, viscera or a combination of these. The present report describes a patient with extensive cutaneous and pulmonary paracoccidioidomycosis, with disseminated papules and pustules, fever and pulmonary symptoms, probably related to immunosuppression caused by a renal carcinoma.

A review of prognostic pathologic features and algorithms for patients treated surgically for renal cell carcinoma.

Accurate subtyping of RCC is critically important and should be considered in algorithms that are developed as prognostic tools for the patient and clinician. The TNM classification, already a powerful prognostic factor, will continue to evolve. The authors recommend that each component of the classification be assessed and reported during pathologic examination. This article also highlighted the importance of assigning a nuclear grade that is based on standardized and reproducible criteria that reflect the heterogeneity of nuclear and nucleolar features within RCC. Lastly, it is increasingly evident that coagulative tumor necrosis and sarcomatoid differentiation are compelling prognostic factors, on par with nuclear grade, and should be assessed routinely. To conclude, the complete list of pathologic features that are evaluated as part of the Mayo Clinic Nephrectomy Registry is presented. The features that are reported routinely in clinical practice also are indicated; this can serve as a guide for the reporting of results from the pathologic examination of RCC.

Can mycoplasma-mediated oncogenesis be responsible for formation of conventional renal cell carcinoma?

OBJECTIVES: To investigate the association between Mycoplasma sp. infection and conventional renal cell carcinoma (RCC). METHODS: Normal kidney, renal intratubular neoplasia, and tumor tissue samples from 33 patients with RCC and 35 healthy controls were studied. Molecular DNA analysis was done after nested polymerase chain reaction performed in two steps with seven primers (four outer and three inner) that can recognize at least 15 different Mycoplasma sp. RESULTS: Mycoplasma sp. DNA was detected in normal kidney, renal intratubular neoplasia, and tumor tissue samples at a ratio of 36%, 67%, and 82%, respectively. In 6 of the 33 patients with RCC, no Mycoplasma sp. was detected from any of the three tissue samples. Mycoplasma sp. DNA was detected in only 5 (14%) of the 35 samples from the control group. CONCLUSIONS: The relationship between mycoplasma infection and conventional RCC has been investigated for the first time, and a significantly high existence of Mycoplasma sp. DNA was found in the tissues of patients with conventional RCC compared with that found in a healthy control group. This suggests that mycoplasma-mediated multistage carcinogenesis may play a role in the development of RCC.

Prognostic significance of tumor thrombus level in patients with renal cell carcinoma and venous tumor thrombus extension. Is all T3b the same?

PURPOSE: We investigated the prognostic significance of venous tumor thrombus extension in patients with renal cell carcinoma with particular emphasis on 2 questions. Does the level of thrombus in the inferior vena cava (IVC) impact long-term survival? Is there a difference in long-term survival when tumor thrombus is in the renal vein versus the IVC for patients classified as T3b by 1997 TNM staging? MATERIALS AND METHODS: Between July 1970 and July 2000, 153 patients underwent surgical resection. Cancer specific survival was determined for different tumor thrombus levels in a retrospective fashion. RESULTS: Mean followup was 60 months with a range of 12 to 221. Level of tumor thrombus was renal vein (in 46), level I (in 68), level II (in 17) and level III (in 22). No demographic differences existed between the different levels including gender, age, perinephric extension, Fuhrman grade, percentage of metastatic disease and tumor size (Fisher's exact test). Patients with evidence of nodal disease or metastasis at surgery were eliminated from cancer specific survival analysis. The overall 10-year cancer specific survival for patients was 30%, 19% and 29% for level I, II and III, respectively. Patient survival at 5 and 10 years was not significantly different between the 3 IVC levels (p = 0.48). Ten-year survival of patients with renal vein involvement (66%) versus level I (29%) was significantly different (p = 0.0001). CONCLUSIONS: The level of tumor thrombus in the IVC does not significantly effect long-term survival. Ten-year survival of patients classified as T3b is statistically different for patients having tumor thrombus in the renal vein compared to level I. Combining these 2 groups as T3b by the 1997 TNM staging may need to be reevaluated.

Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma.

PURPOSE: A strain of Salmonella typhimurium (VNP20009), attenuated by chromosomal deletion of the purI and msbB genes, was found to target to tumor and inhibit tumor growth in mice. These findings led to the present phase I study of the intravenous infusion of VNP20009 to patients with metastatic cancer. PATIENTS AND METHODS: In cohorts consisting of three to six patients, 24 patients with metastatic melanoma and one patient with metastatic renal cell carcinoma received 30-minute intravenous bolus infusions containing 10(6) to 10(9) cfu/m(2) of VNP20009. Patients were evaluated for dose-related toxicities, selective replication within tumors, and antitumor effects. RESULTS: The maximum-tolerated dose was 3 x 10(8) cfu/m(2). Dose-limiting toxicity was observed in patients receiving 1 x 10(9) cfu/m(2), which included thrombocytopenia, anemia, persistent bacteremia, hyperbilirubinemia, diarrhea, vomiting, nausea, elevated alkaline phosphatase, and hypophosphatemia. VNP20009 induced a dose-related increase in the circulation of proinflammatory cytokines, such as interleukin (IL)-1beta, tumor necrosis factor alpha, IL-6, and IL-12. Focal tumor colonization was observed in two patients receiving 1 x 10(9) cfu/m(2) and in one patient receiving 3 x 10(8) cfu/m(2). None of the patients experienced objective tumor regression, including those patients with colonized tumors. CONCLUSION: The VNP20009 strain of Salmonella typhimurium can be safely administered to patients, and at the highest tolerated dose, some tumor colonization was observed. No antitumor effects were seen, and additional studies are required to reduce dose-related toxicity and improve tumor localization.

Fine needle aspiration cytology diagnosis of a fungal lesion of the Verticillium species. A case report.

BACKGROUND: Fine needle aspiration (FNA) cytology has great potential for the diagnosis of fungal lesions and other opportunistic infections, the frequency of which is rising due to immunosuppression, travel and environmental exposure. However, reports on FNA diagnosis of fungal lesions are rare. CASE: A 40-year-old male juvenile diabetic presented with a 5 x 4-cm swelling over the upper part of the left arm. He had a recent history of a left nephrectomy for renal cell carcinoma followed by radiotherapy. At the time of presentation, he was also receiving chemotherapy and interferon alpha-2a for a suspected pulmonary metastasis. FNA smears from the swelling showed an inflammatory exudate rich in neutrophils and a few septate fungal hyphae that branched at acute angle. Gomori's silver methenamine stain and periodic acid-Schiff stain revealed numerous fungal hyphae. The provisional diagnosis based on the cytomorphologic features was aspergillosis. However, culture of the aspirate confirmed the fungus to be of the Verticillium species. The lesion on the left arm responded to antifungal therapy, and the swelling disappeared gradually. CONCLUSION: FNA cytology was very useful in the diagnosis of a rare fungal lesion that was not clinically suspected in spite of the fact that the patient was a highly susceptible candidate for it.