Melanoma mortality is driven by the formation and growth of distant metastases. Here, we interrogated the role of tumor oxidative phosphorylation (OXPHOS) in the formation of distant metastases in melanoma. OXPHOS was the most upregulated metabolic pathway in primary tumors that formed distant metastases in the RCAS-TVA mouse model of spontaneous lung and brain metastases, and in melanoma patients that developed brain or other distant metastases. Knockout of PGC1α in melanocytes in the RCAS-TVA melanoma mouse model had no impact on primary tumor formation, but markedly reduced the incidence of lung and brain metastases. Genetic knockout of a component of electron transport chain complex I, NDUFS4, in B16-F10 and D4M-UV2 murine melanoma cell lines did not impact tumor incidence following subcutaneous, intravenous, or intracranial injection, but decreased tumor burden specifically in the lungs and brain. Together, these data demonstrate that OXPHOS is critical for the formation of metastases in melanoma. STRUCTURED ABSTRACT:Purpose: Melanoma mortality is driven by the formation and growth of distant metastases. However, the process and pathogenesis of melanoma metastasis remain poorly understood. Here, we interrogate the role of tumor oxidative phosphorylation (OXPHOS) in the formation of distant metastases in melanoma.Experimental Design: This study includes (1) new RNA-seq analysis of primary melanomas from patients characterized for distant metastasis events; (2) RNA-seq analysis and functional testing of genetic OXPHOS inhibition (PGC1α KO) the RCAS-TVA model, which is the only existing immunocompetent murine model of autochthonous lung and brain metastasis formation from primary melanoma tumors; and (3) functional experiments of genetic OXPHOS inhibition (NDUFS4 KO) in the B16-F10 and D4M-UV2 murine melanoma cell lines, including evaluation of subcutaneous, lung, and brain metastatic site dependencies.Results: OXPHOS was the most upregulated metabolic pathway in primary tumors that formed distant metastases in the RCAS-TVA mouse model of spontaneous lung and brain metastases, and in melanoma patients that developed brain or other distant metastases. Knockout of PGC1a in melanocytes in the RCAS-TVA melanoma mouse model had no impact on primary tumor formation, but markedly reduced the incidence of lung and brain metastases. Genetic knockout of a component of electron transport chain complex I, NDUFS4, in B16-F10 and D4M-UV2 murine melanoma cell lines did not impact tumor incidence following subcutaneous, intravenous, or intracranial injection, but decreased tumor burden specifically in the lungs and brain.Conclusions: Together, these data demonstrate that OXPHOS is critical for the formation of metastases in melanoma. TRANSLATIONAL RELEVANCE:Melanoma is the most aggressive form of skin cancer. One hallmark of this disease is a high risk of distant metastasis formation. The process and pathogenesis of metastasis in this disease remain poorly understood and there is controversy regarding the role of oxidative phosphorylation (OXPHOS) in melanoma metastasis. This study incorporates RNAseq analysis of primary melanoma tumors from patients characterized for distant metastasis events, RNAseq analysis of the only existing immunocompetent murine model of autochthonous lung and brain metastasis formation from primary melanoma tumors, and functional testing in multiple syngeneic models of melanoma at different tissue sites. This integrated analysis consistently demonstrates that melanoma OXPHOS promotes distant metastasis to the lungs and brain, two of the most common and clinically relevant sites of melanoma metastasis. This improved understanding of tumor OXPHOS may represent novel vulnerabilities for therapeutics development and surveillance/preventative strategies for melanoma metastasis.

Table S1: Clinical Characteristics of Melanoma Brain Metastases, Extracranial Metastases, and Primary Tumors. Tab 1. Clinical data for the MBMs and ECMs from melanoma patients utilized in this manuscript. Tab 2. Clinical data for primary melanomas from melanoma patients utilized in this manuscript.

