Oral Oncology 134 (2022) 106184 Contents lists available at ScienceDirect Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology Development of a mouse model for spontaneous oral squamous cell carcinoma in Fanconi anemia Ricardo Errazquin a, Angustias Page a, b, c, Anna Suñol d, Carmen Segrelles a, b, c, Estela Carrasco d, Jorge Peral c, Alicia Garrido-Aranda e, Sonia Del Marro c, Jessica Ortiz c, Corina Lorz a, b, c, Jordi Minguillon f, g, 1, Jordi Surralles f, g, Cristina Belendez g, h, i, j, Martina Alvarez e, Judith Balmaña d, Ana Bravo k, Angel Ramirez a, b, c, Ramon Garcia-Escudero a, b, c, * a Research Institute Hospital 12 de Octubre (imas12), University Hospital “12 de Octubre”, Av Córdoba s/n, 28041 Madrid, Spain b Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), 28029 Madrid, Spain c Biomedical Oncology Unit, CIEMAT (Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas), Avenida Complutense 40, 28040 Madrid, Spain d Hereditary Cancer Genetics Group and Medical Oncology Department, VHIO, Barcelona, Spain e Centro de Investigaciones Médico-Sanitarias (CIMES), Malaga, Spain f Join Research Unit on Genomic Medicine UAB-Sant Pau Biomedical Research Institute (IIB Sant Pau), Hospital de la Santa Creu i Sant Pau, 08041 Barcelona, Spain g Centro de Investigación Biomédica en Enfermedades Raras (CIBERER), 28029 Madrid, Spain h Pediatric Hematology and Oncology, Hospital General Universitario Gregorio Marañón, Madrid, Spain i Facultad de Medicina, Universidad Complutense de Madrid, Spain j Instituto Investigación Sanitaria Gregorio Marañón, Madrid, Spain k Department of Anatomy, Animal Production and Veterinary Clinical Sciences, Laboratory of Pathology Phenotyping of Genetically Engineered Mice, Faculty of Veterinary Medicine, University of Santiago de Compostela, 27002 Lugo, Spain A R T I C L E I N F O A B S T R A C T Keywords: Fanconi anemia (FA) patients frequently develop oral squamous cell carcinoma (OSCC). This cancer in FA pa­ Fanconi anemia tients is diagnosed within the first 3–4 decades of life, very often preceded by lesions that suffer a malignant Head and neck squamous cell carcinoma transformation. In addition, they respond poorly to current treatments due to toxicity or multiple recurrences. Oral squamous cell carcinoma Translational research on new chemopreventive agents and therapeutic strategies has been unsuccessful partly Mouse model due to scarcity of disease models or failure to fully reproduce the disease. Here we report that Fanca gene Oral mucosa FANCA knockout mice (Fanca-/-) frequently display pre-malignant lesions in the oral cavity. Moreover, when these TP53 animals were crossed with animals having conditional deletion of Trp53 gene in oral mucosa (K14cre;Trp53F2-10/ F2-10 Trp53 ), they spontaneously developed OSCC with high penetrance and a median latency of less than ten months. p53 Tumors were well differentiated and expressed markers of squamous differentiation, such as keratins K5 and Mutation K10. In conclusion, Fanca and Trp53 genes cooperate to suppress oral cancer in mice, and Fanca-/-;K14cre; Trp53F2-10/F2-10 mice constitute the first animal model of spontaneous OSCC in FA. Introduction blood malignancies such as myelodysplastic syndrome (MDS) and leu­ kemia in FA. HSCT has significantly improved life expectancy in FA Fanconi anemia (FA) is a heritable syndrome with predisposition to individuals so that there are now more adults living with FA than chil­ congenital abnormalities, bone marrow failure (BMF), and cancer. In FA dren diagnosed with this syndrome. Unfortunately, a consequence of patients, pathogenic mutations have been described in 23 different FA this achievement has been the identification of solid tumor predisposi­ genes which give rise to Fanconi or Fanconi-like clinical phenotype. tion as the most important health challenge in FA young adults. Patients Most FA children have BMF that can be successfully treated with hem­ have an extraordinarily high lifetime risk of squamous cell carcinomas atopoetic stem cell transplant (HSCT). HSCT has also been used to treat (SCC) of the head and neck, esophagus, vulva, or anus. This risk is * Corresponding author at: Biomedical Oncology Unit, CIEMAT (Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas), Avenida Complutense 40, 28040 Madrid, Spain. E-mail address: ramon.garcia@ciemat.es (R. Garcia-Escudero). 