About 87 % of kidney cancer diagnoses per year is represented by renal cell carcinoma (RCC), which accounted for over 400,000 new cases globally in 2020, making it one of the most prevalent forms of genitourinary cancer and the 14th most commonly diagnosed malignancy worldwide [1]. In more details, RCC encompasses a broad spectrum of histopathological entities, with clear cell RCC (ccRCC) comprising approximately 80 % of cases. The remaining 20 % of cases, collectively known as non-clear cell RCC (nccRCC), includes several variant histologies characterized by distinct molecular and cytogenetic profiles. Among these, the two major subtypes are papillary RCC (pRCC, ∼15 %) and chromophobe RCC (chRCC, ∼5 %) 2, 3. The remaining cases consist of extremely rare subtypes, each with an incidence of ≤ 1 %, as well as unclassified tumors (also known as not-other-specified RCC, NOS RCC), which account for 4–5 % of RCC diagnoses [4].

As for the therapeutic management of this disease, curative surgery remains the gold standard for localized and early-stage RCC, typically through complete or partial nephrectomy and irrespective of histology (ccRCC vs nccRCC) [5]. However, 20–40 % of these patients will experience recurrence or disease progression. The programmed cell death-1 (PD-1) inhibitor pembrolizumab has recently been approved as adjuvant treatment strategy for patients with resected ccRCC at a higher risk of relapse, based on KEYNOTE-564 results 5, 6, 7. Additionally, 25–30 % of patients present with metastatic disease at diagnosis, with a notable lower 5-year overall survival (OS) rate compared to those with localized tumors (12 % vs 93 %) [2].

Over the past decade, the systemic treatment for metastatic RCC (mRCC) has been radically revolutionized by the introduction of the so-called immune-based combinations, which are combinations of immune checkpoint inhibitors (ICIs) alone (PD-1 inhibitors plus cytotoxic T-lymphocyte antigen 4 (CTLA-4) inhibitors such as nivolumab plus ipilimumab) [8] or with tyrosine-kinase inhibitors (TKIs, such as pembrolizumab plus axitinib or lenvatinib, and nivolumab plus cabozantinib) 9, 10, 11. The onset of ICI/ICI and ICI/TKI combinations led to a meaningful improvement in terms of tumor response rates and survival outcomes, never seen before with the use of the previous standard-of-care single agent TKIs [12].

Despite these therapeutic advances, responses to ICI-based treatments are heterogeneous among mRCC patients. To date, the only validated parameter to guide the therapeutic decision-making process is represented by the International Metastatic RCC Database Consortium (IMDC) risk stratification [13]. However, the IMDC risk stratification poses considerable limitations in the current age of immunotherapy, as it was initially developed as a prognostic model during the previous TKIs’ era. As a consequence, the urgent need of validated biomarkers for ICI-based treatments is remarkable to better tailor the available treatment strategies to patients and tumor features, thus avoiding unnecessary toxicities and highlighting those patients with an increased probability of experiencing a durable response to therapies [14]. Moreover, established prognostic factors which may be currently used in the everyday clinical practice during this immunotherapy era are still awaited [14].

Multiple potential biomarkers have been assessed to date as possible prognostic factors or predictors of response to systemic therapies, including driver mutations in gatekeeper-genes involved in RCC biology and progression [14]. Although no single genomic predictor has been identified for mRCC, ongoing research continues to show promise in this area.

Focusing on ccRCC, the loss of chromosome 3p is an early and pivotal oncogenic event, leading to the biallelic inactivation of the Von Hippel Lindau (VHL) tumor suppressor gene, which occurs in over 90 % of cases [15]. Following VHL inactivation, other 3p-located tumor suppressor genes, including polybromo 1 (PBRM1), SET domain containing 2 (SETD2), and BRCA1-associated protein 1 (BAP1), may contribute to disease progression, though their mutations occur later and are less rate-limiting [16]. The loss of chromatin-modifying genes on 3p disrupts both epigenetic and non-epigenetic processes, playing a crucial role in tumorigenesis. Additionally, lysine demethylase 5 C (KDM5C) is the most frequently mutated non-3p gene in ccRCC, highlighting the importance of epigenetics in this cancer [16]. In contrast, the molecular drivers of nccRCC are less understood, apart from the role of the mesenchymal-epithelial transition (MET) gene’s pathway and chromosome 7 in some pRCC cases [17]. While PBRM1, BAP1, and SETD2 mutations are also found in pRCC, they occur at lower frequencies than in ccRCC [18].

