Predictive genomic medicine enlarges the spectrum of predisposing mutations for head and neck cancers via a panel of 56 genes selected for human neoplasia in Southern Italy: a pilot study

Introduction

Squamous cell carcinomas of the head and neck (SCCHN) are malignant tumors arising from mucosal surfaces located in the upper aero-digestive tract (paranasal sinuses, nasopharynx, oropharynx, hypopharynx, larynx, oral cavity, and nostrils) [1], 2]. Squamous cell carcinomas of the head and neck is the seventh most common cancer in Europe; in Italy approximately 9,750 people have been diagnosed in 2023 (7,050 men and 2,700 women), with 3,800 deaths (numbers of cancer from AIOM, 2023). However, these numbers are likely underestimated due to significant underreporting [3]. While tobacco and alcohol use remain the main risks factor in Europe, human papillomavirus (HPV) plays a critical role in the etiology of SCCHN globally. Patients with early-stage SCCHN can be cured with surgery or radiation. Patients with an aggressive disease and those with locally advanced stage tumors, which represent two-thirds of newly diagnosed, are more likely to undergo surgery with a 50 % 5-year overall survival) [4]. Within SCCHN, each tumor location has a different clinical presentation (see above), staging, prognosis and can be treated differently [5].

Oral squamous cell carcinoma (OSCC) is the most common type of SCCHN and represents more than 90 % of all malignancies of the oral cavity. Each year approximately 275,000 cases are newly diagnosed worldwide. When detected at an early stage (T1-T2), OSCC survival rate is up to 80 %, however when detected in later stages (T3-T4), the survival rate significantly drops to 20–30 % [6]. Globally, 275,000 to 300,000 new cases of OCSCC are diagnosed each year with over 150,000 associated deaths [7]. These oral cavity cancers are classified by the involvement of distinct anatomic subsites – notably, the lip, buccal mucosa, gums (or gingiva/alveolar ridge), anterior two-thirds of the tongue, floor of the mouth, hard palate, and retromolar trigone [8]. Subsite involvement may have important prognostic implications, and may influence treatment planning [9]. Subsites distribution varies by geographic location, largely due to environmental exposures such as tobacco and alcohol use [10].

These cancers typically develop silently in their early stages, leading to delayed detection and unfortunately cervical lymph node metastasis can easily occur. Early diagnosis of head and neck cancers plays a crucial role in improving patient outcomes, with significantly, higher chances of successful treatment, in terms of surgery, radiation, and chemotherapy. Early detection also reduces the need for more invasive surgical procedures and minimizes long-term complications. Regular screening (i.e., regular observation of the local sites) and awareness of risk factors, such as smoking, alcohol use, and HPV infection, can help identify potential cancers before symptoms arise., This proactive approach enhances survival rates and preserves a good quality of life.

We designed a panel of 56 genes associated with different types of tumors, primarily targeting DNA damage repair pathways. We tested 56 patients with head and neck tumors at different localizations, as we identified approximately 12.5 % of pathogenic mutations in our neoplasia cases, which is one of the highest reported percentages among studied cohorts of these tumors, thereby suggesting that genetics plays a significant role in OCSCC.

Materials and methods

Results

We identified seven different mutations in six genes implicated in DNA damage repair (Figure 1) in seven unrelated subjects with head and neck tumors in different locations. Three variants were in BRCA genes (two in BRCA2 and one in BRCA1), one in the MUTYH gene, one in BRIP1 and two variants were in FANC family genes, one in FANCC and one in FANCM genes (Table 2).

Figure 1:

Genes involved in DNA damage repair mechanisms present in our panel that can occur through single-strand or double-strand breaks. The six mutated genes are marked in red): MUTYH, involved in base excision repair (BER); BRCA1, BRCA2, and BRIP, involved in homologous recombination (HR); FANCC and FANCM, involved in interstrand crosslink (IC) repair.

These seven patients were followed over time to assess post-surgery effects and radio/chemotherapy treatments. All clinical data are summarized in Table 3.

Among the seven mutations identified, four are variants that cause frameshift: three duplications of one base (in BRCA2 c.4284dupT, p.Gln1429Serfs*9 – rs80359439; BRCA1 c.5266dupC, p.Gln1756Profs*74 – rs80357906 and BRIP1 c.394dupA, p.Thr132Asnfs*10 – rs587781416) and one deletion of two bases (BRCA2 c.658_659delGT, p.Val220Ilefs*4 – rs80359604). Two variants were nonsense with a premature stop codon formation (FANCM c.5791C>T, p.Arg1931* – rs144567652; FANCC c.37C>T, p.Gln13* – rs121917784) and one was a missense mutation in the MUTYH gene known to be pathogenic (c.527A>G, p.Tyr176Cys–rs34612342). Confirmatory Sangers methodology was also conducted for the seven variants found. We identified seven distinct pathogenic mutations across six genes. Among these, three patients harbored mutations in BRCA genes. Of these, two had tonsillar tumors, and one had a cheekbone tumor. All three patients were smokers and/or alcohol consumers. The BRCA genes, which are pivotal in homologous recombination (HR) processes, are well-known for their roles in breast and ovarian cancer susceptibility [12], 13] (see Figure 2A–D). In the pedigree A, no other cancer cases were registered within the family.