1 Present address: Genetics Department (INGEMM), La Paz University Hospital, 28046 Madrid, Spain. https://doi.org/10.1016/j.oraloncology.2022.106184 Received 22 June 2022; Received in revised form 21 September 2022; Accepted 24 September 2022 Available online 30 September 2022 1368-8375/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). R. Errazquin et al. Oral Oncology 134 (2022) 106184 increased by prior HSCT, together with a continuing risk of acute stratified epithelia (such as oral mucosa). We previously reported that myeloid leukemia [1]. Synchronic and metachronic tumors, especially K14cre;Trp53F2-10/F2-10 animals spontaneously develop skin SCC, in the oral cavity, are distressingly common in FA [2]. Besides SCC, demonstrating a predominant tumor suppressor role of p53 in mouse other malignant tumors have also been described in FA, although less epidermis [22]. Furthermore, K14cre;Trp53F2-10/F2-10 mice display oral frequently: lymphoma, liver cancer, brain tumors, and embryonal tu­ cavity tumors when combined with constitutive Akt activity [23] or mors such as Wilms tumor and neuroblastoma. However, the risk of IKKβ overexpression [24]. We then hypothesized that combined loss of these types of tumors is much lower than SCC. Fanca and Trp53 genes in the oral cavity could produce spontaneous oral A hallmark feature of FA is the inability to repair DNA interstrand tumors. Here we describe the phenotype of double Fanca-/-; K14cre; cross-links (ICLs). FA proteins repair ICLs in a common cellular pathway Trp53F2-10/F2-10 mice and we demonstrate that loss of both Fanca and known as the FA pathway or FA/BRCA pathway. A defective FA pathway Trp53 genes in oral mucosa recapitulates human FA HNSCC. leads to the accumulation of genomic aberrations and the emergence of precancerous cells that eventually might progress into invasive carci­ Materials and methods nomas. However, the etiology of SCC in mucosal epithelia in FA is not well understood. Known drivers of head and neck SCC (HNSCC) are Patients smoking and alcohol consumption, but they are less commonly reported in FA than in the general population. Human papillomavirus (HPV) Oral squamous cell carcinomas (OSCCs) from 3 Fanconi anemia infection is another driver of HNSCC. However, some reports suggest patients were analyzed. The study was conducted in accordance with the that HPV may be a major contributor to HNSCC development in patients precepts established in the Declaration of Helsinki, Good Clinical Prac­ with FA [3], whereas other studies dispute these results [4]. HNSCC in tice guidelines, and all applicable regulatory requirements. The ethics FA is difficult to treat, as patients cannot tolerate current therapies, committee of Hospital 12 Octubre approved the study protocol (study including ionizing radiation and platinum-based chemotherapies [5]. In number 18/294). addition, most tumors are detected at advanced stages. Altogether, these events have made cancer the principal cause of early mortality in adults Tumor sequencing from FA patients with FA. Therefore, the discovery of non-genotoxic and efficient treat­ ment opportunities for FA patients is of utmost importance. Deep-sequencing was performed from formalin-fixed paraffin- New therapies against FA HNSCC could be discovered and preclini­ embedded (FFPE) tumor blocks, from 10 to 15 sections of 4 μm thick­ cally tested in adequate disease models such as cellular and animal ness. Briefly, the cancerous tissue on 4–5 slides (4 μm sections) per pa­ models. Tremendous efforts have been made to generate and analyze tient was dissected out. Areas containing >30% tumor cells as whether genetically engineered mouse models (GEMMs) with delete­ determined by an expert pathologist were macrodissected and depar­ rious mutations in FANC genes could model disease features of FA pa­ affinated using Deparaffinization Solution (Qiagen, Hilden, Germany). tients. Thus, lines of mice bearing mutations in Fanca, Fancc, Fancd1, DNA