Figure S1. Biphasic histologic components of sarcomatoid renal cell carcinoma. The macrodissected paired epithelioid or carcinomatous (E) and spindled or sarcomatoid (S) components of clear cell RCC (upper panel), Papillary RCC (middle panel), and chromophobe RCC (lower panel). H&E stain, scale bar 200 ï­m. Figure S2. Sarcomatoid ccRCC shows fewer VHL deletions. (A) Clear cell RCC (H&E stain, scale bar 100 µm) with (B) Fluorescence in situ hybridization (FISH) image showing paired CEN3q signals (green) and a single VHL signal (red). (C) Sarcomatoid ccRCC (H&E stain, scale bar 100 µm) with (D) FISH image showing balanced CEN3q (green) and VHL (red) signals. (E) Box plot showing significantly higher VHL/3q ratios associated with sarcomatoid histology, P<0.008. Figure S3: The smooth scatter plot of signal B versus signal A. Figure S4a: 3p21 and 3p21.1 copy number versus chromosome position. Figure S4b: 3p25 copy number versus chromosome position. Figure S5: Kernel density plots. Each sample per row; left three columns: 3p21; middle three columns: 2q37; and right three columns: 1p1. Figure S6a: Example of more than one peak in the summed signal. Figure S6b: Example of 4 peaks in signals A and B. Figure S7: Density plots of TCGA samples with copy-neutral LOH. Figure S8. VHL and PBRM1 show fewer 2-hit inactivation in sarcomatoid ccRCC. Sarcomatoid ccRCC and ccRCC cases shown in terms of the inactivating "hits" on 3p21-25 genes (VHL, PBRM1, SETD2, BAP1) consisting of mutations or methylation (mutually exclusive for VHL) and deletions. Figure S9. Top activated and inhibited pathways of sarcomatoid samples. Pathways altered by differentially expressed genes between non-sarcomatoid and sarcomatoid samples. The genes selected were differentially expressed in both the TCGA and MD Anderson samples. Figure S10. S- component shows a higher mutational load in sarcomatoid RCC. The total number of non-synonymous mutations in the E- and S- components of sarcomatoid RCC, across all parent RCC subtypes (A) and in clear cell RCC (B).

428 Background: Bilateral TGCT offers a unique opportunity to elucidate a stem cell versus genetic origin of cancer (Coffey, 2008). Comparison of the epigenetics and genetics of disparate tumors in either synchronous or metachronous bilateral TGCT from the same patients is feasible and may be informative. Methods: We examined the clinical characteristics and natural history of 37 patients with bilateral TGCT. We performed reduced representation bisulfite sequencing (RRBS) of FFPE DNA and whole exon sequencing (WES) of 5 of those patients whose tumor samples were available for the study. Results: We identified 37 patients with bilateral TGCT, who underwent their first orchiectomy between January 1984 and April 2022 and the second between August 1997 and April 2022. Thirty-five (95%) of these patients are still alive. Seven patients had synchronous, while 30 had metachronous bilateral TGCT. There were 13 bilateral seminomas, 14 bilateral nonseminomas, and 10 bilateral seminoma and nonseminoma. For those patients with metachronous bilateral TGCT, the median time between the two TGCT was 59 months (range 6-400). WES revealed a mutation in 189 (<1%) out of about 20,000 genes in the 10 paired samples from 5 patients. A total of 2 genes were mutated in more than 1 sample: KIT (n=4), and KRAS (n=2). Although one patient with synchronous bilateral seminomas shared KIT mutation, another did not with KIT and KRAS mutations (Table). RRBS showed a similar methylation profile in the 2 metachronous nonseminomas, which did not share any genetic mutations in the WES study. Conclusions: The clinical course of our patients with synchronous and metachronous bilateral TGCT and the results of our RRBS and WES reaffirmed that a preponderance of TGCT was curable and alluded to whether its epigenetic vs genetic origins might be causative or contributory to its general curability vs potential lethality. Coffey J et al. Somatic KIT mutations occur predominantly in seminoma germ cell tumors and are not predictive of bilateral disease: report of 220 tumors and review of literature. Genes Chromosomes Cancer 2008;47:34-42. [Table: see text]

