Hyperactive RAS-MAPK signaling is recognized as one of the most important events contributing to oncogenesis (1). Its key role in cancer has been established in a broad range of pediatric and adult malignancies, where mutations in proto-oncogenes such as PTPN11, KRAS, NRAS, HRAS, and BRAF, and in the NF1, LZTR1 and CBL tumor suppressor genes represent the most common events (1). While these oncogenic mutations have generally a somatic origin, constitutive variants in the same genes are responsible for a group of multisystemic developmental disorders, the RASopathies, that are characterized by variable predisposition to malignancies (2, 3).

RASopathies exhibit overlapping phenotypes and multi-organ involvement with major shared features including growth failure, distinctive facial gestalt, variable degree of developmental delay/intellectual disability, a wide range of congenital and evolutive cardiac defects, ectodermal manifestations, and muscular-skeletal involvement (3–5). This family of “systemic”, constitutional disorders include Noonan syndrome (NS, MIM #PS163950),, Costello syndrome (CS, MIM #218040), cardiofaciocutaneous syndrome (CFCS, MIM #PS115150), Mazzanti syndrome (MIM #PS607721), Noonan syndrome with multiple lentigines (NSML, MIM #151100), neurofibromatosis type 1 (NF1, MIM #162200), Legius syndrome (LS, MIM #611431), CBL-associated syndrome (CBLS, MIM #613563), and the recently recognized MAPK1-associated syndrome (MIM #619087) and ERF Noonan-like syndrome (3, 6).

Given the critical role of RAS signaling in oncogenesis, RASopathies account for the most prevalent cancer predisposition syndromes (7–9). Several studies have documented a significantly increased, though variable, incidence of both myeloproliferative disorders and solid tumors in affected individuals(10–13), with an overall 10.5-fold increased cancer risk compared to age-matched populations (12). Cancer predisposition is well-established in NF1, CS, CBLS, and certain forms of NS linked to specific PTPN11 pathogenic variants (PVs), whereas both hematologic malignancies and solid tumors are less frequently reported in NS caused by pathogenic variants (PVs) in other disease-causing genes, as well as other RASopathies(8, 14–16)Solid tumors are observed in NF1, CS, and NS, more rarely. In NF1, most tumors involve the nervous system, including gliomas, benign neurofibromas, and malignant peripheral sheath tumors (14). In NS, the most frequent solid tumors reported are low- to high-grade gliomas, neuroblastoma (NB), rhabdomyosarcoma (RMS), and giant cell lesions primarily affecting the jaw(9, 12, 15–24). Whereas in CS, NB, RMS, and transitional cell carcinoma of the bladder (25, 26). Reports of solid tumors in CFCS and other RASopathies remain anecdotal (23, 27–29).

Given the heterogeneity in risk of tumor development, providing appropriate care is challenging. Surveillance protocols vary according to countries, professional’s opinions, and type of genetic alteration (8, 9). The association of a mildly increased cancer risk with specific genetic variants has raised awareness, suggesting the need for a personalized screening to promptly guide accurate diagnostic and therapeutic approaches (9, 25, 30).

We analyzed the prevalence of solid tumors in a large monocentric cohort of individuals with RASopathies and compared these findings with available data from retrospective analyses obtained through a systematic literature review of RASopathy-related cancer-cases. For each syndrome, we evaluated tumor prevalence and types. Moreover, we compared the molecular spectrum of germline variants in RASopathy genes in patients with and without co-occurrence of cancer to identify variants putatively associated with cancer predisposition.

Data were both retrospectively and prospectively collected and stored in a dedicated database. All medical records were reviewed, and a questionnaire was administered during the last clinical evaluation to assess: i) personal history of solid tumor; ii) family history of cancer in first- and second-degree relatives in patients who had developed tumors; iii) risk factors associated to solid tumors development. Information on age of tumor onset, type of tumor (macroscopic diagnosis and histological characterization) and treatment was also collected. Each enrolled patient was examined by experienced pediatricians and geneticists and underwent active surveillance according to the available guidelines (8, 31–33), expert consensus for solid tumor development in RASopathies(9, 34, 35) and tailored surveillance protocol according to local institutional expert consensus (Supplementary Materials Methodology). (26)

Germline genetic assessment was performed on DNA extracted from peripheral blood samples using parallel sequencing using customized gene panels upon clinical suspicion of RASopathy. MLPA was performed to exclude structural variants involving RASopathy tumor suppressor genes. Sequencing data processing, and variant call and annotation were performed using an in-house implemented bioinformatics pipeline. The gene panels were designed and updated during the timeframe of the study according to ClinGen guidelines and literature (see Supplementary Materials Methodology).

