Mutational profile of primary clear cell renal cell carcinoma predicts recurrence and potential candidacy for adjuvant immune checkpoint inhibition

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Vlachostergios" }, { "@type": "Person", "name": "Maria Papathanassiou" }, { "@type": "Person", "name": "Maria Anagnostou" }, { "@type": "Person", "name": "Eleni Thodou" }, { "@type": "Person", "name": "Ioannis Tamposis" }, { "@type": "Person", "name": "Lampros Mitrakas" }, { "@type": "Person", "name": "Ioannis Zachos" }, { "@type": "Person", "name": "George K. Koukoulis" }, { "@type": "Person", "name": "Maria Samara" }, { "@type": "Person", "name": "Vassilios Tzortzis" } ], "publisher": { "@type": "Organization", "name": "F1000Research", "logo": { "@type": "ImageObject", "url": "https://f1000research.com/img/AMP/F1000Research_image.png", "height": 480, "width": 60 } }, "image": { "@type": "ImageObject", "url": "https://f1000research.com/img/AMP/F1000Research_image.png", "height": 1200, "width": 150 }, "description": " Background The risk of recurrence after nephrectomy for primary clear cell renal cell carcinoma (ccRCC) is estimated in daily practice solely based on clinical criteria. The aim of this study was to assess the prognostic relevance of common somatic mutations with respect to tumor aggressiveness and outcomes of ccRCC patients after definitive treatment. Methods Primary tumors from 37 patients with ccRCC who underwent radical nephrectomy were analyzed for presence of somatic mutations using a 15-gene targeted next-generation sequencing (NGS) panel. Associations to histopathologic characteristics and outcomes were investigated in the study cohort (n=37) and validated in The Cancer Genome Atlas (TCGA) ccRCC cohort (n=451). Results VHL was the most frequently mutated gene (51%), followed by PBRM1 (27%), BAP1 (13%), SETD2 (13%), KDM5C (5%), ATM (5%), MTOR (5%), and PTEN (3%). One-third of patients did not have any somatic mutations within the 15-gene panel. The vast majority of tumors harboring no mutations at all or VHL-only mutations (51%) were more frequently of smaller size (pT1-2) and earlier stage (I/II), whereas presence of any other gene mutations in various combinations with or without VHL was enriched in larger (pT3) and higher stage tumors (III) (p=0.02). No recurrences were noted in patients with unmutated tumors or VHL-only mutations as opposed to three relapses in patients with non-VHL somatic mutations (p=0.06). Presence of somatic mutations in PBRM1, BAP1, SETD2, KDM5C, ATM, MTOR, or PTEN genes in 451 TCGA ccRCC patients was associated with a significantly shorter disease-free survival (DFS) compared to those with unaltered tumors (q=0.01). Conclusions Preliminary findings from this ongoing study support the prognostic value of non-VHL mutations including PBRM1, BAP1, SETD2, KDM5C, ATM, MTOR, and PTEN in primary ccRCC tumors as surrogates of earlier recurrence and potential selection for adjuvant immune checkpoint inhibition. " } { "@context": "http://schema.org", "@type": "BreadcrumbList", "itemListElement": [ { "@type": "ListItem", "position": "1", "item": { "@id": "https://f1000research.com/", "name": "Home" } }, { "@type": "ListItem", "position": "2", "item": { "@id": "https://f1000research.com/browse/articles", "name": "Browse" } }, { "@type": "ListItem", "position": "3", "item": { "@id": "https://f1000research.com/articles/12-918/v2", "name": "Mutational profile of primary clear cell renal cell carcinoma predicts..." } } ] } Home Browse Mutational profile of primary clear cell renal cell carcinoma predicts... ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article Vlachostergios PJ, Papathanassiou M, Anagnostou M et al. Mutational profile of primary clear cell renal cell carcinoma predicts recurrence and potential candidacy for adjuvant immune checkpoint inhibition [version 2; peer review: 2 approved] . F1000Research 2024, 12 :918 ( https://doi.org/10.12688/f1000research.136087.2 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Brief Report Revised Mutational profile of primary clear cell renal cell carcinoma predicts recurrence and potential candidacy for adjuvant immune checkpoint inhibition [version 2; peer review: 2 approved] Panagiotis J. Vlachostergios https://orcid.org/0000-0002-1704-1517 1-3 , Maria Papathanassiou 4 , Maria Anagnostou 4 , [...] Eleni Thodou 4 , Ioannis Tamposis https://orcid.org/0000-0001-9247-6871 5 , Lampros Mitrakas 1 , Ioannis Zachos 1 , George K. Koukoulis 4 , Maria Samara 4 , Vassilios Tzortzis 1 Panagiotis J. Vlachostergios https://orcid.org/0000-0002-1704-1517 1-3 , Maria Papathanassiou 4 , [...] Maria Anagnostou 4 , Eleni Thodou 4 , Ioannis Tamposis https://orcid.org/0000-0001-9247-6871 5 , Lampros Mitrakas 1 , Ioannis Zachos 1 , George K. Koukoulis 4 , Maria Samara 4 , Vassilios Tzortzis 1 PUBLISHED 14 Jun 2024 Author details Author details 1 Urology, University of Thessaly, Faculty of Medicine, School of Health Sciences, University Hospital of Larissa, Larissa, Thessalia, Greece 2 Medical Oncology, IASO Thessalias Hospital, Larissa, Thessalia, Greece 3 Hematology & Medical Oncology, Weill Cornell Medicine, New York, New York, USA 4 Pathology, University of Thessaly, Faculty of Medicine, School of Health Sciences, Larissa, Greece 5 Computer Science and Biomedical Informatics, University of Thessaly, Lamia, Greece Panagiotis J. Vlachostergios Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Maria Papathanassiou Roles: Data Curation, Formal Analysis, Funding Acquisition, Investigation, Writing – Original Draft Preparation Maria Anagnostou Roles: Data Curation, Investigation, Writing – Original Draft Preparation Eleni Thodou Roles: Investigation, Writing – Review & Editing Ioannis Tamposis Roles: Funding Acquisition, Investigation, Methodology, Resources, Software, Writing – Review & Editing Lampros Mitrakas Roles: Investigation, Methodology, Writing – Review & Editing Ioannis Zachos Roles: Investigation, Resources, Writing – Review & Editing George K. Koukoulis Roles: Investigation, Resources, Writing – Review & Editing Maria Samara Roles: Conceptualization, Funding Acquisition, Investigation, Project Administration, Resources, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing Vassilios Tzortzis Roles: Conceptualization, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Supervision, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the HEAL1000 gateway. This article is included in the Oncology gateway. Abstract Background The risk of recurrence after nephrectomy for primary clear cell renal cell carcinoma (ccRCC) is estimated in daily practice solely based on clinical criteria. The aim of this study was to assess the prognostic relevance of common somatic mutations with respect to tumor aggressiveness and outcomes of ccRCC patients after definitive treatment. Methods Primary tumors from 37 patients with ccRCC who underwent radical nephrectomy were analyzed for presence of somatic mutations using a 15-gene targeted next-generation sequencing (NGS) panel. Associations to histopathologic characteristics and outcomes were investigated in the study cohort (n=37) and validated in The Cancer Genome Atlas (TCGA) ccRCC cohort (n=451). Results VHL was the most frequently mutated gene (51%), followed by PBRM1 (27%), BAP1 (13%), SETD2 (13%), KDM5C (5%), ATM (5%), MTOR (5%), and PTEN (3%). One-third of patients did not have any somatic mutations within the 15-gene panel. The vast majority of tumors harboring no mutations at all or VHL-only mutations (51%) were more frequently of smaller size (pT1-2) and earlier stage (I/II), whereas presence of any other gene mutations in various combinations with or without VHL was enriched in larger (pT3) and higher stage tumors (III) (p=0.02). No recurrences were noted in patients with unmutated tumors or VHL -only mutations as opposed to three relapses in patients with non- VHL somatic mutations (p=0.06). Presence of somatic mutations in PBRM1, BAP1, SETD2, KDM5C, ATM, MTOR , or PTEN genes in 451 TCGA ccRCC patients was associated with a significantly shorter disease-free survival (DFS) compared to those with unaltered tumors (q=0.01). Conclusions Preliminary findings from this ongoing study support the prognostic value of non- VHL mutations including PBRM1, BAP1, SETD2, KDM5C, ATM, MTOR , and PTEN in primary ccRCC tumors as surrogates of earlier recurrence and potential selection for adjuvant immune checkpoint inhibition. READ ALL READ LESS Keywords clear cell, kidney cancer, renal cell carcinoma, recurrence, immune checkpoint inhibition, genomics, mutational profile, next generation sequencing Corresponding Author(s) Panagiotis J. Vlachostergios ( [email protected] ) Close Corresponding author: Panagiotis J. Vlachostergios Competing interests: No competing interests were disclosed. Grant information: This work was funded by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH—CREATE—INNOVATE, project code: T2EDK-03079. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2024 Vlachostergios PJ et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Vlachostergios PJ, Papathanassiou M, Anagnostou M et al. Mutational profile of primary clear cell renal cell carcinoma predicts recurrence and potential candidacy for adjuvant immune checkpoint inhibition [version 2; peer review: 2 approved] . F1000Research 2024, 12 :918 ( https://doi.org/10.12688/f1000research.136087.2 ) First published: 01 Aug 2023, 12 :918 ( https://doi.org/10.12688/f1000research.136087.1 ) Latest published: 14 Jun 2024, 12 :918 ( https://doi.org/10.12688/f1000research.136087.2 ) Revised Amendments from Version 1 We have now addressed all reviewers' comments, particularly pertaining to the biological rationale of the study, the potential for predicting response to immunotherapy and guiding current practice, as well as study limitations. We have now addressed all reviewers' comments, particularly pertaining to the biological rationale of the study, the potential for predicting response to immunotherapy and guiding current practice, as well as study limitations. See the authors' detailed response to the review by Athanasia Pavlopoulou See the authors' detailed response to the review by Konstantinos Kamposioras READ REVIEWER RESPONSES Introduction Renal cell carcinoma (RCC) is a heterogenous group of kidney cancers originating from the nephron. 1 Renal cell carcinoma ranks among the ten most frequently diagnosed malignancies worldwide, with an estimated 400.000 new diagnoses and more than 170.000 deaths annually. 2 , 3 Clear cell RCC (ccRCC) is the most common RCC subtype, accounting for about 70-75% of cases and has a distinct molecular profile. 