was extracted and purified with GeneRead DNA FFPE Kit (Qiagen, Fancd2, Fance, Fancf, Fancg, Fanci, Fancl, Fancm, Fancn, Fanco, or Fancp Hilden, Germany). DNA concentration was quantified using Qubit™ ds have been produced [6–8]. To date, all FA mouse models display DNA High-Sensitive Assay kit (Thermo Fisher Scientific, Waltham, MA, reduced fertility and cells derived from them are sensitive to ICLs. Em­ USA) on the Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, bryonic and perinatal lethality has been observed in most FA models, USA). All library preparation was performed manually for Oncomine which could be influenced by the mouse genetic background. Some Comprehensive Assay v3 (OCAv3) (Thermo Fisher Scientific, Waltham, models display other minor developmental abnormalities, such as MA, USA) according to manufacturer’s instructions MAN0015885. microphthalmia in Fanca [9], Fancc [10], Fancd2 [11], Fanci [7], and Multiplex PCR amplification was conducted using a DNA concentration Fancp [12]. The life-threatening anemia that FA patients suffer is not of approximately 20 ng as input. For sequencing, prepared libraries were modelled in FA mice, although some defects are present, such as lower loaded according to manufacturer’s instructions (Ion 540™-Chef, blood cellularity in Fancp [12] or reduced proliferation in hypomorphic MAN0010851) onto Ion 540™ Chips (Thermo Fisher Scientific, Wal­ Fancd1 [13]. Furthermore, the high incidence of BMF, leukemia and tham, MA, USA) and prepared using the Ion Chef™ System. Sequencing SCC, accompanied with early mortality in adults with FA, is not well was performed using the Ion GeneStudio™ S5 Prime System (Thermo recapitulated in FA mice. Long-term survival has been reported in Fancc- Fisher Scientific, Waltham, MA, USA). The data was mapped to the , Fancd2-, Fancf-, Fancm- and Fancp-deficient mice. However, increased human genome assembly 19, embedded as the standard reference incidence of various solid tumors exists in Fanca-, Fancf-, Fancd2- and genome in the Ion Reporter™ Software (v. 5.14) (Thermo Fisher Sci­ Fancm-mice. Strikingly, none of these animals develop SCC in the oral entific, Waltham, MA, USA). We used the Ion Reporter™ Software for cavity, esophagus or anogenital tissues. These findings demonstrate that initial automated analysis, and Oncomine Comprehensive v3-w4.0- the loss of a single Fanc gene is not a sufficient condition to model early DNA-Single Sample as analysis workflow. Additionally, coverage anal­ cancer appearance or SCC development in GEMMs. Double-mutant mice ysis reports from the Ion Reporter™ Software providing measurements have been investigated to uncover genetic interactions with other of mapped reads, mean depth, uniformity, and alignment over a target cellular pathways leading to oncogenesis. Cooperation in tumor sup­ region were used as quality assessment of the sequencing reactions. pression clearly exists between the FA pathway and p53. Accelerated tumorigenesis is present in double Fancc-/-;Trp53-/- [14] and Fancd2-/-; Mice Trp53-/- [15], although these mice do not show increased SCC formation in oral and anogenital tissues, thus precluding their use as animal Fanca-/- animals were maintained in FVB/NJ background and ob­ models of SCC in the appropriate tissue of origin. Fancd2-/- animals, tained from the laboratory of Dr. Paula Rio (Biomedical Innovation Unit, when crossed with K14cre;HPVE6 or K14cre;HPVE6E7, and upon treat­ CIEMAT, Madrid, Spain) [25]. Trp53F2-10/F2-10 and K14cre mice were ment with oral carcinogen 4-NQO, developed HNSCC [16,17]. In these maintained in a mixed FVB/NJ × DBA/2J × C57BL/6J background. animals, no tumor formation was reported in the absence of 4-NQO. Animals were genotyped by PCR using specific primers as previously Most HNSCCs from FA patients display alterations typically found in described [22,25,26]. All animals showing tumors, or significant HPV-negative HNSCC tumors from the general population, such as morbidity, over a period of 24 months were sacrificed for necropsy. mutations in the TP53 gene [18–21]. Here, we also detected mutations Histopathological analysis was systematically performed on