Bibliographic search was performed following the 2020 Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and checklist for reporting this review. In detail, a comprehensive literature review was done separately for NS, CFCS, and CS. The literature search was performed between May 2023 and December 2023 on PubMed using specific queries (see Supplementary Materials Methodology).

We evaluated articles obtained from the latest systematic reviews on cancer in RASopathies to identify any potentially overlooked papers during the PubMed search. Abstracts were read to evaluate the relevance to the research topic. For publications, the following year limits were applied: CS (1977–2023), NS (2001–2023), NSML (2002–2023), and CFCS (2006–2023).

Results considered eligible for evaluation were finally exported into two datasets (Microsoft Excel files) to identify duplicates and unique records (Supplementary Fig. 1).

Data extraction and analysis were performed by four researchers assigned to the two different main topics (NS and CFC/CS). Working in pairs (CR and SM, VT and GV, respectively), four reviewers independently completed the assessment for each topic and two senior reviewers reconciled disagreements (CL and SM). The following information was extracted from the included articles: first author, publication year, publication source, study type, number of patients with NS, CS or CFCS, number of patients with cancer, type of cancer, and cancer histology (when available). Moreover, when provided, the following additional patient data were gathered: gender, vital status, age at diagnosis and eventually death, diagnostic method, genetic testing, and identified variants, when available.

Qualitative variables were presented as absolute frequencies and percentages, and proportions were compared using either the Chi-square or Fisher’s exact test, as appropriate. Quantitative variables were described as medians with interquartile ranges (first and third quartiles). All statistical tests were two-sided, with a p-value < 0.05 considered statistically significant. The analyses were conducted using Stata software (StataCorp. 2023. Stata Statistical Software: Release 18. College Station, TX: StataCorp LP).

A cohort comprising 138 individuals with RASopathies (Fondazione Policlinico Gemelli cohort, FPG cohort, hereafter) was included in the study. Details on the gene involved, age at diagnosis, method used for tumor diagnosis, histology, treatment, and outcome are reported in Table 1, while demographic data about FPG cohort are detailed in Table 2.

In the NS cohort, which also includes NS-like phenotypes associated with pathogenic variants in SHOC2 and ERF, ~ 10.8% of patients (8/74) were diagnosed with a solid tumor (Table 1–2 and Fig. 1A-C). As expected, PTPN11 was the most frequently implicated gene. Notably, in one family (NS#1 and NS#4), the mother and her daughter, who shared the p.Glu139Asp amino acid substitution, developed solid tumors. In detail, the daughter had a retropharyngeal ganglioneuroblastoma and a low-grade pilocytic astrocytoma, while a melanoma in situ was diagnosed in the mother. In this cohort, a statistically significant association between p.Glu139Asp and solid tumors (p < 0.02;) was observed. CNS tumor progression was observed in two separate patients affected with a low grade pilocytic astrocytoma and an unclassified astrocytic tumor, respectively (Table 1). In one case the relapse occurred following chemotherapy treatment, and in the other one during active surveillance (NS#1 and NS#2). See Table 1 for all tumors detected in the NS cohort.

Approximately 47.8% (11/23) of the CS patients in the FPG cohort developed a benign or malignant neoplasia. Notably, five subjects showed the occurrence of multiple primary tumors in different districts (CS#1, CS#2, CS#4, CS#5, CS#6) (Fig. 2A-B, Table 1–2 and Supplementary Table 2). Bladder tumors [7 urothelial carcinoma (G1-2) and 7 papillary urothelial neoplasia of low malignant potential-PUNLMP] were the most frequent neoplasia observed in the CS cohort. Spontaneous regression of adrenal neuroblastoma was recorded for CS#2. CS#5 presented with multiple neoplasms involving the bladder (PUNLMP), and breast (relapse of intraductal breast neoplasia detected after 11 years from surgical intervention). All patients were alive at last follow-up, except for CS#5 who passed unexpectedly due to sudden cardiac death at the age of 28 years.

Finally, solid tumors were reported in ~ 7.3% of CFCS cases in the FPG cohort (3/41 (Table 1–2). Two patients carried a pathogenic variant in BRAF and developed relapsing neoplastic lesions. The first subject, a male who harbored the p.Thr470Pro amino acid substitution, developed giant cell lesions of the jaw at 8 years of age, which recurred at 14 years of age. The second patient, a female carrier of the p.Lys499Asn change, developed a pylocytic astrocytoma at 13 years of age, which recurred twice, at 15 and 18 years of age. The third individual, carrier of the p.Tyr130Cys variant in the MAP2K1 gene, developed multiple peripheral nerve sheath tumors at 17 years of age (Table 1–2, Suppl. Table 3).