Figure 2:

Pedigrees of the families of the seven mutation carriers identified. (A) The index case, diagnosed with cheekbone cancer (TC_15), carries a BRCA2 mutation. (B) The proband (TC_18), diagnosed with parotid cancer, is a carrier of a MUTYH gene mutation. (C) The proband (TC_27), diagnosed with tonsil cancer, carries a BRCA1 mutation. (D) A female (TC_31) diagnosed with tonsil cancer carries a BRCA2 mutation. (E) A male patient (TC_36) diagnosed with tongue cancer carries a BRIP gene mutation. (F) A male patient (TC_51) diagnosed with tongue cancer carries a FANCM gene mutation. (G) A male patient (TC_52) diagnosed with cheekbone cancer carries a FANCC gene mutation.

One patient with a parotid gland tumor carried a pathogenic variant in the MUTYH gene and reported a history of smoking. MUTYH is involved in the base excision repair (BER) pathway, particularly in correcting oxidative DNA damage, and is known to interact with BRCA1 in broader DNA repair mechanisms (see Figure 2B).

Finally, we detected three mutations in genes associated with the Fanconi anemia (FA) pathway: BRIP1 (FANCJ), FANCM, and FANCC. Two of these patients had tongue cancer, and the third had a cheek tumor (see Figure 2E–G). All three patients were smokers. The FA pathway is integral to DNA damage repair, particularly to coordinate the repair of inter-strand crosslinks and to support HR.

Discussion

The study of DNA mutations is becoming increasingly important, as it enables the detection of predisposing mutations in a genome, even in newborns. In addition, later mutations, often caused by environmental factors, can trigger the first signs of abnormal cell proliferation, potentially leading to cancer [14]. These mutations can be risk factors for tumor development. Moreover, pathogenic mutations, identified through bioinformatic tools and software, can serve as diagnostic markers and may help in tumor staging, including the assessment of metastatic spread [15]. The identification of pathogenic mutations also guides more effective therapeutic approaches, such as surgery and chemotherapy [16]. Furthermore, monitoring circulating tumor DNA (ctDNA) is a valuable method to detect early relapse of cancer [17], [18], [19]. In summary, mutations that predispose individuals to cancer are crucial to understand both cancer susceptibility and the natural history of the neoplastic process.

Most SCCHN are considered sporadic and are commonly associated to established risk factors, such as tobacco use, alcohol consumption, and HPV infection [10], 20], 21]. A 2015 study by the International Head and Neck Cancer Epidemiology (INHANCE) consortium [22] identified a correlation between family history of cancer, early onset, and SCCHN, particularly in younger individuals and smokers [23]. Despite these findings, genetic predisposition and the interaction of risk factors in SCCHN remain underexplored, especially when compared to other cancers such as breast or colon cancer. Although early-onset SCCHN is relatively uncommon, its incidence has been increasing in recent years also in very young subjects.

Several recent studies have investigated the presence of germline variants in patients with head and neck cancers. Variants in the FANCG, CDKN2A, and TPP genes, which had previously been associated with these types of tumors, were found [23], along with pathogenic variants in BRCA2, ARID1A, ATM, and BRCA1 [24]. In 2023, Brake et al. analyzed 200 patients and identified 22 pathogenic variants (10.5 %). Among these, 11 were in high- or moderate-penetrance genes, predominantly PMS2 and HOXB13, while the remaining 11 were in low-penetrance genes, including MUTYH, WNR, and RECQL4. Notably, their study revealed that nearly 95 % of the variants identified would not have been detected using the current guidelines for genetic testing in SCCHN [25].

Our findings are in strong agreement with those of Brake et al. and suggest that the current selection criteria for genetic testing related to oncological predisposition should be revised, as it has been the case for other types of cancers. According to existing guidelines [26], genetic testing is generally not recommended for patients with head and neck cancer. Our findings, however, highlight the importance of genetic testing in these patients, suggesting that such an approach could offer valuable insights and contribute to early diagnosis, particularly in families with a known history of SCCHN.