the esoph­ in TP53 in all five SCCs analyzed from the oral cavity of three FA pa­ agus and the oral cavity, including lips, palate, tongue, and oral mucosa. tients. In order to develop a mouse model of FA oral SCC, we decided to Overt tumors existing in other organs were also sampled for histopa­ cross Fanca-/- animals with K14cre;Trp53F2-10/F2-10 mice lacking p53 in thology. All mice husbandry and experimental procedures were 2 R. Errazquin et al. Oral Oncology 134 (2022) 106184 performed according to European and Spanish regulations and were animals, but not in WT or Fanca-/- (hereinafter referred Fanca) ones approved by the local Animal Ethical Committee and competent au­ (Table 1). OSCCs were located in the lips, tongue, and oral mucosa thority (code PROEX006/18). (Fig. 1A). The incidence of OSCCs was significantly higher in Fanca/ p53EPI than in p53EPI: 91% versus 58% (p < 0.05, Fisher’s exact test) Histopathology and Immunohistochemistry (Table 1). It is of note that double mutant Fanca/p53EPI mice developed OSCC much earlier than p53EPI as analyzed by a Kaplan-Meier plot Mouse tissues were fixed in 4% buffered formalin and embedded in (median latencies of 303 versus 432 days, respectively) (Fig. 1B). Tu­ paraffin. Sections (5 µm) were stained with H&E or processed for im­ mors in the tongue and in the oral mucosa were more frequent in double munostaining. Sections were incubated with primary antibodies and mutant mice (Fig. 1A), which are the prevalent locations of HNSCC in FA thereafter with biotinylated secondary antibodies (Jackson Immunor­ patients [1,28]. We did not find differences in OSCC incidences between esearch Laboratory, Ely, UK). Primary antibodies used were: anti- males and females (Supplementary Table 6). Carcinomas were well Keratin 5 polyclonal antibody, clone Poly19055 (BioLegend); anti- differentiated and frequently infiltrating (Fig. 2). Tumors in the lips Keratin 10 antibody, clone Poly19054 (BioLegend). Immunoreactivity were normally located at the mucocutaneous junction, which is a tran­ was revealed using the ABC-peroxidase system and the DAB substrate kit sitional region from oral mucosa to skin (Fig. 2A–C). Some animals (Vector Laboratories; Burlingame, CA, USA), and the sections were developed more than one tumor (multicentric SCCs) (Fig. 2D–F). counterstained with hematoxylin. Anti-Cytokeratin 5 antibody was Therefore, Fanca and p53 cooperate to suppress SCC in the oral cavity in purchased from Santa Cruz (sc-32721). Control experiments without the mice. Fanca/p53EPI animals constitute the first animal model of spon­ primary antibody gave no signal. taneous HNSCC in FA. OSCC from FA patients and mice express squamous differentiation Survival curves markers Tumor-free survival curves were obtained with Prism software SCC formation involves deregulation of normal cell differentiation. (Graphpad Software, Inc., San Diego, CA, https://www.graphpad.com). As expected, immunodetection of differentiation biomarkers in tumors Statistical significance of survival between genotypes was calculated from FA patients (Fig. 3) showed expansion of K5-expressing cells from with the log-rank test yielding a p-value. the basal location into suprabasal compartment in malignant cells (Fig. 3B and C). K10, a marker of suprabasal cells in epidermis that is Results ectopically expressed in human oral epithelial dysplasia and in skin and oral SCCs [29], was also detected in malignant cells (Fig. 3D and E). We Oral squamous carcinomas from Fanconi anemia patients display found similar K5 and K10 staining patterns in Fanca/p53EPI mice SCCs mutations in TP53 (Fig 4), supporting the double mutant animals as a valid model for the human disease. We performed deep-sequencing of DNA from cancerous areas of five FFPE blocks from three FA patients who had been diagnosed with Other malignancies in p53EPI and Fanca/p53EPI mice squamous cell carcinoma in the oral cavity and treated surgically. Pa­ tient clinical and sample information is shown in Supplementary We previously reported spontaneous skin SCC development in FVB/ Table 1. Genes included in the sequencing panel are frequently mutated NJ p53EPI mice [22], with