The RAS-MAPK pathway plays a central role in tumorigenesis, with somatic mutations in the three members of the RAS family of proto-oncogenes involved in nearly one-third of human cancers (42). The occurrence of cancer in RASopathies has been supported by numerous case reports and some case series in the literature (7, 15).

Kratz and colleagues reported a cumulative cancer incidence of ~ 4% in NS and ~ 15% in CS within the first two decades of life, with a peak during adolescence (7). These data were retrospectively collected from a cohort of 1,941 individuals with a clinical diagnosis of RASopathy (7). Considering the very low number of tumors reported in the CFCS group, a precise estimate of the tumor risk in this condition could not be established. More recently, the same research group investigated the presence of malignancies in a cohort of 735 RASopathy patients. In that study, molecularly confirmed cases collected from 25 laboratories performing genetic screening in Germany were matched with those of the German Childhood Cancer Registry (GCCR), showing that the overall cancer risk during childhood was 10.5-fold higher compared to the general population (8). More specifically, individuals with NS and CS showed an 8-fold and 42.4-fold increased risk, respectively, compared to age-matched populations (8). No tumors were documented in 53 subjects of the cohort with CFCS as well as in a single CBLS patient. The study provided an enormous contribution in stimulating awareness on performing cancer-screening programs in patients with RASopathies. At the same time, it was limited by the exclusion of subjects older than 14 years and the lack of information from patient medical records, since the case-identifying resource was a childhood cancer registry. In addition, patients carrying variants in RASopathy-genes identified after 2015, such as RIT1, RRAS2, MRAS, MAPK1, YWHAZ, LZTR1, were not included. A recent review by the same authors confirmed that children with RASopathies, except those with LS, face a significantly increased cancer risk compared to the age-matched general population, and provided updated consensus recommendations for cancer surveillance in these categories (9, 30).

Considering the limited data available in the literature, a major goal of the present study was to define the prevalence and spectrum of solid tumors in a large and unselected monocentric cohort of RASopathy patients (FPG cohort), not including NF1 and LS cases. We also aimed to compare the findings of the FPG cohort with those reported in literature, by providing a systematic review.

In the FPG cohort, at least one solid tumor was documented in 10.8% of individuals with NS, 47.8% of those with CS, and 7.3% of those with CFCS, significantly higher compared to those previously reported (7, 12, 30). The discrepancy might stem from both center-dependent (e.g., advancements in diagnostic techniques) and center-independent (e.g., implementation of surveillance protocols) factors. Consistently with previous findings, we observed an early age at tumor diagnosis in patients with RASopathies, with a median age of 13.5 years in CS and 19 years in NS (7), likely due to the intrinsic oncogenic potential of hyperactive RAS-MAPK signaling. Interestingly, in the FPG cohort 34.7% and 5.4% of patients with CS and NS, respectively, developed at least one tumor in the first two decades of life; the number of CFCS patients with cancer was too low to draw any definitive conclusion.

Regarding the tumor spectrum in RASopathies reported in literature, PTPN11-positive NS cases have primarily been linked to malignant tumors of the nervous system (including DNET, glioma, and NB) whereas SOS1-positive NS cases have been predominantly associated with RMS and giant cell lesions of the jaw (7, 8, 12, 15). We documented low-grade gliomas as the most common malignancy observed in the PTPN11-NS sub-cohort (9.3%), while no cases of DNET were documented, and only one patient developed NB (i.e. retropharyngeal ganglioneuroblastoma). Only 1 patient with PTPN11-NS developed a high-grade glioma requiring chemotherapy (temozolomide) and radiotherapy (54 Gray).

The most updated recommendations concerning tumor screening for NS proposed by Perrino et al., do not suggest specific screening for non-hematologic malignancy (9). However, given the high prevalence of low CNS tumors reported to date also in our FPG cohort, a personalized screening protocol may be discussed in future dedicated consensus.

In the SOS1-related NS sub-cohort, RMS was not reported.

A giant cell lesion of the jaw was diagnosed in one NS patient carrying a RIT1 variant (p.Ala57Gly). Notably, to the best of our knowledge, this is the first case in literature reported with this association. Literature data support the evidence that in CS, RMS is the most common tumor of childhood with average onset age of six years of age, followed by NB and bladder neoplasia from childhood through adolescence (7, 11, 25, 26). While most malignancies are diagnosed during childhood, transitional cell carcinoma of the bladder has been reported exclusively in adulthood (43).