BACKGROUND:Lymph node invasion is associated with poor outcome in patients with renal cell carcinoma (RCC).PATIENTS AND METHODS:Patients with RCC within a single center from 2001 to 2018 were retrospectively obtained from the Chang Gung Research Database. Patient gender, physical status, Charlson Comorbidity Index, tumor side, histology, age at diagnosis, and body mass index (BMI) were compared. The overall survival (OS) and cancer-specific survival (CSS) of each group were estimated using the Kaplan-Meier method. Log-rank tests were used to compare between the subgroups.RESULTS AND CONCLUSIONS:A total of 335 patients were enrolled, of whom 76 had pT3N0M0, 29 had pT1-3N1M0, 104 had T1-4N0M1, and 126 had T1-4N1M1 disease. Significant OS difference was noted between pT3N0M0 and pT1-3N1M0 groups with 12.08 years [95% confidence interval (CI), 8.33-15.84] versus 2.58 years (95% CI, 1.32-3.85), respectively (P < 0.005). No significant difference was observed in OS between pT1-3N1M0 and T1-4N0M1 groups with 2.58 years (95% CI, 1.32-3.85) versus 2.50 years (95% CI, 1.85-3.15, P = 0.72). The OS of N1M1 group was worse than that of N0M1 group with 1.00 year (95% CI, 0.74-1.26) versus 2.50 years (95% CI, 1.85-3.15, P < 0.05). Similar results were also observed in CSS. In summary, we claim that RCC with lymph node (LN) invasion should be reclassified as stage IV disease in terms of survival outcome.

Table S2: Differentially Expressed Genes in Patient-Matched Melanoma Brain and Extracranial Metastases. Comprehensive list of the 494 differentially expressed genes (adjusted p<0.05) between 35 MBMs and 42 patient-matched ECMs (from 29 patients). Results acquired via EdgeR-limma-voom R packages. Gene names are listed according to HGNC nomenclature. "Log2FC" is the estimate of the log2-fold-change of the gene in MBMs relative to patient-matched ECMs. "AveExr" is the average log2-expression for the gene across all samples. "t" is the moderated t-statistic. "P.Value" is the nominal p-value. "Adj.P.Val" is the p-value corrected for multiple testing via Benjamini-Hochberg procedure. "B" is the log-odds that the gene is differentially expressed.

Supplemental Methods 1. Exome sequencing pipeline Supplemental Methods 2. RNA seq pipeline Supplemental Methods 3. DNA methylation profiling pipeline Supplemental Methods 4. Fluorescence in situ hybridization Supplemental Methods 5. TCGA samples with possible copy neutral loss of heterozygosity in 3p21 and 3p25 regions

Table S3: Pathway Analysis of Intracranial and Subcutaneous Melanoma Xenografts. Results of EGSEA analyses of ICr (n=3) vs. SQ (n=3) tumors of 4 melanoma cell lines implanted into CD-1 nude mice. RNA-seq data were processed via Xenome to filter out contaminating mouse stromal signatures. "Gene Set" lists the database (MSigDB) and the specific collection of gene sets (Hallmark and c2: Curated) included in each analysis. Median rank was used to order the top 20 gene sets in each analysis. All gene sets listed have an adjusted p-value<0.001. Gene sets up-regulated in ICr tumors are shown in red. Gene sets down-regulated in ICr tumors are shown in blue. Of note, we identified no upregulation of nervous system gene sets in ICr xenografts and instead observed highly significant upregulation of numerous OXPHOS-related gene sets.