1 , 4 Localized ccRCC is treated with surgical resection, either partial or radical nephrectomy. Nevertheless, one-third of these patients experience recurrence. 5 So far, ccRCC recurrence, disease progression and mortality are being predicted using clinicopathological criteria. 6 While various recurrence models have been proposed, they only marginally outperformed standard staging. 7 Further, they demonstrate statistically significant variability in their predictive ability over time, rendering implementation into clinical practice and clinical trial design challenging. 7 Immune checkpoint inhibition with the use of pembrolizumab, a monoclonal antibody against programmed death-1 (PD-1), is approved as adjuvant therapy for patients with resected ccRCC who have a high risk of recurrence. 8 This was based on results of a phase III randomized double-blind study comparing pembrolizumab with placebo, which demonstrated a significant improvement in disease-free survival (DFS). 9 Patient selection criteria for a high risk of recurrence included tumor stage II with nuclear grade 4 or sarcomatoid differentiation, tumor stage III or higher, regional lymph-node metastasis, or stage M1 without evidence of disease after combined nephrectomy and metastasectomy either concurrently or within a year from primary tumor resection. 9 What currently remains an unmet need is the ability to predict which patients with ccRCC will relapse using a single or composite molecular biomarker that would be more directly related with tumor biology. In this ongoing prospective study, we examined the mutational profile of patients with non-metastatic ccRCC who underwent nephrectomy, followed by observation or adjuvant immunotherapy with pembrolizumab depending on established clinical and histopathological criteria. We studied associations of mutated genes with high-risk features and assessed the prognostic relevance of somatic mutations with regard to DFS after nephrectomy with or without adjuvant immunotherapy. Methods Ethical considerations The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board and Ethics Committee of Faculty of Medicine, University of Thessaly (3214/29.07.2016) on 29 July 2016. Study design This was a prospective single-center cohort study of patients with a diagnosis of ccRCC who underwent radical nephrectomy followed by observation or adjuvant immune checkpoint inhibition with pembrolizumab at the University Hospital of Larissa between December 2020 and February 2023. Eligible subjects included patients of ≥18 years of age, with histologically confirmed non-metastatic ccRCC, without prior systemic therapy for RCC. Patients were eligible if they had an intermediate-to-high or high risk of recurrence based on histopathological features including pT2 with grade 4 or sarcomatoid differentiation, pT3 or pT4 with any grade, or any pT and grade with presence of positive lymph nodes (N+). Subjects unable to provide consent, those with low risk of recurrence or subjects receiving steroids at a daily dose above 10mg of prednisone for an active autoimmune or other condition were excluded from the study. The primary endpoint of the study was the percentage of patients without disease recurrence. Data collection Fresh frozen tissue samples were acquired from patients. Tumor tissue was acquired after surgical resection, cut in 5 mg cubes and stored in stabilization solution (RNA later, Thermo Fisher Scientific) at -80 o C freezer, after written informed consent was obtained. DNA extraction and quantification was performed from 5 mg of fresh-frozen tissue. Library preparation was conducted with the use of DNA AmpliSeq for Ion Torrent, with an input of 40 ng DNA per sample. Sample libraries were quantified with the use of Qubit (Thermo-Fisher Scientific) and real-time polymerase chain reaction (PCR), then sequenced on Ion Torrent S5 sequencer, using the Oncomine Kidney panel (Thermo-Fisher Scientific). Library amplification was conducted using the Library PLUS for Ion Torrent kit (Thermo Fisher). Library amplification included enzyme activation at 99 o C for 2 minutes, 16 cycles of denaturation at 99 o C for 15 seconds and annealing and extension steps at 60 o C for 4 minutes and a final hold step at 10 o C. The commercially available Oncomine TM Kidney Panel (Thermo Fisher Scientific) provided the primer pairs used for library preparation. qPCR was conducted using the Ion Universal Library Quantitation Kit (Thermo Fisher Scientific). qPCR steps include an initial step of incubation at 50 o C for 2 minutes, a polymerase activation step at 95 o C for 2 minutes, 40 cycles of 95 o C for 15 seconds and 60 o C for 1 minute and a final hold step at 10 o C. 10 Clinicopathological characteristics recorded for the analysis included patient age, sex, ISUP grade, tumor diameter, T-stage, presence of vascular invasion, presence of sarcomatoid differentiation, presence of necrosis, AJCC stage, and emergence of recurrent disease during follow up. Nephrectomy surgical specimens were reviewed by two independent pathologists from our institution. A publicly available database, cBioportal for Cancer Genomics (accessed on 24 May 2023), was used to query DNA sequencing data for mutations in a prospective multicenter cohort from The Cancer Genome Atlas (TCGA ) including 451 patients with ccRCC (accessed on 24 May 2023). Data analysis The following genes were analyzed for presence of somatic mutations: ATM, BAP1, KDM5C, MET, MTOR, NF2, PBRM1, PIK3CA, PTEN, SETD2, SMARCB1, TP53, TSC1, TSC2 , and VHL. Variant calling was performed using the Ion Reporter Software (Thermo-Fisher Scientific). The Pearson’s Chi squared test was used to determine whether there was a statistically significant difference in clinicopathological characteristics and emergence of recurrence between subgroups of patients with distinct mutational profiles (unmutated or VHL -only mutated versus other gene mutations). Time-to-event outcomes (DFS) were estimated using the Kaplan-Meier method. Multiple hypothesis test correction was applied using the Benjamini–Hochberg method. All tests were two-sided, and p and q values ≤0.05 were considered statistically significant. The IBM SPSS v.22 software was used for the analysis. Results Mutations in non- VHL genes are associated with more aggressive disease We first assessed the frequency of mutations in primary ccRCC tumors. In the discovery cohort (n=37), patients’ clinical and histopathological characteristics are described in Table 1 . VHL was the most frequently mutated gene (n=19; 51%), followed by PBRM1 (n=10; 27%), BAP1 (n=5; 13%), SETD2 (n=5; 13%), KDM5C (n=2, 5%), ATM (n=2, 5%), MTOR (n=2, 5%), and PTEN (n=1, 3%) ( Table 2 ). Variant types per gene are listed in the data file. 35 11 patients (30%) did not have any somatic mutations within the 15-gene targeted panel. Table 1. Clinical and histopathological characteristics of ccRCC patients (discovery cohort, n=37). Characteristic Number (%) Age median (range) 63 (42-87) Sex males 28 (75) Tumor diameter (cm) median (range) 5.6 (1.6 – 15) Pathological T stage T1 21 (57) T2 6 (16) T3 10 (27) ISUP grade 2 13 (35) 3 15 (41) 4 8 (24) Vascular invasion 5 (14) Necrosis 13 (35) Sarcomatoid differentiation 0 AJCC TNM stage I 21 (57) II 6 (16) III 10 (27) Table 2. Frequency of mutated genes in primary ccRCC tumors (discovery cohort, n=37). Gene Frequency, n (%) VHL 19 (51) PBRM1 10 (27) BAP1 5 (13) SETD2 5 (13) KDM5C 2 (5) ATM 2 (5) MTOR 2 (5) PTEN 1 (3) Tumors harboring no mutations at all or only VHL mutations (n=19, 51%) were associated with smaller size (pT1-2 n=17, 89%) and earlier stage (I/II n=17; 89%), whereas presence of any other gene mutations in various combinations with or without VHL was enriched in larger (pT3, n=8; 44%; p=0.02) and more advanced tumors (III, n=8; 44%; p=0.02) ( Table 3 ). There was also a trend towards higher frequency of ISUP grade, vascular invasion, and necrosis in these tumors ( Table 3 ). Table 3. Associations between histopathological characteristics and mutational profile (discovery cohort, n=37). Variable Mutated genes P value None or VHL -only Non- VHL Pathological T stage 0.02 T1/T2 17 (89) 8 (44) T3 2 (11) 10 (56) AJCC TNM stage 0.02 I/II 17 (89) 8 (44) III 2 (11) 10 (56) ISUP grade 0.93 2/3 15 (79) 14 (78) 4 4 (21) 4 (22) Necrosis 0.64 no 13 (68) 11 (61) yes 6 (32) 7 (39) Vascular invasion 0.58 no 17 (89) 15 (83) yes 2 (11) 3 (17) In the validation TCGA cohort (n=451), similar mutations frequencies were noted, including VHL in 50% of patients/samples, PBRRM1 in 29%, SETD2 in 11%, BAP1 in 9%, MTOR in 7%, KDM5C in 6%, PTEN in 4%, and ATM in 2.7%, respectively ( Figure 1 ). Larger and higher stage tumors, particularly T3a, T3b, and stage III tended to have a higher frequency of non-VHL mutations (p=0.282; q=0.437) ( Figure 2 ). Figure 1. Frequency of mutated genes in primary ccRCC tumors (validation cohort, n=451). Figure 2. Associations between histopathological characteristics and mutational profile (validation cohort, n=451). Mutations in non- VHL genes predict recurrence No recurrences were noted in patients with unmutated tumors or VHL -only mutations whereas three patients (17%) with other somatic mutations relapsed (p=0.06) ( Table 4 ). Another three patients received and completed adjuvant PD-1 inhibition with pembrolizumab until present, none of whom recurred despite the presence of PBRM1 and SETD2 mutations in 2/3 and 1/3, respectively. Presence of somatic mutations in PBRM1, BAP1, SETD2, KDM5C, ATM, MTOR , or PTEN genes in 451 TCGA ccRCC patients was associated with a significantly shorter DFS compared to those with unaltered tumors (p<0.001; q=0.01) ( Figure 3 ). Table 4. Associations of mutational profile (none or VHL-only versus non-VHL mutated genes) and disease recurrence. Outcome Mutated genes P value None or VHL -only Non- VHL Recurrence 0.06 no 19 (100) 15 (83) yes 0 (0) 3 (17) Figure 3. Kaplan-Meier analysis of DFS in patients with unmutated or VHL -only versus non- VHL mutated tumors (validation cohort, n=451). Discussion Cancer is a disease caused by the accumulation of genomic alterations. 11 This is a multistep process during which certain somatic mutations confer a survival advantage by activating signaling pathways that are associated with several hallmarks of cancer, including proliferative signaling, evasion of growth suppression, resistance to cell death, replicative immortality, angiogenesis, invasion and metastasis, reprogramming of energy metabolism and evasion of immune recognition and destruction. 12 Since these biological principles apply to the majority of solid tumors, including RCCs, we hypothesized that the higher the number of pathogenic mutations within a primary renal tumor, the more aggressive clinical behavior could be observed. The tumor suppressor VHL is the most frequently mutated gene in ccRCC and is a major player in renal cell carcinogenesis. However, VHL mutations alone are insufficient to drive disease progression, and have no prognostic or predictive value. 