Extensive research has demonstrated that DNA damage repair (DDR) mechanisms play a critical role in the initiation, progression, prognosis, and treatment of SCCHN [27]. Moreover, recurrence rates remain high, approximately 50 %, in SCCHN patients treated with radiotherapy and chemotherapy [28]. This underscores the need for alternative and earlier approaches, potentially involving molecularly targeted therapies to target specific molecular alterations [29], 30]. In this context, the use of a “universal” panel that includes a few dozens of genes can be an invaluable tool for initial screening across a wide range of cancer cases (i.e. colon cancer, breast cancer, etc.). The panel would provide a broad overview of genetic alterations across various cancer types and could help identify individuals who carry predisposing mutations and require closer and more frequent monitoring. The main limitation of studies on SCCHN lies in the diverse range of tumor localizations, which makes the assembling of large and uniform patient cohorts challenging [31]. This limitation was also evident in our study, where we enrolled 56 patients with tumors at different anatomical sites; nonetheless, we believe that this pilot study paves the way to several new insights. Taken together, these genes act in complementary DNA repair networks, principally safeguarding genomic stability. In detail: BRCA1 and BRCA2 genes are implicated and are central components of the homologous recombination pathway, orchestrating the recognition and the repair of DNA double-strand breaks, as well as ensuring replication fork stability. MUTYH gene, is related to base excision repair (BER) pathway, preventing mutagenesis arising from oxidative DNA damages. BRIP1 gene, is a helicase interacting with BRCA1, FANCM and FANCC, members of the Fanconi anemia pathway, which contribute to the resolution of interstrand crosslinks and the stabilization of replication forks. Mutations affecting these pathways can, therefore, compromise the ability of the cell to properly repair the majority of DNA damages, increasing genomic instability and promoting tumorigenesis process [32], [33], [34]. The presence of such alterations in head and neck cancers – despite the absence of current predictive medicine guidelines and extensive and recent studies in the literature – highlight also in our study, that tumor predisposition profile is comparable to other malignancies where targeted approaches are already in place or clinical trials are being conducted. This biological rationale reinforces the potential value of implementing genetic screening especially for considering different therapeutic strategies, including PARP inhibitors currently used in tumors with homologous recombination deficiencies (as breast and ovarian cancers) [35].

The discovery of pathogenic variants in the BRCA genes in patients with SCCHN raises important considerations. First, it prompts the question of whether PARP inhibitors, already successfully used in breast and ovarian cancers, might be effective in the treatment of head and neck cancers in patients carrying BRCA mutations.

In addition, the identification of BRCA genes carriers prompts the need for targeted surveillance protocols for individuals carrying these variants. Notably, currently there are no guidelines for monitoring head and neck cancers in BRCA mutation carriers. This raises important questions about how to effectively monitor these individuals given the increased risk for developing SCCHN. Moreover, the targeted surveillance should also extend to carriers of other genetic variants, thereby suggesting the need for a broader, more tailored approach to monitoring cancer risk in genetically predisposed cohorts. In this context, families with a history of neoplasia should be monitored more frequently to enable earlier detection and intervention.

Conclusions

Although SCCHNs have traditionally been seen as rarely linked to germline mutations – largely due to strong lifestyle-related risk factors such as smoking, alcohol use, drug use, and HPV infection – our pilot study suggests that their heritability warrants further investigation. In our cohort of 56 patients, we found seven pathogenic (12.5 % of the cases). This proportion is even higher than those reported for more extensively studied cancers, like breast and colon cancer.

These findings underscore the urgent need to further investigate the genetic contribution to the etiology of SCCHN and the complex interplay between genetic predisposition and environmental exposures. Further research in this area could increase our understanding of SCCHN pathogenesis and pave the way for more personalized prevention and treatment strategies. In particular, identifying the key driver mutations in SCCHN in larger case series may allow targeted therapies, contributing to a more tailored and effective management of this type of cancer. Recent advances in molecular biology have deepened the role of smallRNAs as long non-coding RNAs (lncRNAs) [36], [37], [38]; or miRNAs; furthermore, the deregulation of miRNAs has been proposed in the pathogenesis and in the clinical management of head and neck squamous cell carcinoma (SCCHN), also as potential biomarker [39]. As discussed by Jiang et al., lncRNAs regulate critical oncogenic pathways such as PI3K/AKT and Wnt/β-catenin, driving tumor initiation and progression [40]. Expanding on this, Tran et al. emphasized the potential of targeting lncRNAs as novel therapeutic strategies, with several candidates already under clinical evaluation as biomarkers (as ELDR, MALAT1, NEAT1, HOTAIR, and UCA1) [41]. In parallel, Pandruvada et al. placed these findings within the broader context of molecular medicine, suggest that integrating lncRNA biology into clinical practice may enhance personalized approaches and open new therapeutic strategies for patients with head and neck cancers although further studies are needed [30].