aggressive behavior and molecular signatures in solid tumors (Supplementary Table 2). Interestingly, mutations in of poor prognostic human cancer [30]. In addition, we and others re­ TP53 previously reported to be oncogenic drivers were detected in all ported the development of mammary carcinomas in p53EPI mice five tumor samples analyzed (Supplementary Table 3 and Supplemen­ [31,32]. As expected, we found skin and mammary tumors both in tary Table 4), as well as mutations and copy number variants in other p53EPI and in double Fanca/p53EPI mice (Supplementary Fig. 1). Skin genes (Supplementary Table 4 and 5). In addition, we also found the tumors were mainly SCCs, although basal cell carcinomas (BCC), seba­ germline mutations in the FANCA gene that had previously been diag­ ceous gland carcinomas and fibrosarcomas were also diagnosed. Some nosed in all three FA patients [27] (Supplementary Table 3). These re­ p53EPI and Fanca/p53EPI animals were sacrificed as they developed overt sults are in line with the frequent detection of TP53 alterations in SCC thymic lymphomas (Supplementary Fig. 1). These findings suggest that from FA patients [19–21]. Therefore, animal models resembling germ­ Fanca/p53EPI mice also constitute a model for FA-associated cancer in line FANCA and somatic TP53 mutations might model SCC in FA. locations other than head and neck. Fanca/p53EPI and p53EPI animals develop spontaneous squamous cell Fanca mice display frequent pre-tumoral lesions in the oral cavity carcinomas in the oral cavity (OSCC) FA patients display a very high incidence of premalignant, non- Mice of the indicated genotypes were monitored for a period of 24 invasive lesions in the oral cavity, which are difficult to prevent, di­ months, and spontaneous lesions in the oral cavity were analyzed. We agnose and treat. We performed a careful inspection of histology sam­ found OSCC in K14cre;Trp53F2-10/F2-10 (hereinafter referred p53EPI) and ples from oral tissues collected at the time of animal sacrifice to detect Fanca-/-;K14cre;Trp53F2-10/F2-10 (hereinafter referred Fanca/p53EPI) possible lesions indicative of cellular transformation. Interestingly, most Fanca mice developed aberrant tissue phenotypes in the oral cavity Table 1 (including atypia, acantolysis, picnosis or vacuolization) associated with Incidences of oral SCC in animal genotypes. hyperplasia or, more importantly, carcinoma in situ (CIS), which rep­ Genotype Group size, n Oral SCC, n (%) resents the highest atypia of premalignant epithelial cells (Supplemen­ Control 12 0 (0) tary Table 7 and Fig. 5). Incidences were significantly higher than in Fanca 16 0 (0) wild type (WT) animals (p-val < 0.05, Fisher’s exact test), even though p53EPI 12 7 (58) mean age at the analysis was very similar in both genotypes (WT = 18 Fanca/p53EPI 11 10 (91) months versus Fanca = 19.4 months). Lesions were observed in the lips, NOTE: The difference in OSCC incidence between p53EPI and Fanca/p53EPI palate, tongue, oral mucosa, and esophagus (Fig. 5 and 6), and were also groups was statistically significant (p-val < 0.05). Statistical comparisons were found in p53EPI and, mainly, in double Fanca/p53EPI. Some animals had performed using the Fisher’s exact test. more than one (multicentric) pre-tumoral lesion. Globally, the results 3 R. Errazquin et al. Oral Oncology 134 (2022) 106184 Figure 1. Fanca and Trp53 genes cooperate to suppress oral SCC in mice. Mice from the indicated genotypes were long-term monitored (24 months), sacrificed at the observance of tumor/s development or significant morbidity, and analyzed histologically. (A) Percentages of animals in p53EPI and Fanca/p53EPI genotypes having oral SCC, shown by sub-anatomical location. (B) A Kaplan-Meier plot of the percentage of oral SCC free survival is shown. Note that Fanca loss accelerates SCC development in p53EPI animals (Fanca/p53EPI versus Fanca). Statistical significance between curves was determined using log rank test. ***P < 0.001, ****P < 0.0001, P > 0.05 (NS). Figure 3. Oral SCCs in FA patients express K5 and K10 squamous differ­ entiation keratin markers. (A) H&E staining of a well differentiated SCC in the retromolar trigone