With regard to chromosome 3p genes, VHL was certainly one of the primaries focuses of translational research in ccRCC due to its common mutations in these patients. As a part of an E3 ubiquitin ligase complex, VHL plays a key role in cellular oxygen sensing by binding hydroxylated hypoxia-inducible factor-α (HIFα), marking it for degradation. Loss-of-function mutations in VHL prevent this process, causing HIFα accumulation and persistent activation of hypoxia response genes, even in normal oxygen conditions, thus encouraging blood vessel formation, cancer cells proliferation and metastatic dissemination [19]. In 2019, a sequencing analysis of ccRCC tumor samples from an institutional database revealed improved progression-free survival (PFS) in VHL-mutated patients treated with first line TKIs, suggesting a favorable response to targeted therapy [20]. On the other hand, VHL mutations did not reliably predict responses to immunotherapy in retrospective analyses of mRCC patients treated with later-line nivolumab 21, 22. While studies regarding the prognostic role of VHL mutations in mRCC are conflicting [14], only few inconsistent data exist on the efficacy of this gene alteration as predictor of response to ICI/TKI combinations 23, 24.

PBRM1 encodes the BRG1-associated factor (BAF180), which acts as a subunit of the SWI/SNF-chromatin remodeling complex, also being the second most frequently mutated gene in ccRCC (with a mutational frequency of about 20–40 %) [16]. Inactivating mutations of PBRM1 were shown to have a favorable prognostic role among mRCC patients treated with TKIs [25], while several studies suggest promising potential for using PBRM1 status as a biomarker for predicting response to ICIs 22, 26, 27. Nonetheless, the current results do not support the use of this mutation as a validated marker for immune-based combinations and more robust data is still awaited.

As well as PBRM1, SETD2, and BAP1 also work as epigenetic regulators [16]. BAP1 mutations were shown to be related to poor prognosis in patients with localized and metastatic RCC [14]. No evidence exists regarding a potential correlation between BAP1 mutations and response to immunotherapy in mRCC, while BAP1-mutated patients have shown lower survival benefits with TKIs compared to non-mutated patients 14, 28. Notably, BAP1 and PBRM1 mutations are mutually exclusive.

As for SETD2, this 3p-located gene encodes a H3 lysine 36 histone methyltransferases, involved in several chromatin-regulated processes, and its role as a marker of poor prognosis is nowadays well established [29]. Interestingly, a 2021 study analyzed a pan-cancer cohort from The Cancer Genome Atlas (TCGA) and found a significant correlation between SETD2 mutations and overall response rates (ORR) in patients treated with ICIs, suggesting SETD2 as a potential tissue-agnostic predictive biomarker for immunotherapy [30]. Conversely, data regarding the potential role of SETD2 as a predictor of response to targeted therapies is inconsistent and controversial 14, 29.

The available results on the efficacy of BAP1 or SETD2 mutations as predictive biomarkers for mRCC patients treated with immune-based combinations are limited and primarily derived from phase III pivotal studies’ biomarkers analyses, which remain inconclusive 14, 23, 24. Consequently, ongoing research aims to obtain more robust evidence on this topic.

Recently, a hypothesis-generating exploratory analysis conducted on a small cohort of mRCC patients treated with ICI/ICI or ICI/TKI combinations from our Center showed a possible negative prognostic role of SETD2 mutations in this setting, also highlighting that concomitant VHL and PBRM1 alterations could act as a predictor for ICI/TKI efficacy [31].

The aim of this retrospective study is to evaluate tissue-based biomarkers that may be associated with prognosis or may predict a response to available first line systemic therapy, focusing on chromosome 3p gene mutations.