Objective: Mexican Americans are disproportionally affected by non-alcoholic fatty liver disease (NAFLD), liver fibrosis and hepatocellular carcinoma. Noninvasive means to identify those in this population at high risk for these diseases are urgently needed.Approach: The Cameron County Hispanic Cohort (CCHC) is a population-based cohort with high rates of obesity (51%), type 2 diabetes (28%) and NAFLD (49%). In a subgroup of 564 CCHC subjects, we evaluated 339 genetic variants previously reported to be associated with liver injury markers aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in United Kingdom and Japanese cohorts.Results: Association was confirmed for 86 variants. Among them, 27 had higher effect allele frequency in the CCHC than in the United Kingdom and Japanese cohorts, and 16 had stronger associations with AST and ALT than rs738409 (PNPLA3). These included rs17710008 (MYCT1), rs2519093 (ABO), rs1801690 (APOH), rs10409243 (S1PR2), rs1800759 (LOC100507053) and rs2491441 (RGL1), which were also associated with steatosis and/or liver fibrosis measured by vibration-controlled transient elastography. Main contributors to advanced fibrosis risk were rs11240351 (CNTN2), rs1800759 (LOC100507053), rs738409 (PNPLA3) and rs1801690 (APOH), with advanced fibrosis detected in 37.5% of subjects with 3 of these 4 variants [AOR = 11.6 (95% CI) = 3.8–35.3]. AST- and ALT-associated variants implicated distinct pathways (ethanol and galactose degradation versus antigen presentation and B cell development). Finally, 8 variants, including rs62292950 (DNAJC13), were associated with gut microbiome changes.Conclusion: These genotype-phenotype findings may have utility in risk modeling and disease prevention in this high-risk population.

Immune checkpoint blockade (ICB) has revolutionized cancer treatment, yet quality of life and continuation of therapy can be constrained by immune-related adverse events (irAEs). Limited understanding of irAE mechanisms hampers development of approaches to mitigate their damage. To address this, we examined whether mice gained sensitivity to anti-CTLA-4 (αCTLA-4)–mediated toxicity upon disruption of gut homeostatic immunity. We found αCTLA-4 drove increased inflammation and colonic tissue damage in mice with genetic predisposition to intestinal inflammation, acute gastrointestinal infection, transplantation with a dysbiotic fecal microbiome, or dextran sodium sulfate administration. We identified an immune signature of αCTLA-4–mediated irAEs, including colonic neutrophil accumulation and systemic interleukin-6 (IL-6) release. IL-6 blockade combined with antibiotic treatment reduced intestinal damage and improved αCTLA-4 therapeutic efficacy in inflammation-prone mice. Intestinal immune signatures were validated in biopsies from patients with ICB colitis. Our work provides new preclinical models of αCTLA-4 intestinal irAEs, mechanistic insights into irAE development, and potential approaches to enhance ICB efficacy while mitigating irAEs.

The gut microbiome composition varies across ethnicities and geographical locations, yet studies on diabetes-associated microbiome changes specific to high-risk Mexican Americans are lacking. Here, we aimed to identify specific alterations associated with diabetes in this population, as well as host genetic factors that may explain increased disease susceptibility in this ethnic group.

A stem cell theory of cancer considers genetic makeup in the proper cellular context. It is a unified theory of cancer that unites the genome with the epigenome, links the intracellular with the extracellular, and connects the cellular constituents and compartments with the microenvironment. Although it allies with genomic medicine, it is better aligned with integrated medicine. In this perspective, we focus on translational research in cancer care. We expose some intrinsic fallacies in translational research when it relates to the basic principles of the scientific method in the care of patients with genomic medicine versus integrated medicine. We postulate that genomic medicine may be at the root of many failed efforts in drug development and data reproducibility. We propose an alternate heuristic approach that may expedite the development of safe and effective treatments and minimize the generation of unproductive pharmaceutical products and nonreproducible experimental results. Importantly, a heuristic approach emphasizes the role of a pertinent scientific theory and distinguishes therapy development from drug development, such that we discover not only useful drugs but also better ways to use them in order to optimize patient care and maximize clinical outcomes.