of tumor #FA903-17, with a “horny pearl” or concentric foci of squamous differentiation (asterisk in A). K5 and K10 are expressed in malignant cells of the #FA903-17 SCC shown in (A) (B and D), and in a tongue SCC (#FA310-18) from another FA patient (C and E). showed that Fanca gene mutation is associated with development of premalignant lesions in the oral mucosa of transgenic mice. EPI EPI Figure 2. Histopathology of oral SCC in p53 and Fanca/p53 mice. (A) Well differentiated SCC at the mucocutaneous junction of the lips in a p53EPI Discussion mouse. The tumor infiltrates deep into the subcutaneous muscle (black arrows). Observe the mucosal (left dotted box) and epidermal (right dotted box) epithelia surrounding the carcinoma. (B) and (C) are magnifications of the Fanconi anemia patients display an exacerbated susceptibility to dotted boxes in (A), where mucosal and cutaneous junctions are shown, develop HNSCC early in life, mainly in the oral cavity. Due to their respectively. (D) Well differentiated SCCs developing at the oral mucosa and sensitivity to DNA damaging agents, treatments based on radiotherapy the epidermis enclosing the lips (dotted boxes) in a Fanca/p53EPI animal. (E) and chemotherapy should be avoided. When compared with non-FA and (F) are magnifications of the dotted boxes in (D), where mucosal and patients, poorer survival after cancer development can be explained cutaneous (yellow arrow) SCCs are shown. partially by this suboptimal treatment. Therefore, modeling the disease in animals is important to understand the oncogenic mechanisms, discover cancer drivers and biomarkers, and perform preclinical tests of new therapies. Here, we report the first mouse model of spontaneous 4 R. Errazquin et al. Oral Oncology 134 (2022) 106184 observation (up to 24 months of age). Therefore, additional genetic events are needed. We have found TP53 mutations in all five HNSCC from three FA patients (Supplementary Table 3 and 4), which is in agreement with the high frequency of mutations or deletions in TP53 described in HPV-negative, FA [18–21] and non-FA patients [34–36]. In addition, TP53 mutations are early events during HPV-negative HNSCC carcinogenesis in non-FA patients [37,38]. These findings together with the switch from premalignant to malignant oral lesions observed in Fanca animals when crossed with p53EPI (Figs. 1 and 2) point to an essential contribution of Trp53 in early oral carcinogenesis in FA in­ dividuals. We propose that SCC development in FA individuals is a sequential process in which DNA instability produced by germline mu­ tations in Fanconi genes might eventually give rise to alterations affecting additional cancer genes. Such double mutant cells might ac­ quire a selective advantage over neighbor cells and expand to produce transformed clones with differential invasive capabilities. Cooperation between Trp53 and FA genes has already been described: tumor appearance in full body deletion in Trp53 (Trp53-/- mutant mice) is accelerated when combined with loss of Brca2 (Fancd1), Figure 4. Oral SCC in Fanca/p53EPI mice express K5 and K10 squamous differentiation keratin markers. (A) and (B) H&E staining of well differen­ tiated SCC in the oral mucosa (A) and tongue (B) of two different Fanca/p53EPI animals (#319 and #276). Both are well differentiated SCCs since “horny pearls” or concentric foci of squamous differentiation are evident in the tumors (asterisks in A and B). K5 and K10 expression is present in hyperplastic and malignant cells of the SCCs shown in A) (C and D) and B) (E and F). SCC in the oral cavity with genetic depletion of a Fanconi pathway gene. FA patients display numerous pre-malignant lesions in the oral cavity, which are difficult to diagnose and treat [2]. These lesions may lead to carcinoma development, and new methods to predict their ma­ lignant transformation are needed to improve early detection of the malignant disease [33]. We found that Fanca animals develop hyper­ Figure 6. Histology of carcinoma in-situ (CIS) in Fanca mice. CIS prema­ plastic and CIS lesions that might resemble patient pre-malignant lignant changes found in the epithelia of the oral cavity of