13 , 14 Mounting evidence has revealed an emerging role of other genes, heavily involved in chromatin rearrangement and epigenetic DNA modifications, including PBRM1, SETD2, BAP1 , and KDM5C in ccRCC progression. 15 , 16 Inactivating mutations or/and low expression of these genes in primary renal tumors have been associated with poor outcomes. 17 , 23 Interestingly, many of these frequently non- VHL genes, including PBRM1, BAP1, SETD2 , have been investigated in the metastatic setting as potential predictors of response or resistance to immune checkpoint inhibitors (ICIs). For example, mutated PBRM1 was included in a composite biomarker together with tumor infiltrating lymphocytes (TILs) and absence of necrosis predicting a favorable response to ICIs. 24 , 25 Likewise, SETD2 loss results in greater vulnerability to immune checkpoint blockade compared to SETD2-proficient tumors, in vitro and in vivo . 26 BAP1 -mutated tumors, while they portend a worse prognosis, they are more likely to be PD-L1 positive and demonstrate a more inflamed immune microenvironment suggesting that immune-targeting approaches could benefit these patients. 27 Another DNA damage response and repair gene, ATM, was the second most frequently altered gene in ICI-responders with advanced RCC. 28 Pancancer analyses of tumors from ICI-treated patients suggested that those with KDM5C alterations have a substantially higher tumor mutational burden (TMB) level and a significantly higher level of CD8+ T cell infiltration and T effector signature which were associated with prolonged OS compared to the wild-type group. 29 Expression of PTEN/PI3K/mTOR pathway genes was significantly associated with numerous immune cells and immune-evasive mediators such as CD274/PD-L1, TGFBR1, CSF1R and PDCD1 in patients with ccRCC, 30 suggesting that alterations in this pathway could also play a role in shaping these patients’ outcomes after treatment with ICIs. Our ongoing prospective study examined the mutational profile of patients with ccRCC on primary tumors after nephrectomy followed by observation or adjuvant immunotherapy with pembrolizumab and assessed for associations of mutated genes with high-risk features and DFS. We hypothesized that primary RCC tumors either umutated or harboring VHL mutations might be associated with a more benign clinical course after nephrectomy compared to those with a burden of mutations in other, non-VHL genes. In this preliminary analysis of the first 37 patients, non- VHL mutations, including mutations in PBRM1, SETD2, BAP1, KDM5C, MTOR, PTEN , or ATM genes in a targeted 15-gene NGS panel were significantly associated with more aggressive histopathological characteristics including larger size, and higher stage. Presence of mutations in any of those genes as opposed to completely unmutated or VHL -only mutated tumors was also associated with higher ISUP grade, necrosis, sarcomatoid differentiation, vascular invasion and predicted recurrence. In ccRCC tumors from TCGA, non- VHL mutations in the same genes were also associated with high-risk features and predicted a significantly shorter DFS compared to VHL -only mutations or complete absence thereof. Due to the retrospective nature of previous studies and lack of reproducibility, particularly across immunohistochemical assessments 31 , 32 in an era when observation was the only available modality post-operatively even in high-risk patients, there has been a paucity of data to support testing of these genes as a molecular tool to assist in selection of patients who might benefit from adjuvant therapy. Our ongoing prospective study addresses this gap by demonstrating that patients who had either no mutation or mutations in the most frequently altered gene, VHL , were more likely to have smaller tumors and experienced a more benign course without relapse, compared to those patients with tumors that harbored mutations in other genes, including PBRM1, BAP1, SETD2, KDM5C, ATM, MTOR , or PTEN. Thus, this study suggests that patients with mutations in these high-risk genes might be more suitable candidates and should be prioritized for post-operative immunotherapy with pembrolizumab, which has demonstrated clinical benefit in PFS and OS and is currently approved as adjuvant treatment. 33 Our findings are in line with a previous work with slightly different design, whereby primary RCC tumors were segregated into VHL +0, VHL +1, VHL +2, and VHL +≥3 mutations. 33 In both the discovery and validation cohorts of the study, those patients with a VHL+0 tumor had longer 5-year DFS and were proposed as candidates for surveillance. Conversely, patients with VHL+2 and VHL+≥3 tumors experienced shorter DFS rates of less than 50% and were deemed candidates for adjuvant therapy. 34 Our study was limited by small size and relatively short follow-up of patients. Additionally, the lack of transcriptional or/and epigenetic analyses is another limitation of our study. Nevertheless, presence of an early “signal” of high-risk genes in this preliminary report will be further studied in additional patients being accrued as part of this ongoing prospective study. Collectively, our study combined with emerging evidence on the genomic landscape of RCC might open new avenues for both prognostication and better selection of a subgroup of patients with RCC that could benefit from adjuvant anti-PD1 immunotherapy. Data availability Underlying data figshare: F1000Res_Vlachostergios et al_raw data (subm).xlsx. https://doi.org/10.6084/m9.figshare.23697252.v1 . 23 Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). Data from the validation TCGA cohort analyzed in this study are available from cbioportal.org , http://www.cbioportal.org/study/summary?id=kirc_tcga . References 1. 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Cancer Res. 2023; 29 :1220–1231. PubMed Abstract | Publisher Full Text | Free Full Text 35. Vlachostergios PJ: F1000Res_Vlachostergios et al_raw data (subm).xlsx. figshare. Figure. 2023. Publisher Full Text Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 01 Aug 2023 ADD YOUR COMMENT Comment Author details Author details 1 Urology, University of Thessaly, Faculty of Medicine, School of Health Sciences, University Hospital of Larissa, Larissa, Thessalia, Greece 2 Medical Oncology, IASO Thessalias Hospital, Larissa, Thessalia, Greece 3 Hematology & Medical Oncology, Weill Cornell Medicine, New York, New York, USA 4 Pathology, University of Thessaly, Faculty of Medicine, School of Health Sciences, Larissa, Greece 5 Computer Science and Biomedical Informatics, University of Thessaly, Lamia, Greece Panagiotis J. Vlachostergios Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Maria Papathanassiou Roles: Data Curation, Formal Analysis, Funding Acquisition, Investigation, Writing – Original Draft Preparation Maria Anagnostou Roles: Data Curation, Investigation, Writing – Original Draft Preparation Eleni Thodou Roles: Investigation, Writing – Review & Editing Ioannis Tamposis Roles: Funding Acquisition, Investigation, Methodology, Resources, Software, Writing – Review & Editing Lampros Mitrakas Roles: Investigation, Methodology, Writing – Review & Editing Ioannis Zachos Roles: Investigation, Resources, Writing – Review & Editing George K. Koukoulis Roles: Investigation, Resources, Writing – Review & Editing Maria Samara Roles: Conceptualization, Funding Acquisition, Investigation, Project Administration, Resources, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing Vassilios Tzortzis Roles: Conceptualization, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Supervision, Writing – Review & Editing Competing interests No competing interests were disclosed. Grant information This work was funded by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH—CREATE—INNOVATE, project code: T2EDK-03079. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (2) version 2 Revised Published: 14 Jun 2024, 12:918 https://doi.org/10.12688/f1000research.136087.2 version 1 Published: 01 Aug 2023, 12:918 https://doi.org/10.12688/f1000research.136087.1 Copyright © 2024 Vlachostergios PJ et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Vlachostergios PJ, Papathanassiou M, Anagnostou M et al. Mutational profile of primary clear cell renal cell carcinoma predicts recurrence and potential candidacy for adjuvant immune checkpoint inhibition [version 2; peer review: 2 approved] . F1000Research 2024, 12 :918 ( https://doi.org/10.12688/f1000research.136087.2 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 2 VERSION 2 PUBLISHED 14 Jun 2024 Revised Views 0 Cite How to cite this report: Kamposioras K. Reviewer Report For: Mutational profile of primary clear cell renal cell carcinoma predicts recurrence and potential candidacy for adjuvant immune checkpoint inhibition [version 2; peer review: 2 approved] . F1000Research 2024, 12 :918 ( https://doi.org/10.5256/f1000research.163600.r291419 ) The direct URL for this report is: https://f1000research.com/articles/12-918/v2#referee-response-291419 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 25 Jun 2024 Konstantinos Kamposioras , Department of Medical Oncology, The Christie NHS Foundation Trust, Manchester, UK Approved VIEWS 0 https://doi.org/10.5256/f1000research.163600.r291419 The authors have dealt with the ... Continue reading READ ALL The authors have dealt with the points raised in an appropriate manner. Competing Interests: No competing interests were disclosed. Reviewer Expertise: Medical Oncology, Clinical biomarkers I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Kamposioras K. Reviewer Report For: Mutational profile of primary clear cell renal cell carcinoma predicts recurrence and potential candidacy for adjuvant immune checkpoint inhibition [version 2; peer review: 2 approved] . F1000Research 2024, 12 :918 ( https://doi.org/10.5256/f1000research.163600.r291419 ) The direct URL for this report is: https://f1000research.com/articles/12-918/v2#referee-response-291419 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Version 1 VERSION 1 PUBLISHED 01 Aug 2023 Views 0 Cite How to cite this report: Pavlopoulou A. Reviewer Report For: Mutational profile of primary clear cell renal cell carcinoma predicts recurrence and potential candidacy for adjuvant immune checkpoint inhibition [version 2; peer review: 2 approved] . F1000Research 2024, 12 :918 ( https://doi.org/10.5256/f1000research.149223.r228073 ) The direct URL for this report is: https://f1000research.com/articles/12-918/v1#referee-response-228073 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 09 Mar 2024 Athanasia Pavlopoulou , Dokuz Eylül University, Izmir, İzmir, Turkey Approved VIEWS 0 https://doi.org/10.5256/f1000research.149223.r228073 The authors conducted a study of the molecular profile of RCC primary tumors in a small cohort of patients and examined whether particular gene mutations were associated with clinicopathological characteristics and outcomes of patients during a short follow up period. ... Continue reading READ ALL The authors conducted a study of the molecular profile of RCC primary tumors in a small cohort of patients and examined whether particular gene mutations were associated with clinicopathological characteristics and outcomes of patients during a short follow up period. The following points need to be clarified: - how do the specific genes examined in this study relate to RCC prognosis in the literature? - the lack of transcriptional or epigenetic analyses should be discussed as a limitation of the study. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Molecular Biology; Computational Biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Pavlopoulou A. Reviewer Report For: Mutational profile of primary clear cell renal cell carcinoma predicts recurrence and potential candidacy for adjuvant immune checkpoint inhibition [version 2; peer review: 2 approved] . F1000Research 2024, 12 :918 ( https://doi.org/10.5256/f1000research.149223.r228073 ) The direct URL for this report is: https://f1000research.com/articles/12-918/v1#referee-response-228073 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 14 Jun 2024 Panagiotis J. Vlachostergios , Hematology & Medical Oncology, Weill Cornell Medicine, New York, USA 14 Jun 2024 Author Response Reviewer 2 The authors conducted a study of the molecular profile of RCC primary tumors in a small cohort of patients and examined whether particular gene mutations were associated with ... Continue reading Reviewer 2 The authors conducted a study of the molecular profile of RCC primary tumors in a small cohort of patients and examined whether particular gene mutations were associated with clinicopathological characteristics and outcomes of patients during a short follow up period. The following points need to be clarified: 1. How do the specific genes examined in this study relate to RCC prognosis in the literature? Response : We appreciate the Reviewer’s comment. We have indicated in the Discussion section that: ”Inactivating mutations or/and low expression of these genes in primary renal tumors have been associated with poor outcomes. 14 – 20 ” 2. The lack of transcriptional or epigenetic analyses should be discussed as a limitation of the study. Response: We appreciate the Reviewer’s comment. We have now discussed the lack of transcriptional or/and epigenetic analyses as a limitation of our study, in the Discussion section. All edits in the revised manuscript are highlighted with ”Track changes” tool of MS Word. Reviewer 2 The authors conducted a study of the molecular profile of RCC primary tumors in a small cohort of patients and examined whether particular gene mutations were associated with clinicopathological characteristics and outcomes of patients during a short follow up period. The following points need to be clarified: 1. How do the specific genes examined in this study relate to RCC prognosis in the literature? Response : We appreciate the Reviewer’s comment. We have indicated in the Discussion section that: ”Inactivating mutations or/and low expression of these genes in primary renal tumors have been associated with poor outcomes. 14 – 20 ” 2. The lack of transcriptional or epigenetic analyses should be discussed as a limitation of the study. Response: We appreciate the Reviewer’s comment. We have now discussed the lack of transcriptional or/and epigenetic analyses as a limitation of our study, in the Discussion section. All edits in the revised manuscript are highlighted with ”Track changes” tool of MS Word. Competing Interests: None. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 14 Jun 2024 Panagiotis J. Vlachostergios , Hematology & Medical Oncology, Weill Cornell Medicine, New York, USA 14 Jun 2024 Author Response Reviewer 2 The authors conducted a study of the molecular profile of RCC primary tumors in a small cohort of patients and examined whether particular gene mutations were associated with ... Continue reading Reviewer 2 The authors conducted a study of the molecular profile of RCC primary tumors in a small cohort of patients and examined whether particular gene mutations were associated with clinicopathological characteristics and outcomes of patients during a short follow up period. The following points need to be clarified: 1. How do the specific genes examined in this study relate to RCC prognosis in the literature? Response : We appreciate the Reviewer’s comment. We have indicated in the Discussion section that: ”Inactivating mutations or/and low expression of these genes in primary renal tumors have been associated with poor outcomes. 14 – 20 ” 2. The lack of transcriptional or epigenetic analyses should be discussed as a limitation of the study. Response: We appreciate the Reviewer’s comment. We have now discussed the lack of transcriptional or/and epigenetic analyses as a limitation of our study, in the Discussion section. All edits in the revised manuscript are highlighted with ”Track changes” tool of MS Word. Reviewer 2 The authors conducted a study of the molecular profile of RCC primary tumors in a small cohort of patients and examined whether particular gene mutations were associated with clinicopathological characteristics and outcomes of patients during a short follow up period. The following points need to be clarified: 1. How do the specific genes examined in this study relate to RCC prognosis in the literature? Response : We appreciate the Reviewer’s comment. We have indicated in the Discussion section that: ”Inactivating mutations or/and low expression of these genes in primary renal tumors have been associated with poor outcomes. 14 – 20 ” 2. The lack of transcriptional or epigenetic analyses should be discussed as a limitation of the study. Response: We appreciate the Reviewer’s comment. We have now discussed the lack of transcriptional or/and epigenetic analyses as a limitation of our study, in the Discussion section. All edits in the revised manuscript are highlighted with ”Track changes” tool of MS Word. Competing Interests: None. Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Kamposioras K. Reviewer Report For: Mutational profile of primary clear cell renal cell carcinoma predicts recurrence and potential candidacy for adjuvant immune checkpoint inhibition [version 2; peer review: 2 approved] . F1000Research 2024, 12 :918 ( https://doi.org/10.5256/f1000research.149223.r192983 ) The direct URL for this report is: https://f1000research.com/articles/12-918/v1#referee-response-192983 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 22 Aug 2023 Konstantinos Kamposioras , Department of Medical Oncology, The Christie NHS Foundation Trust, Manchester, UK Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.149223.r192983 I would like to congratulate the authors on their excellent work. However, there are a few points that need further explanation. The authors evaluated a number of genes in relation to clinical outcomes in a small number ... Continue reading READ ALL I would like to congratulate the authors on their excellent work. However, there are a few points that need further explanation. The authors evaluated a number of genes in relation to clinical outcomes in a small number of cases. I would like to see the authors explain the biological rationale behind the two different molecular signatures and how this might relate to immunotherapy response, as suggested in the Discussion section. I would also like to know how this study compares with previously published reports and if there is any consistency in the literature with regard to their findings. How do the authors see these findings changing or guiding clinical practice? Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Medical Oncology, Clinical biomarkers I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Kamposioras K. Reviewer Report For: Mutational profile of primary clear cell renal cell carcinoma predicts recurrence and potential candidacy for adjuvant immune checkpoint inhibition [version 2; peer review: 2 approved] . F1000Research 2024, 12 :918 ( https://doi.org/10.5256/f1000research.149223.r192983 ) The direct URL for this report is: https://f1000research.com/articles/12-918/v1#referee-response-192983 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 14 Jun 2024 Panagiotis J. Vlachostergios , Hematology & Medical Oncology, Weill Cornell Medicine, New York, USA 14 Jun 2024 Author Response Reviewer 1 I would like to congratulate the authors on their excellent work. However, there are a few points that need further explanation. The authors evaluated a number of ... Continue reading Reviewer 1 I would like to congratulate the authors on their excellent work. However, there are a few points that need further explanation. The authors evaluated a number of genes in relation to clinical outcomes in a small number of cases. 1. I would like to see the authors explain the biological rationale behind the two different molecular signatures and how this might relate to immunotherapy response, as suggested in the Discussion section. Response: We appreciate the Reviewer’s comment. We have added a detailed discussion on the biological rationale of our study and how the selected genes may relate to response to immune checkpoint inhibition, as follows: “Cancer is a disease caused by the accumulation of genomic alterations. 11 This is a multistep process during which certain somatic mutations confer a survival advantage by activating signaling pathways that are associated with several hallmarks of cancer, including proliferative signaling, evasion of growth suppression, resistance to cell death, replicative immortality, angiogenesis, invasion and metastasis, reprogramming of energy metabolism and evasion of immune recognition and destruction. 12 Since these biological principles apply to the majority of solid tumors, including RCCs, we hypothesized that the higher the number of pathogenic mutations within a primary renal tumor, the more aggressive clinical behavior could be observed. The tumor suppressor VHL is the most frequently mutated gene in ccRCC and is a major player in renal cell carcinogenesis. However, VHL mutations alone are insufficient to drive disease progression, and have no prognostic or predictive value. 