BACKGROUND AND AIMS:Hispanics are disproportionately affected by NAFLD, liver fibrosis, cirrhosis, and HCC. Preventive strategies and noninvasive means to identify those in this population at high risk for liver fibrosis, are urgently needed. We aimed to characterize the gut microbiome signatures and related biological functions associated with liver fibrosis in Hispanics and identify environmental and genetic factors affecting them. APPROACH AND RESULTS:Subjects of the population-based Cameron County Hispanic Cohort (CCHC; n = 217) were screened by vibration-controlled transient elastography (FibroScan). Among them, 144 (66.7%) had steatosis and 28 (13.0%) had liver fibrosis. The gut microbiome of subjects with liver fibrosis was enriched with immunogenic commensals (e.g., Prevotella copri, Holdemanella, Clostridiaceae 1) and depleted of Bacteroides caccae, Parabacteroides distasonis, Enterobacter, and Marinifilaceae. The liver fibrosis-associated metagenome was characterized by changes in the urea cycle, L-citrulline biosynthesis and creatinine degradation pathways, and altered synthesis of B vitamins and lipoic acid. These metagenomic changes strongly correlated with the depletion of Parabacteroides distasonis and enrichment of Prevotella and Holdemanella. Liver fibrosis was also associated with depletion of bacterial pathways related to L-fucose biosynthesis. Alcohol consumption, even moderate, was associated with high Prevotella abundance. The single-nucleotide polymorphisms rs3769502 and rs7573751 in the NCK adaptor protein 2 (NCK2) gene positively associated with high Prevotella abundance. CONCLUSION:Hispanics with liver fibrosis display microbiome profiles and associated functional changes that may promote oxidative stress and a proinflammatory environment. These microbiome signatures, together with NCK2 polymorphisms, may have utility in risk modeling and disease prevention in this high-risk population.

Introduction: MBMs are a deadly complication of melanoma. We previously showed that OXPHOS is increased in MBMs, but the roles of OXPHOS in MBM formation and growth are unknown. Methods: To determine if OXPHOS predicts formation of MBMs, RNA-sequencing (RNAseq) was performed on primary melanomas from patients (pts) diagnosed with MBM (P_MBM; n=19) and from pts diagnosed with metastases (mets) solely to non-CNS sites within 3-18 months of initial diagnosis (P_ECM; n=16), as well as pts who did not develop any mets within 5 years (P_NoMet; n=19). To functionally test the role of OXPHOS in brain and lung met formation and maintenance, brain-tropic YUMM5.2-Br3 cells were treated in vitro with the direct OXPHOS inhibitor IACS-010759 (100 nM) or vehicle control for 12 hours, and then injected into the left ventricle (intracardiac; ICa) in C57BL/6 mice. Mice were randomized to in vivo treatment with vehicle or IACS-010759 (7.5 mg/kg daily) for 14 days. Mice were euthanized and ex vivo bioluminescence imaging (BLI) was performed to evaluate the extent of brain and lung mets. We also evaluated A375P and G361 human melanoma cell lines stably transfected with shRNAs targeting the OXPHOS mediator PGC1α (shPGC1α) or scrambled control (shScr). Cells were injected ICa, intracranially (ICr) or subcutaneously (SQ) in CD-1 nude mice. Lung and brain tissues were harvested for histological analysis of mets once mice became moribund. SQ tumors were measured twice weekly to assess tumor growth. Mice with ICr injection were monitored for survival and euthanized once moribund. Results: Pathway analysis of the primary tumors from pts demonstrated elevated KEGG OXPHOS gene set expression in P_MBM and P_ECM groups compared to P_NoMet (FDR q-val= 0.048 and Conclusions: OXPHOS was associated with increased risk of mets in pts, but not specifically MBMs. In mice, OXPHOS inhibition did not prevent MBM formation, but reduced the growth of established MBMs. Thus, OXPHOS is critical to MBM maintenance. Citation Format: Grant M. Fischer, Renato A. Guerrieri, Qianghua Hu, Fernando C. Carapeto, Aron Y. Joon, Vashisht Y. Gopal, Barbara Knighton, Wanleng Deng, Lauren E. Haydu, Jeffrey E. Gershenwald, Alexander J. Lazar, Michael T. Tetzlaff, Michael A. Davies. The role of oxidative phosphorylation (OXPHOS) in melanoma brain metastasis (MBM) formation and growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2844.