In the CS FPG cohort, asymptomatic bladder tumors were the most common neoplasia, followed by breast intraductal papillomas, NB, and RMS. (43, 44)

PUNLMP and low-grade bladder carcinoma have been diagnosed in individuals as young as 10 and 12 years, respectively, underscores the importance of active surveillance (26, 30) in line with recent recommendations (4, 9, 25).

Although somatic mutations in HRAS are well-established as playing a pivotal role in embryonal RMS (7, 25), the latter being the most reported CS-associated neoplasia in literature, in the FPG cohort RMS was detected only in one patient (vaginal embryonal RMS). Despite the differences in the prevalence of tumors between CS-FPG cohort and literature, the existing surveillance protocols for these solid tumors remain strongly recommended, with clinical evaluation and routine ultrasound monitoring being the gold standard investigation (25, 30, 33).

Of note, six patients in the CS FPG cohort developed more than one tumor. Among them, two patients experienced at least one relapse (1 intraductal breast papillary neoplasia, 1 urothelial carcinoma) and one patient experienced spontaneous regression of NB (Table 1).

Another major finding of this study was the identification of candidate variants that might be associated with a higher risk of solid tumor development. These include p.Asp106Ala and p.Ser502Thr in PTPN11, p.Glu846Lys in SOS1, p.Gly12Arg in NRAS, and p.Gly12Ala in HRAS. Interestingly, PTPN11 variants associated with solid tumors differ from the mutational hotspots identified in PTPN11-related NS/JMML (16,18,51) or in sporadic cancer (COSMIC database). Noteworthy, we identified a mother and daughter carrying the p.Glu139Asp variant in PTPN11, who developed in situ melanoma and multiple tumors of the central nervous system, respectively.

Among RAS gene variants, the p.Gly12Arg substitution in NRAS is a well-established oncogenic change that frequently occurs as a somatic event in cancer, particularly in hematologic malignancies (COSMIC database). Based on available data, among the six amino acid substitutions resulting from single-base changes at this codon, p.Gly12Arg has the lowest prevalence in cancer (Gly12Asp > Gly12Ser > Gly12Cys > Gly12Ala > Gly12Val > Gly12Arg), suggesting a relatively mild oncogenic potential. However, when p.Gly12Arg occurs as a constitutional variant, it confers an increased cancer susceptibility (25). Similarly, the p.Gly12Ala substitution in HRAS represents the rarest variant found in sporadic cancer among missense substitutions at this codon (Gly12Val > Gly12Ser > Gly12Asp > Gly12Cys > Gly12Arg > Gly12Ala). Notably, our single patient carrying this variant has not developed any solid tumor to date (last follow up performed at age 12 years).

Finally, no systematic studies investigated the prevalence of cancer in CFCS, with only anecdotal reports documenting cases with hepatoblastoma, granuloma, chondroblastoma, and neoplasia of the jaw (9, 23, 27). In our CFCS cohort, solid tumors were observed in 7.3% of individuals, including relapsing lesions in one patient and one with recurrent tumor. Despite the distinctive skin manifestations of this condition, no cases of melanoma or skin tumors have been reported either in the literature or in CFCS-FPG cohort. (35)

Based on a large, molecularly confirmed monocentric cohort of individuals with RASopathies monitored for solid tumors, this study provides important insights into tumor risk and surveillance. Comparison with literature data highlights a strong association between NS and low-grade CNS tumors, as well as between CS and bladder tumors. Differences in tumor prevalence may reflect variability in surveillance protocols at our institution. Consistent with previous reports, we observed an earlier onset of solid neoplasms in NS and CS compared with the general population. While the number of reported tumors in the FPG cohort alone was insufficient to establish definitive genotype-phenotype correlations, integration with literature data allowed identification of potential high-risk variants associated with increased tumor susceptibility. Notably, we report for the first time a giant cell lesion of the jaw in a NS patient carrying a RIT1 variant, and relapsing neoplasia in two CFCS individuals. These findings emphasize the need for multicentric studies to generate real-world data on tumor prevalence in RASopathies, which could inform personalized surveillance strategies to optimize cancer prevention while minimizing unnecessary interventions. Moreover, molecular characterization of tumors may be critical for understanding the interaction between constitutional RASopathy variants and secondary somatic alterations in cancer development.