13,14 Mounting evidence has revealed an emerging role of other genes, heavily involved in chromatin rearrangement and epigenetic DNA modifications, including PBRM1, SETD2, BAP1, and KDM5C in ccRCC progression. 15 , 16 Inactivating mutations or/and low expression of these genes in primary renal tumors have been associated with poor outcomes. 17 – 23 Interestingly, many of these frequently non-VHL genes, including PBRM1, BAP1, SETD2, have been investigated in the metastatic setting as potential predictors of response or resistance to immune checkpoint inhibitors (ICIs). For example, mutated PBRM1 was included in a composite biomarker together with tumor infiltrating lymphocytes (TILs) and absence of necrosis predicting a favorable response to ICIs. 24,25 Likewise, SETD2 loss results in greater vulnerability to immune checkpoint blockade compared to SETD2-proficient tumors, in vitro and in vivo. 26 BAP1-mutated tumors, while they portend a worse prognosis, they are more likely to be PD-L1 positive and demonstrate a more inflamed immune microenvironment suggesting that immune-targeting approaches could benefit these patients. 27 Another DNA damage response and repair gene, ATM, was the second most frequently altered gene in ICI-responders with advanced RCC. 28 Pancancer analyses of tumors from ICI-treated patients suggested that those with KDM5C alterations have a substantially higher tumor mutational burden (TMB) level and a significantly higher level of CD8+ T cell infiltration and T effector signature which were associated with prolonged OS compared to the wild-type group. 29 Expression of PTEN/PI3K/mTOR pathway genes was significantly associated with numerous immune cells and immune-evasive mediators such as CD274/PD-L1, TGFBR1, CSF1R and PDCD1 in patients with ccRCC, 30 suggesting that alterations in this pathway could also play a role in shaping these patients’ outcomes after treatment with ICIs. Our ongoing prospective study examined the mutational profile of patients with ccRCC on primary tumors after nephrectomy followed by observation or adjuvant immunotherapy with pembrolizumab and assessed for associations of mutated genes with high-risk features and DFS. We hypothesized that primary RCC tumors either umutated or harboring VHL mutations might be associated with a more benign clinical course after nephrectomy compared to those with a burden of mutations in other, non-VHL genes.“ References (new): 11. Meyerson M, Gabriel S, Getz G: Advances in understanding cancer genomes through second-generation sequencing. Nat Rev Genet. 2010;11:685-696. 10.1038/nrg2841 12. Hanahan D, Weinberg RA: Hallmarks of cancer: the next generation. Cell. 2011; 144:646-674. 10.1016/j.cell.2011.02.013. 14. Kim BJ, Kim JH, Kim HS, Zang DY: Prognostic and predictive value of VHL gene alteration in renal cell carcinoma: a meta-analysis and review. Oncotarget. 2017;8:13979-13985. 10.18632/oncotarget.14704. PMC5355155. 24. Deutsch JS, Lipson EJ, Danilova L, et al: Combinatorial biomarker for predicting outcomes to anti-PD-1 therapy in patients with metastatic clear cell renal cell carcinoma. Cell Rep Med. 2023; 4:100947. 10.1016/j.xcrm.2023.100947. PMC9975323. 25. Wang N, Qin Y, Du F, Wang X, Song C: Prevalence of SWI/SNF genomic alterations in cancer and association with the response to immune checkpoint inhibitors: A systematic review and meta-analysis. Gene. 2022; 834:146638. 10.1016/j.gene.2022.146638. 26. Liu XD, Zhang YT, McGrail DJ, et al: SETD2 Loss and ATR Inhibition Synergize to Promote cGAS Signaling and Immunotherapy Response in Renal Cell Carcinoma. Clin Cancer Res. 2023; 29:4002-4015. 10.1158/1078-0432.CCR-23-1003. PMC10592192. 27. Kapur P, Rajaram S, Brugarolas J. The expanding role of BAP1 in clear cell renal cell carcinoma. Hum Pathol. 2023; 133:22-31. 10.1016/j.humpath.2022.07.022. PMC9898467. 28. Ged Y, Chaim JL, DiNatale RG, et al: DNA damage repair pathway alterations in metastatic clear cell renal cell carcinoma and implications on systemic therapy. J Immunother Cancer. 2020;8:e000230. 10.1136/jitc-2019-000230. PMC7311069. 29. Chen XJ, Ren AQ, Zheng L, Zheng ED: Predictive Value of KDM5C Alterations for Immune Checkpoint Inhibitors Treatment Outcomes in Patients With Cancer. Front Immunol. 2021;12:664847. 10.3389/fimmu.2021.664847. PMC8089485. 30. Li N, Chen J, Liu Q, et al: Prognostic significance and tumor-immune infiltration of mTOR in clear cell renal cell carcinoma. PeerJ. 2021;9:e11901. 10.7717/peerj.11901. PMC8378334. 2. I would also like to know how this study compares with previously published reports and if there is any consistency in the literature with regard to their findings. Response: We have added relevant literature that is in concordance with our study findings, as follows: “Our findings are in line with a previous work with slightly different design, whereby primary RCC tumors were segregated into VHL+0, VHL+1, VHL+2, and VHL+≥3 mutations.33 In both the discovery and validation cohorts of the study, those patients with a VHL+0 tumor had longer 5-year DFS and were proposed as candidates for surveillance. Conversely, patients with VHL+2 and VHL+≥3 tumors experienced shorter DFS rates of less than 50% and were deemed candidates for adjuvant therapy. 33 ” Reference (new): 33. Vasudev NS, Scelo G, Glennon KI, et al: Application of Genomic Sequencing to Refine Patient Stratification for Adjuvant Therapy in Renal Cell Carcinoma. Clin Cancer Res. 2023;29:1220-1231. 10.1158/1078-0432.CCR-22-1936. PMC10068441. 3. How do the authors see these findings changing or guiding clinical practice? Response: We have now added a paragraph discussing the potential implications of our study for changing or guiding clinical practice, as follows: ”Collectively, our study combined with emerging evidence on the genomic landscape of RCC might open new avenues for both prognostication and better selection of a subgroup of patients with RCC that could benefit from adjuvant anti-PD1 immunotherapy.” Reviewer 1 I would like to congratulate the authors on their excellent work. However, there are a few points that need further explanation. The authors evaluated a number of genes in relation to clinical outcomes in a small number of cases. 1. I would like to see the authors explain the biological rationale behind the two different molecular signatures and how this might relate to immunotherapy response, as suggested in the Discussion section. Response: We appreciate the Reviewer’s comment. We have added a detailed discussion on the biological rationale of our study and how the selected genes may relate to response to immune checkpoint inhibition, as follows: “Cancer is a disease caused by the accumulation of genomic alterations. 11 This is a multistep process during which certain somatic mutations confer a survival advantage by activating signaling pathways that are associated with several hallmarks of cancer, including proliferative signaling, evasion of growth suppression, resistance to cell death, replicative immortality, angiogenesis, invasion and metastasis, reprogramming of energy metabolism and evasion of immune recognition and destruction. 12 Since these biological principles apply to the majority of solid tumors, including RCCs, we hypothesized that the higher the number of pathogenic mutations within a primary renal tumor, the more aggressive clinical behavior could be observed. The tumor suppressor VHL is the most frequently mutated gene in ccRCC and is a major player in renal cell carcinogenesis. However, VHL mutations alone are insufficient to drive disease progression, and have no prognostic or predictive value. 13,14 Mounting evidence has revealed an emerging role of other genes, heavily involved in chromatin rearrangement and epigenetic DNA modifications, including PBRM1, SETD2, BAP1, and KDM5C in ccRCC progression. 15 , 16 Inactivating mutations or/and low expression of these genes in primary renal tumors have been associated with poor outcomes. 17 – 23 Interestingly, many of these frequently non-VHL genes, including PBRM1, BAP1, SETD2, have been investigated in the metastatic setting as potential predictors of response or resistance to immune checkpoint inhibitors (ICIs). For example, mutated PBRM1 was included in a composite biomarker together with tumor infiltrating lymphocytes (TILs) and absence of necrosis predicting a favorable response to ICIs. 24,25 Likewise, SETD2 loss results in greater vulnerability to immune checkpoint blockade compared to SETD2-proficient tumors, in vitro and in vivo. 26 BAP1-mutated tumors, while they portend a worse prognosis, they are more likely to be PD-L1 positive and demonstrate a more inflamed immune microenvironment suggesting that immune-targeting approaches could benefit these patients. 27 Another DNA damage response and repair gene, ATM, was the second most frequently altered gene in ICI-responders with advanced RCC. 28 Pancancer analyses of tumors from ICI-treated patients suggested that those with KDM5C alterations have a substantially higher tumor mutational burden (TMB) level and a significantly higher level of CD8+ T cell infiltration and T effector signature which were associated with prolonged OS compared to the wild-type group. 29 Expression of PTEN/PI3K/mTOR pathway genes was significantly associated with numerous immune cells and immune-evasive mediators such as CD274/PD-L1, TGFBR1, CSF1R and PDCD1 in patients with ccRCC, 30 suggesting that alterations in this pathway could also play a role in shaping these patients’ outcomes after treatment with ICIs. Our ongoing prospective study examined the mutational profile of patients with ccRCC on primary tumors after nephrectomy followed by observation or adjuvant immunotherapy with pembrolizumab and assessed for associations of mutated genes with high-risk features and DFS. We hypothesized that primary RCC tumors either umutated or harboring VHL mutations might be associated with a more benign clinical course after nephrectomy compared to those with a burden of mutations in other, non-VHL genes.“ References (new): 11. Meyerson M, Gabriel S, Getz G: Advances in understanding cancer genomes through second-generation sequencing. Nat Rev Genet. 2010;11:685-696. 10.1038/nrg2841 12. Hanahan D, Weinberg RA: Hallmarks of cancer: the next generation. Cell. 2011; 144:646-674. 10.1016/j.cell.2011.02.013. 14. Kim BJ, Kim JH, Kim HS, Zang DY: Prognostic and predictive value of VHL gene alteration in renal cell carcinoma: a meta-analysis and review. Oncotarget. 2017;8:13979-13985. 10.18632/oncotarget.14704. PMC5355155. 24. Deutsch JS, Lipson EJ, Danilova L, et al: Combinatorial biomarker for predicting outcomes to anti-PD-1 therapy in patients with metastatic clear cell renal cell carcinoma. Cell Rep Med. 2023; 4:100947. 