Background. Recently, we showed that melanoma brain metastases (MBMs) are characterized by increased utilization of the oxidative phosphorylation (OXPHOS) metabolic pathway compared to melanoma extracranial metastases (ECMs). MBM growth was inhibited by a potent direct OXPHOS inhibitor, but observed toxicities support the need to identify alternative therapeutic strategies. Thus, we explored the features associated with OXPHOS to improve our understanding of the pathogenesis and potential therapeutic vulnerabilities of MBMs. Methods. We applied an OXPHOS gene signature to our cohort of surgically resected MBMs that had undergone RNA-sequencing (RNA-seq) (n = 88). Clustering by curated gene sets identified MBMs with significant enrichment (High-OXPHOS; n = 21) and depletion (Low-OXPHOS; n = 25) of OXPHOS genes. Clinical data, RNA-seq analysis, and immunohistochemistry were utilized to identify significant clinical, molecular, metabolic, and immune associations with OXPHOS in MBMs. Preclinical models were used to further compare melanomas with High- and Low-OXPHOS and for functional validation. Results. High-OXPHOS MBMs were associated with shorter survival from craniotomy compared to Low-OXPHOS MBMs. High-OXPHOS MBMs exhibited an increase in glutamine metabolism, and treatment with the glutaminase inhibitor CB839 improved survival in mice with MAPKi-resistant, High-OXPHOS intracranial xenografts. High-OXPHOS MBMs also exhibited a transcriptional signature of deficient immune activation, which was reversed in B16-F10 intracranial tumors with metformin treatment, an OXPHOS inhibitor. Conclusions. OXPHOS is associated with distinct clinical, molecular, metabolic, and immune phenotypes in MBMs. These associations suggest rational therapeutic strategies for further testing to improve outcomes in MBM patients.

You have accessJournal of UrologyPenile & Testicular Cancer: Penile & Testicular Cancer I (MP11)1 Apr 2020MP11-17 ORIGIN OF GERM CELL TUMORS: SOMATIC TRANSFORMATION AND PRESENCE OF CANCER STEM-LIKE CELLS IN TERATOMA Eric Umbreit*, Christopher Wood, Andrew McIntosh, Mary Beth Westerman, Daniel Shapiro, Aron Joon, Miao Zhang, Jose Karam, and Shi-ming Tu Eric Umbreit*Eric Umbreit* More articles by this author , Christopher WoodChristopher Wood More articles by this author , Andrew McIntoshAndrew McIntosh More articles by this author , Mary Beth WestermanMary Beth Westerman More articles by this author , Daniel ShapiroDaniel Shapiro More articles by this author , Aron JoonAron Joon More articles by this author , Miao ZhangMiao Zhang More articles by this author , Jose KaramJose Karam More articles by this author , and Shi-ming TuShi-ming Tu More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000831.017AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Controversy surrounds the genesis of germ cell-associated teratoma and the origin of its ability to transform into malignancy. It is unknown whether a teratoma dedifferentiates from a progeny cell into a progenitor cell or teratoma develops from cancer stem cells, enabling it to differentiate into multiple lineages. We hypothesize that somatic transformation occurs by differentiation of a progenitor stem-like cell embedded in the teratoma. METHODS: Two investigations where explored to further evaluate this hypothesis: (1) develop a post-chemotherapy teratoma primary culture and demonstrate a stem cell-like progenitor cell embedded within teratoma derived from surgically resected specimens, and (2) evaluate the genetic and molecular profiles of metastatic teratoma and corresponding malignant transformation. Flow cytometry was performed on live cells for cell-surface stem-ness markers to identify a potential cancer stem cell within teratoma. Second, archival tumor tissue from 7 patients with teratoma and its associated somatically transformed tumor was used for DNA and RNA sequencing. RESULTS: Teratoma cells were successfully grown in primary culture from two patients. Cells were confirmed as teratoma by final pathology and primary culture analysis for i12p, SALL4, OCT3 and NANOG. By flow cytometry, cell lines were found to have cells positive for the stem-ness markers CD24, CD26, CD34, CD44, CD90, CD117, Cripto-1, SSEA-1, and SSEA-4. In the second investigation arm using the T200 panel for DNA analysis, we identified 58 genes harboring mutations of which 24 genes indicated discordance between teratoma and somatic transformation. None of the 24 genes were consistently altered between patients, thus, teratoma and transformed tissue had similar genetic profiles. RNA analysis revealed 7 significant genes with increased expression in teratoma tissue compared to somatic transformation, suggesting a different epigenetic profile between tissue types. CONCLUSIONS: For the first time ever reported, we successfully created primary culture teratoma cell lines. In addition, we have begun to identify cancer stem-like cells within mature teratoma. By demonstrating that metastatic teratoma and their corresponding somatic transformations had similar if not identical genetic profiles, we hypothesize they have a common clonal origin. We propose an alternative hypothesis in which teratoma contains a stem-cell entity that could both differentiate into a teratoma or any other tumor phenotype. Source of Funding: None. © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e142-e142 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Eric Umbreit* More articles by this author Christopher Wood More articles by this author Andrew McIntosh More articles by this author Mary Beth Westerman More articles by this author Daniel Shapiro More articles by this author Aron Joon More articles by this author Miao Zhang More articles by this author Jose Karam More articles by this author Shi-ming Tu More articles by this author Expand All Advertisement PDF downloadLoading ...

ABSTRACT Elevated serum lactate dehydrogenase (sLDH) is associated with poor clinical outcomes in patients with stage IV metastatic melanoma (MM). It is currently unknown if sLDH elevation correlates with distinct molecular, metabolic, or immune features of melanoma metastases. The identification of such features may identify rational therapeutic strategies for patients with elevated sLDH. Thus, we obtained sLDH levels for melanoma patients with metastases who had undergone molecular and/or immune profiling. Our analysis of multi‐omics data from independent cohorts of melanoma metastases showed that elevated sLDH was not significantly associated with differences in immune cell infiltrate, point mutations, DNA copy number variations, promoter methylation, RNA expression, or protein expression in melanoma metastases. The only significant association observed for elevated sLDH was with the number of metastatic sites of disease. Our data support that sLDH correlates with disease burden, but not specific molecular or immunological phenotypes, in metastatic melanoma.