10.1016/j.xcrm.2023.100947. PMC9975323. 25. Wang N, Qin Y, Du F, Wang X, Song C: Prevalence of SWI/SNF genomic alterations in cancer and association with the response to immune checkpoint inhibitors: A systematic review and meta-analysis. Gene. 2022; 834:146638. 10.1016/j.gene.2022.146638. 26. Liu XD, Zhang YT, McGrail DJ, et al: SETD2 Loss and ATR Inhibition Synergize to Promote cGAS Signaling and Immunotherapy Response in Renal Cell Carcinoma. Clin Cancer Res. 2023; 29:4002-4015. 10.1158/1078-0432.CCR-23-1003. PMC10592192. 27. Kapur P, Rajaram S, Brugarolas J. The expanding role of BAP1 in clear cell renal cell carcinoma. Hum Pathol. 2023; 133:22-31. 10.1016/j.humpath.2022.07.022. PMC9898467. 28. Ged Y, Chaim JL, DiNatale RG, et al: DNA damage repair pathway alterations in metastatic clear cell renal cell carcinoma and implications on systemic therapy. J Immunother Cancer. 2020;8:e000230. 10.1136/jitc-2019-000230. PMC7311069. 29. Chen XJ, Ren AQ, Zheng L, Zheng ED: Predictive Value of KDM5C Alterations for Immune Checkpoint Inhibitors Treatment Outcomes in Patients With Cancer. Front Immunol. 2021;12:664847. 10.3389/fimmu.2021.664847. PMC8089485. 30. Li N, Chen J, Liu Q, et al: Prognostic significance and tumor-immune infiltration of mTOR in clear cell renal cell carcinoma. PeerJ. 2021;9:e11901. 10.7717/peerj.11901. PMC8378334. 2. I would also like to know how this study compares with previously published reports and if there is any consistency in the literature with regard to their findings. Response: We have added relevant literature that is in concordance with our study findings, as follows: “Our findings are in line with a previous work with slightly different design, whereby primary RCC tumors were segregated into VHL+0, VHL+1, VHL+2, and VHL+≥3 mutations.33 In both the discovery and validation cohorts of the study, those patients with a VHL+0 tumor had longer 5-year DFS and were proposed as candidates for surveillance. Conversely, patients with VHL+2 and VHL+≥3 tumors experienced shorter DFS rates of less than 50% and were deemed candidates for adjuvant therapy. 33 ” Reference (new): 33. Vasudev NS, Scelo G, Glennon KI, et al: Application of Genomic Sequencing to Refine Patient Stratification for Adjuvant Therapy in Renal Cell Carcinoma. Clin Cancer Res. 2023;29:1220-1231. 10.1158/1078-0432.CCR-22-1936. PMC10068441. 3. How do the authors see these findings changing or guiding clinical practice? Response: We have now added a paragraph discussing the potential implications of our study for changing or guiding clinical practice, as follows: ”Collectively, our study combined with emerging evidence on the genomic landscape of RCC might open new avenues for both prognostication and better selection of a subgroup of patients with RCC that could benefit from adjuvant anti-PD1 immunotherapy.” Competing Interests: None. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 14 Jun 2024 Panagiotis J. Vlachostergios , Hematology & Medical Oncology, Weill Cornell Medicine, New York, USA 14 Jun 2024 Author Response Reviewer 1 I would like to congratulate the authors on their excellent work. However, there are a few points that need further explanation. The authors evaluated a number of ... Continue reading Reviewer 1 I would like to congratulate the authors on their excellent work. However, there are a few points that need further explanation. The authors evaluated a number of genes in relation to clinical outcomes in a small number of cases. 1. I would like to see the authors explain the biological rationale behind the two different molecular signatures and how this might relate to immunotherapy response, as suggested in the Discussion section. Response: We appreciate the Reviewer’s comment. We have added a detailed discussion on the biological rationale of our study and how the selected genes may relate to response to immune checkpoint inhibition, as follows: “Cancer is a disease caused by the accumulation of genomic alterations. 11 This is a multistep process during which certain somatic mutations confer a survival advantage by activating signaling pathways that are associated with several hallmarks of cancer, including proliferative signaling, evasion of growth suppression, resistance to cell death, replicative immortality, angiogenesis, invasion and metastasis, reprogramming of energy metabolism and evasion of immune recognition and destruction. 12 Since these biological principles apply to the majority of solid tumors, including RCCs, we hypothesized that the higher the number of pathogenic mutations within a primary renal tumor, the more aggressive clinical behavior could be observed. The tumor suppressor VHL is the most frequently mutated gene in ccRCC and is a major player in renal cell carcinogenesis. However, VHL mutations alone are insufficient to drive disease progression, and have no prognostic or predictive value. 13,14 Mounting evidence has revealed an emerging role of other genes, heavily involved in chromatin rearrangement and epigenetic DNA modifications, including PBRM1, SETD2, BAP1, and KDM5C in ccRCC progression. 15 , 16 Inactivating mutations or/and low expression of these genes in primary renal tumors have been associated with poor outcomes. 17 – 23 Interestingly, many of these frequently non-VHL genes, including PBRM1, BAP1, SETD2, have been investigated in the metastatic setting as potential predictors of response or resistance to immune checkpoint inhibitors (ICIs). For example, mutated PBRM1 was included in a composite biomarker together with tumor infiltrating lymphocytes (TILs) and absence of necrosis predicting a favorable response to ICIs. 24,25 Likewise, SETD2 loss results in greater vulnerability to immune checkpoint blockade compared to SETD2-proficient tumors, in vitro and in vivo. 26 BAP1-mutated tumors, while they portend a worse prognosis, they are more likely to be PD-L1 positive and demonstrate a more inflamed immune microenvironment suggesting that immune-targeting approaches could benefit these patients. 27 Another DNA damage response and repair gene, ATM, was the second most frequently altered gene in ICI-responders with advanced RCC. 28 Pancancer analyses of tumors from ICI-treated patients suggested that those with KDM5C alterations have a substantially higher tumor mutational burden (TMB) level and a significantly higher level of CD8+ T cell infiltration and T effector signature which were associated with prolonged OS compared to the wild-type group. 29 Expression of PTEN/PI3K/mTOR pathway genes was significantly associated with numerous immune cells and immune-evasive mediators such as CD274/PD-L1, TGFBR1, CSF1R and PDCD1 in patients with ccRCC, 30 suggesting that alterations in this pathway could also play a role in shaping these patients’ outcomes after treatment with ICIs. Our ongoing prospective study examined the mutational profile of patients with ccRCC on primary tumors after nephrectomy followed by observation or adjuvant immunotherapy with pembrolizumab and assessed for associations of mutated genes with high-risk features and DFS. We hypothesized that primary RCC tumors either umutated or harboring VHL mutations might be associated with a more benign clinical course after nephrectomy compared to those with a burden of mutations in other, non-VHL genes.“ References (new): 11. Meyerson M, Gabriel S, Getz G: Advances in understanding cancer genomes through second-generation sequencing. Nat Rev Genet. 2010;11:685-696. 10.1038/nrg2841 12. Hanahan D, Weinberg RA: Hallmarks of cancer: the next generation. Cell. 2011; 144:646-674. 10.1016/j.cell.2011.02.013. 14. Kim BJ, Kim JH, Kim HS, Zang DY: Prognostic and predictive value of VHL gene alteration in renal cell carcinoma: a meta-analysis and review. Oncotarget. 2017;8:13979-13985. 10.18632/oncotarget.14704. PMC5355155. 24. Deutsch JS, Lipson EJ, Danilova L, et al: Combinatorial biomarker for predicting outcomes to anti-PD-1 therapy in patients with metastatic clear cell renal cell carcinoma. Cell Rep Med. 2023; 4:100947. 10.1016/j.xcrm.2023.100947. PMC9975323. 25. Wang N, Qin Y, Du F, Wang X, Song C: Prevalence of SWI/SNF genomic alterations in cancer and association with the response to immune checkpoint inhibitors: A systematic review and meta-analysis. Gene. 2022; 834:146638. 10.1016/j.gene.2022.146638. 26. Liu XD, Zhang YT, McGrail DJ, et al: SETD2 Loss and ATR Inhibition Synergize to Promote cGAS Signaling and Immunotherapy Response in Renal Cell Carcinoma. Clin Cancer Res. 2023; 29:4002-4015. 10.1158/1078-0432.CCR-23-1003. PMC10592192. 27. Kapur P, Rajaram S, Brugarolas J. The expanding role of BAP1 in clear cell renal cell carcinoma. Hum Pathol. 2023; 133:22-31. 10.1016/j.humpath.2022.07.022. PMC9898467. 28. Ged Y, Chaim JL, DiNatale RG, et al: DNA damage repair pathway alterations in metastatic clear cell renal cell carcinoma and implications on systemic therapy. J Immunother Cancer. 2020;8:e000230. 10.1136/jitc-2019-000230. PMC7311069. 29. Chen XJ, Ren AQ, Zheng L, Zheng ED: Predictive Value of KDM5C Alterations for Immune Checkpoint Inhibitors Treatment Outcomes in Patients With Cancer. Front Immunol. 2021;12:664847. 10.3389/fimmu.2021.664847. PMC8089485. 30. Li N, Chen J, Liu Q, et al: Prognostic significance and tumor-immune infiltration of mTOR in clear cell renal cell carcinoma. PeerJ. 2021;9:e11901. 10.7717/peerj.11901. PMC8378334. 2. I would also like to know how this study compares with previously published reports and if there is any consistency in the literature with regard to their findings. Response: We have added relevant literature that is in concordance with our study findings, as follows: “Our findings are in line with a previous work with slightly different design, whereby primary RCC tumors were segregated into VHL+0, VHL+1, VHL+2, and VHL+≥3 mutations.33 In both the discovery and validation cohorts of the study, those patients with a VHL+0 tumor had longer 5-year DFS and were proposed as candidates for surveillance. Conversely, patients with VHL+2 and VHL+≥3 tumors experienced shorter DFS rates of less than 50% and were deemed candidates for adjuvant therapy. 33 ” Reference (new): 33. Vasudev NS, Scelo G, Glennon KI, et al: Application of Genomic Sequencing to Refine Patient Stratification for Adjuvant Therapy in Renal Cell Carcinoma. Clin Cancer Res. 2023;29:1220-1231. 10.1158/1078-0432.CCR-22-1936. PMC10068441. 3. How do the authors see these findings changing or guiding clinical practice? Response: We have now added a paragraph discussing the potential implications of our study for changing or guiding clinical practice, as follows: ”Collectively, our study combined with emerging evidence on the genomic landscape of RCC might open new avenues for both prognostication and better selection of a subgroup of patients with RCC that could benefit from adjuvant anti-PD1 immunotherapy.” Reviewer 1 I would like to congratulate the authors on their excellent work. However, there are a few points that need further explanation. The authors evaluated a number of genes in relation to clinical outcomes in a small number of cases. 1. I would like to see the authors explain the biological rationale behind the two different molecular signatures and how this might relate to immunotherapy response, as suggested in the Discussion section. Response: We appreciate the Reviewer’s comment. We have added a detailed discussion on the biological rationale of our study and how the selected genes may relate to response to immune checkpoint inhibition, as follows: “Cancer is a disease caused by the accumulation of genomic alterations. 11 This is a multistep process during which certain somatic mutations confer a survival advantage by activating signaling pathways that are associated with several hallmarks of cancer, including proliferative signaling, evasion of growth suppression, resistance to cell death, replicative immortality, angiogenesis, invasion and metastasis, reprogramming of energy metabolism and evasion of immune recognition and destruction. 12 Since these biological principles apply to the majority of solid tumors, including RCCs, we hypothesized that the higher the number of pathogenic mutations within a primary renal tumor, the more aggressive clinical behavior could be observed. The tumor suppressor VHL is the most frequently mutated gene in ccRCC and is a major player in renal cell carcinogenesis. However, VHL mutations alone are insufficient to drive disease progression, and have no prognostic or predictive value. 13,14 Mounting evidence has revealed an emerging role of other genes, heavily involved in chromatin rearrangement and epigenetic DNA modifications, including PBRM1, SETD2, BAP1, and KDM5C in ccRCC progression. 15 , 16 Inactivating mutations or/and low expression of these genes in primary renal tumors have been associated with poor outcomes. 17 – 23 Interestingly, many of these frequently non-VHL genes, including PBRM1, BAP1, SETD2, have been investigated in the metastatic setting as potential predictors of response or resistance to immune checkpoint inhibitors (ICIs). For example, mutated PBRM1 was included in a composite biomarker together with tumor infiltrating lymphocytes (TILs) and absence of necrosis predicting a favorable response to ICIs. 24,25 Likewise, SETD2 loss results in greater vulnerability to immune checkpoint blockade compared to SETD2-proficient tumors, in vitro and in vivo. 26 BAP1-mutated tumors, while they portend a worse prognosis, they are more likely to be PD-L1 positive and demonstrate a more inflamed immune microenvironment suggesting that immune-targeting approaches could benefit these patients. 27 Another DNA damage response and repair gene, ATM, was the second most frequently altered gene in ICI-responders with advanced RCC. 28 Pancancer analyses of tumors from ICI-treated patients suggested that those with KDM5C alterations have a substantially higher tumor mutational burden (TMB) level and a significantly higher level of CD8+ T cell infiltration and T effector signature which were associated with prolonged OS compared to the wild-type group. 29 Expression of PTEN/PI3K/mTOR pathway genes was significantly associated with numerous immune cells and immune-evasive mediators such as CD274/PD-L1, TGFBR1, CSF1R and PDCD1 in patients with ccRCC, 30 suggesting that alterations in this pathway could also play a role in shaping these patients’ outcomes after treatment with ICIs. Our ongoing prospective study examined the mutational profile of patients with ccRCC on primary tumors after nephrectomy followed by observation or adjuvant immunotherapy with pembrolizumab and assessed for associations of mutated genes with high-risk features and DFS. We hypothesized that primary RCC tumors either umutated or harboring VHL mutations might be associated with a more benign clinical course after nephrectomy compared to those with a burden of mutations in other, non-VHL genes.“ References (new): 11. Meyerson M, Gabriel S, Getz G: Advances in understanding cancer genomes through second-generation sequencing. Nat Rev Genet. 2010;11:685-696. 10.1038/nrg2841 12. Hanahan D, Weinberg RA: Hallmarks of cancer: the next generation. Cell. 2011; 144:646-674. 10.1016/j.cell.2011.02.013. 14. Kim BJ, Kim JH, Kim HS, Zang DY: Prognostic and predictive value of VHL gene alteration in renal cell carcinoma: a meta-analysis and review. Oncotarget. 2017;8:13979-13985. 10.18632/oncotarget.14704. PMC5355155. 24. Deutsch JS, Lipson EJ, Danilova L, et al: Combinatorial biomarker for predicting outcomes to anti-PD-1 therapy in patients with metastatic clear cell renal cell carcinoma. Cell Rep Med. 2023; 4:100947. 10.1016/j.xcrm.2023.100947. PMC9975323. 25. Wang N, Qin Y, Du F, Wang X, Song C: Prevalence of SWI/SNF genomic alterations in cancer and association with the response to immune checkpoint inhibitors: A systematic review and meta-analysis. Gene. 2022; 834:146638. 10.1016/j.gene.2022.146638. 26. Liu XD, Zhang YT, McGrail DJ, et al: SETD2 Loss and ATR Inhibition Synergize to Promote cGAS Signaling and Immunotherapy Response in Renal Cell Carcinoma. Clin Cancer Res. 2023; 29:4002-4015. 10.1158/1078-0432.CCR-23-1003. PMC10592192. 27. Kapur P, Rajaram S, Brugarolas J. The expanding role of BAP1 in clear cell renal cell carcinoma. Hum Pathol. 2023; 133:22-31. 10.1016/j.humpath.2022.07.022. PMC9898467. 28. Ged Y, Chaim JL, DiNatale RG, et al: DNA damage repair pathway alterations in metastatic clear cell renal cell carcinoma and implications on systemic therapy. J Immunother Cancer. 2020;8:e000230. 10.1136/jitc-2019-000230. PMC7311069. 29. Chen XJ, Ren AQ, Zheng L, Zheng ED: Predictive Value of KDM5C Alterations for Immune Checkpoint Inhibitors Treatment Outcomes in Patients With Cancer. Front Immunol. 2021;12:664847. 10.3389/fimmu.2021.664847. PMC8089485. 30. Li N, Chen J, Liu Q, et al: Prognostic significance and tumor-immune infiltration of mTOR in clear cell renal cell carcinoma. PeerJ. 2021;9:e11901. 10.7717/peerj.11901. PMC8378334. 2. I would also like to know how this study compares with previously published reports and if there is any consistency in the literature with regard to their findings. Response: We have added relevant literature that is in concordance with our study findings, as follows: “Our findings are in line with a previous work with slightly different design, whereby primary RCC tumors were segregated into VHL+0, VHL+1, VHL+2, and VHL+≥3 mutations.33 In both the discovery and validation cohorts of the study, those patients with a VHL+0 tumor had longer 5-year DFS and were proposed as candidates for surveillance. Conversely, patients with VHL+2 and VHL+≥3 tumors experienced shorter DFS rates of less than 50% and were deemed candidates for adjuvant therapy. 33 ” Reference (new): 33. Vasudev NS, Scelo G, Glennon KI, et al: Application of Genomic Sequencing to Refine Patient Stratification for Adjuvant Therapy in Renal Cell Carcinoma. Clin Cancer Res. 2023;29:1220-1231. 10.1158/1078-0432.CCR-22-1936. PMC10068441. 3. How do the authors see these findings changing or guiding clinical practice? Response: We have now added a paragraph discussing the potential implications of our study for changing or guiding clinical practice, as follows: ”Collectively, our study combined with emerging evidence on the genomic landscape of RCC might open new avenues for both prognostication and better selection of a subgroup of patients with RCC that could benefit from adjuvant anti-PD1 immunotherapy.” Competing Interests: None. Close Report a concern COMMENT ON THIS REPORT Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 01 Aug 2023 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 Version 2 (revision) 14 Jun 24 read Version 1 01 Aug 23 read read Konstantinos Kamposioras , The Christie NHS Foundation Trust, Manchester, UK Athanasia Pavlopoulou , Dokuz Eylül University, Izmir, Turkey Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2024 Kamposioras K. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 25 Jun 2024 | for Version 2 Konstantinos Kamposioras , Department of Medical Oncology, The Christie NHS Foundation Trust, Manchester, UK 0 Views copyright © 2024 Kamposioras K. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The authors have dealt with the points raised in an appropriate manner. Competing Interests No competing interests were disclosed. Reviewer Expertise Medical Oncology, Clinical biomarkers I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Kamposioras K. Peer Review Report For: Mutational profile of primary clear cell renal cell carcinoma predicts recurrence and potential candidacy for adjuvant immune checkpoint inhibition [version 2; peer review: 2 approved] . F1000Research 2024, 12 :918 ( https://doi.org/10.5256/f1000research.163600.r291419) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-918/v2#referee-response-291419 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2024 Pavlopoulou A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 09 Mar 2024 | for Version 1 Athanasia Pavlopoulou , Dokuz Eylül University, Izmir, İzmir, Turkey 0 Views copyright © 2024 Pavlopoulou A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The authors conducted a study of the molecular profile of RCC primary tumors in a small cohort of patients and examined whether particular gene mutations were associated with clinicopathological characteristics and outcomes of patients during a short follow up period. The following points need to be clarified: - how do the specific genes examined in this study relate to RCC prognosis in the literature? - the lack of transcriptional or epigenetic analyses should be discussed as a limitation of the study. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise Molecular Biology; Computational Biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (1) Author Response 14 Jun 2024 Panagiotis J. Vlachostergios, Hematology & Medical Oncology, Weill Cornell Medicine, New York, USA Reviewer 2 The authors conducted a study of the molecular profile of RCC primary tumors in a small cohort of patients and examined whether particular gene mutations were associated with clinicopathological characteristics and outcomes of patients during a short follow up period. The following points need to be clarified: 1. How do the specific genes examined in this study relate to RCC prognosis in the literature? Response : We appreciate the Reviewer’s comment. We have indicated in the Discussion section that: ”Inactivating mutations or/and low expression of these genes in primary renal tumors have been associated with poor outcomes. 