Background: Elevated serum LDH is a poor prognostic factor in pts with MM. It is currently unknown if serum LDH levels correlate with molecular or immune differences in tumors, including LDH expression. We analyzed resected melanoma metastases to identify features that correlate with serum LDH elevation. Methods: Metastases with serum LDH levels measured within 30 days of surgery were included in the study: (1) a TMA of resected stage IV metastases from MD Anderson (MDACC) (n=207); (2) resected stage IV metastases from MDACC with previously acquired RNA-sequencing (RNA-seq) and CD8+ immunohistochemistry (IHC) data (n=24); (3) publicly available data for regional metastases from the melanoma TCGA (n=104) from MDACC and the Melanoma Institute of Australia. IHC was performed for LDHA, LDHB, PTEN, PD-L1, MITF, and Ki67 on the stage IV TMA, and publicly available data for DNA, RNA, and proteins for the TCGA samples were downloaded. Data were analyzed to identify features that differed between tumors with elevated and normal serum LDH levels. TCGA and stage IV tumors expressing the 25% highest and lowest LDHA and LDHB by RNA-seq were also compared. Results: IHC of the stage IV TMA identified no significant associations with serum LDH levels, including tumor LDHA (p=0.69) and LDHB (p=0.93) expression. Serum LDH was elevated in 33.3% of stage IV metastases with RNA-seq data and 21.6 % of TCGA regional metastases, which by Ensemble of Gene Set Enrichment Analyses (EGSEA) correlated with decreased expression of interferon alpha (q=8.26 × 10−14 and q=2.31 × 10−14), interferon gamma (q=9.46 × 10−14 and q=2.34 × 10−19), and inflammatory response (q= 1.26 × 10−20 and q=4.17 × 10−28) gene sets. Fewer (p=0.0464) CD8+ T cells were detected in stage IV tumors from pts with elevated serum LDH. Comparison of stage IV and TCGA tumors with high vs. low LDHA and LDHB mRNA expression levels did not correlate with serum LDH status, but significant enrichment of oxidative phosphorylation (q=6.32 × 10−21 and q=5.69 × 10−19), pyruvate metabolism (q= 7.90 × 10−16 and q=1.78 × 10−14), and citrate cycle (q= 1.51 × 10−06 and q=5.44 × 10−06) gene sets were detected in LDHA-high tumors. Increased LDHB expression correlated with enrichment of citrate cycle (q= 4.04 × 10−16 and q= 1.68 × 10−14), pyruvate metabolism (q= 2.34 × 10−11 and q=1.50 × 10−10), and glycolysis (q= 2.92 × 10−9 and q=1.55 × 10−8) gene sets. Conclusions: Elevated serum LDH levels correlated with decreased expression of immune response genes, but not LDHA or LDHB expression, in melanoma regional and distant metastases. Elevated serum LDH also correlated with decreased CD8+ T cell infiltrates by IHC. LDHA and LDHB expression levels in regional metastases correlated with distinct metabolic pathways, but not serum LDH levels. Citation Format: Fernando Cintra Lopes Carapet, Grant Fischer, Aron Joon, Huiqin Chen, Lauren Haydu, Sandra Lee, Melissa Saul, Savi Appana, John Thompson, Graham Mann, Jennifer McQuade, Alexander Lazar, Michael Tetzlaff, John Kirkwood, Richard Scolyer, Georgina Long, Michael Davies. Analysis of molecular and immune features that correlate with serum lactate dehydrogenase (LDH) levels in patients (pts) with metastatic melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A04.

Although genetic changes may be pivotal in the origin of cancer, cellular context is paramount. This is particularly relevant in a progenitor germ cell tumor and its differentiated mature teratoma counterpart when it concerns tumor heterogeneity and cancer dormancy in subsequent second malignancies (subsequent malignant neoplasms (SMNs)). From our tumor registry database, we identified 655 testicular germ cell tumor (TGCT) patients who developed SMNs between January 1990 and September 2018. Of the 113 solid organ SMNs, 42 had sufficient tumor tissue available for fluorescence in situ hybridization (FISH) analysis of isochromosome 12p [i(12p)]. We identified seven additional patients for targeted DNA and RNA sequencing of teratomas and adjacent somatic transformation. Finally, we established cell lines from freshly resected post-chemotherapy teratomas and evaluated the cells for stemness expression by flow cytometry and by the formation of teratomas in a xenograft model. In our cohort, SMNs comprising non-germ cell tumors occurred about 18 years after a diagnosis of TGCT. Of the 42 SMNs examined, 5 (12%) contained i(12p) and 16 (38%) had 12p gain. When comparing a teratoma and adjacent somatic transformation, targeted DNA and RNA sequencing demonstrated high concordance. Studies of post-chemotherapy teratoma-derived cell lines revealed cancer-initiating cells expressing multipotency as well as early differentiation markers. For the first time, we demonstrated the prevalence of i(12p) in SMNs and the presence of progenitor cells embedded within mature teratomas after chemotherapy. Our findings suggest a progenitor stem-like cell of origin in SMN and TGCT and highlight the importance of cellular context in this disease.