14 – 20 ” 2. The lack of transcriptional or epigenetic analyses should be discussed as a limitation of the study. Response: We appreciate the Reviewer’s comment. We have now discussed the lack of transcriptional or/and epigenetic analyses as a limitation of our study, in the Discussion section. All edits in the revised manuscript are highlighted with ”Track changes” tool of MS Word. View more View less Competing Interests None. reply Respond Report a concern Pavlopoulou A. Peer Review Report For: Mutational profile of primary clear cell renal cell carcinoma predicts recurrence and potential candidacy for adjuvant immune checkpoint inhibition [version 2; peer review: 2 approved] . F1000Research 2024, 12 :918 ( https://doi.org/10.5256/f1000research.149223.r228073) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-918/v1#referee-response-228073 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2023 Kamposioras K. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 22 Aug 2023 | for Version 1 Konstantinos Kamposioras , Department of Medical Oncology, The Christie NHS Foundation Trust, Manchester, UK 0 Views copyright © 2023 Kamposioras K. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions I would like to congratulate the authors on their excellent work. However, there are a few points that need further explanation. The authors evaluated a number of genes in relation to clinical outcomes in a small number of cases. I would like to see the authors explain the biological rationale behind the two different molecular signatures and how this might relate to immunotherapy response, as suggested in the Discussion section. I would also like to know how this study compares with previously published reports and if there is any consistency in the literature with regard to their findings. How do the authors see these findings changing or guiding clinical practice? Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise Medical Oncology, Clinical biomarkers I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 14 Jun 2024 Panagiotis J. Vlachostergios, Hematology & Medical Oncology, Weill Cornell Medicine, New York, USA Reviewer 1 I would like to congratulate the authors on their excellent work. However, there are a few points that need further explanation. The authors evaluated a number of genes in relation to clinical outcomes in a small number of cases. 1. I would like to see the authors explain the biological rationale behind the two different molecular signatures and how this might relate to immunotherapy response, as suggested in the Discussion section. Response: We appreciate the Reviewer’s comment. We have added a detailed discussion on the biological rationale of our study and how the selected genes may relate to response to immune checkpoint inhibition, as follows: “Cancer is a disease caused by the accumulation of genomic alterations. 11 This is a multistep process during which certain somatic mutations confer a survival advantage by activating signaling pathways that are associated with several hallmarks of cancer, including proliferative signaling, evasion of growth suppression, resistance to cell death, replicative immortality, angiogenesis, invasion and metastasis, reprogramming of energy metabolism and evasion of immune recognition and destruction. 12 Since these biological principles apply to the majority of solid tumors, including RCCs, we hypothesized that the higher the number of pathogenic mutations within a primary renal tumor, the more aggressive clinical behavior could be observed. The tumor suppressor VHL is the most frequently mutated gene in ccRCC and is a major player in renal cell carcinogenesis. However, VHL mutations alone are insufficient to drive disease progression, and have no prognostic or predictive value. 13,14 Mounting evidence has revealed an emerging role of other genes, heavily involved in chromatin rearrangement and epigenetic DNA modifications, including PBRM1, SETD2, BAP1, and KDM5C in ccRCC progression. 15 , 16 Inactivating mutations or/and low expression of these genes in primary renal tumors have been associated with poor outcomes. 17 – 23 Interestingly, many of these frequently non-VHL genes, including PBRM1, BAP1, SETD2, have been investigated in the metastatic setting as potential predictors of response or resistance to immune checkpoint inhibitors (ICIs). For example, mutated PBRM1 was included in a composite biomarker together with tumor infiltrating lymphocytes (TILs) and absence of necrosis predicting a favorable response to ICIs. 24,25 Likewise, SETD2 loss results in greater vulnerability to immune checkpoint blockade compared to SETD2-proficient tumors, in vitro and in vivo. 26 BAP1-mutated tumors, while they portend a worse prognosis, they are more likely to be PD-L1 positive and demonstrate a more inflamed immune microenvironment suggesting that immune-targeting approaches could benefit these patients. 27 Another DNA damage response and repair gene, ATM, was the second most frequently altered gene in ICI-responders with advanced RCC. 28 Pancancer analyses of tumors from ICI-treated patients suggested that those with KDM5C alterations have a substantially higher tumor mutational burden (TMB) level and a significantly higher level of CD8+ T cell infiltration and T effector signature which were associated with prolonged OS compared to the wild-type group. 29 Expression of PTEN/PI3K/mTOR pathway genes was significantly associated with numerous immune cells and immune-evasive mediators such as CD274/PD-L1, TGFBR1, CSF1R and PDCD1 in patients with ccRCC, 30 suggesting that alterations in this pathway could also play a role in shaping these patients’ outcomes after treatment with ICIs. Our ongoing prospective study examined the mutational profile of patients with ccRCC on primary tumors after nephrectomy followed by observation or adjuvant immunotherapy with pembrolizumab and assessed for associations of mutated genes with high-risk features and DFS. We hypothesized that primary RCC tumors either umutated or harboring VHL mutations might be associated with a more benign clinical course after nephrectomy compared to those with a burden of mutations in other, non-VHL genes.“ References (new): 11. Meyerson M, Gabriel S, Getz G: Advances in understanding cancer genomes through second-generation sequencing. Nat Rev Genet. 2010;11:685-696. 10.1038/nrg2841 12. Hanahan D, Weinberg RA: Hallmarks of cancer: the next generation. Cell. 2011; 144:646-674. 10.1016/j.cell.2011.02.013. 14. Kim BJ, Kim JH, Kim HS, Zang DY: Prognostic and predictive value of VHL gene alteration in renal cell carcinoma: a meta-analysis and review. Oncotarget. 2017;8:13979-13985. 10.18632/oncotarget.14704. PMC5355155. 24. Deutsch JS, Lipson EJ, Danilova L, et al: Combinatorial biomarker for predicting outcomes to anti-PD-1 therapy in patients with metastatic clear cell renal cell carcinoma. Cell Rep Med. 2023; 4:100947. 10.1016/j.xcrm.2023.100947. PMC9975323. 25. Wang N, Qin Y, Du F, Wang X, Song C: Prevalence of SWI/SNF genomic alterations in cancer and association with the response to immune checkpoint inhibitors: A systematic review and meta-analysis. Gene. 2022; 834:146638. 10.1016/j.gene.2022.146638. 26. Liu XD, Zhang YT, McGrail DJ, et al: SETD2 Loss and ATR Inhibition Synergize to Promote cGAS Signaling and Immunotherapy Response in Renal Cell Carcinoma. Clin Cancer Res. 2023; 29:4002-4015. 10.1158/1078-0432.CCR-23-1003. PMC10592192. 27. Kapur P, Rajaram S, Brugarolas J. The expanding role of BAP1 in clear cell renal cell carcinoma. Hum Pathol. 2023; 133:22-31. 10.1016/j.humpath.2022.07.022. PMC9898467. 28. Ged Y, Chaim JL, DiNatale RG, et al: DNA damage repair pathway alterations in metastatic clear cell renal cell carcinoma and implications on systemic therapy. J Immunother Cancer. 2020;8:e000230. 10.1136/jitc-2019-000230. PMC7311069. 29. Chen XJ, Ren AQ, Zheng L, Zheng ED: Predictive Value of KDM5C Alterations for Immune Checkpoint Inhibitors Treatment Outcomes in Patients With Cancer. Front Immunol. 2021;12:664847. 10.3389/fimmu.2021.664847. PMC8089485. 30. Li N, Chen J, Liu Q, et al: Prognostic significance and tumor-immune infiltration of mTOR in clear cell renal cell carcinoma. PeerJ. 2021;9:e11901. 10.7717/peerj.11901. PMC8378334. 2. I would also like to know how this study compares with previously published reports and if there is any consistency in the literature with regard to their findings. Response: We have added relevant literature that is in concordance with our study findings, as follows: “Our findings are in line with a previous work with slightly different design, whereby primary RCC tumors were segregated into VHL+0, VHL+1, VHL+2, and VHL+≥3 mutations.33 In both the discovery and validation cohorts of the study, those patients with a VHL+0 tumor had longer 5-year DFS and were proposed as candidates for surveillance. Conversely, patients with VHL+2 and VHL+≥3 tumors experienced shorter DFS rates of less than 50% and were deemed candidates for adjuvant therapy. 33 ” Reference (new): 33. Vasudev NS, Scelo G, Glennon KI, et al: Application of Genomic Sequencing to Refine Patient Stratification for Adjuvant Therapy in Renal Cell Carcinoma. Clin Cancer Res. 2023;29:1220-1231. 10.1158/1078-0432.CCR-22-1936. PMC10068441. 3. How do the authors see these findings changing or guiding clinical practice? Response: We have now added a paragraph discussing the potential implications of our study for changing or guiding clinical practice, as follows: ”Collectively, our study combined with emerging evidence on the genomic landscape of RCC might open new avenues for both prognostication and better selection of a subgroup of patients with RCC that could benefit from adjuvant anti-PD1 immunotherapy.” View more View less Competing Interests None. reply Respond Report a concern Kamposioras K. Peer Review Report For: Mutational profile of primary clear cell renal cell carcinoma predicts recurrence and potential candidacy for adjuvant immune checkpoint inhibition [version 2; peer review: 2 approved] . F1000Research 2024, 12 :918 ( https://doi.org/10.5256/f1000research.149223.r192983) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-918/v1#referee-response-192983 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions Adjust parameters to alter display View on desktop for interactive features Includes Interactive Elements View on desktop for interactive features Competing Interests Policy Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality. 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