different Fanca an­ changes (Supplementary Table 7, Figures 5, and 6). Further analysis of imals. (A) Soft palate with a focus of lichenoid inflammation, pseudocarcinomatous basal hyperplasia and severe atypia of the epithelium, the lesions in Fanca mice and comparison with clinical samples might characterized by nuclear pyknosis, increase of the nuclear:cytoplasm ratio, and help to understand the mechanism of development. In addition, new loss of nuclear polarity of keratinocytes (square yellow bracket). (B) Pseudo­ chemopreventive and therapeutic approaches targeting these lesions carcinomatous basal hyperplasia and severe atypia of the epithelium in the hard could be tested in a preclinical setting. palate. (C) Foci of severe atypia and epithelial hyperplasia associated to Our results support the hypothesis that loss of Fanca gene alone does lichenoid inflammation in the labial mucosa. (D) Foci of extreme atypia with not promote the development of SCCs, even at prolonged times of epithelium vacuolization of the dorsal tongue filiform papillae (square bracket). Figure 5. Fanca gene prevents the development of hyperplastic and CIS lesions in the oral cavity. Percentages of animals in all genotypes (WT, Fanca, p53EPI and Fanca/p53EPI) showing hyperplastic (A) or CIS (B) changes in the oral cavity, shown by sub-anatomical location. 5 R. Errazquin et al. Oral Oncology 134 (2022) 106184 Fancd2 or Fancc [14,15,26]. Cells from Fanca-/- animals activate p53 [3] Kutler DI. 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[14] Freie B, Li X, Ciccone SLM, Nawa K, Cooper S, Vogelweid C, et al. Fanconi anemia Declaration of Competing Interest type C and p53 cooperate in apoptosis and tumorigenesis. Blood 2003;102(12): 4146–52. [15] Houghtaling S, Granville L, Akkari Y, Torimaru Y, Olson S, Finegold M, et al. The authors declare that they have no known competing financial Heterozygosity for p53 (Trp53+/-) accelerates epithelial tumor formation in interests or personal relationships that could have appeared to influence fanconi anemia complementation group D2 (Fancd2) knockout mice. Cancer Res the work reported in this paper. 2005;65:85–91. [16] Park JW, Shin M-K, Pitot HC, Lambert PF, Pagano J. High incidence of HPV- associated head and neck cancers in FA deficient mice is associated with E7’s Acknowledgements induction of DNA damage through its inactivation of pocket proteins. PLoS ONE 2013;8(9):e75056. [17] Park JW, Pitot HC, Strati K, Spardy N, Duensing S, Grompe M, et al. Deficiencies in The authors thank J. Bueren, P. Rio, M. Garin, and R. Gargini for the Fanconi anemia DNA damage response pathway increase sensitivity to HPV- helpful discussions. The authors are also indebted to the patients with associated head and neck cancer. Cancer Res 2010;70:9959–68. FA, their families and clinicians from the Fundacion Anemia de Fanconi [18] Montanuy H, Martinez-Barriocanal A, Casado JA, Rovirosa L, Ramirez MJ, Nieto R, et al. Gefitinib and afatinib show potential efficacy for Fanconi anemia-related (Spain). The authors also thank P. Hernández and F. Sanchez-Sierra for head and neck cancer. Clin Cancer Res 2020;26:3044–57. their excellent assistance with the histological processing of the samples, [19] Roohollahi K, de Jong Y, Pai G, Zaini MA, de Lint K, Sie D, et al. BIRC2-BIRC3 and the personnel of the CIEMAT Animal Unit for the care of the mice amplification: a potentially druggable feature of a subset of head and neck cancers used in this work. in patients with Fanconi anemia. Sci Rep 2022;12(1):45. [20] van Zeeburg HJ, Snijders PJ, Pals G, Hermsen MA, Rooimans MA, Bagby G, et al. Generation and molecular characterization of head and neck squamous cell lines of Funding fanconi anemia patients. Cancer Res 2005;65:1271–6. [21] Webster ALH, Sanders MA, Patel K, Dietrich R, Noonan RJ, Lach FP, et al. Fanconi anemia pathway deficiency drives copy number variation in squamous cell This study has been funded by Instituto de Salud Carlos III (ISCIII) carcinomas. bioRxiv. 2021:2021.08.14.456365. through the projects CB16/12/00228/CIBERONC, PI18/00263 and [22] Martinez-Cruz AB, Santos M, Lara MF, Segrelles C, Ruiz S, Moral M, et al. Spontaneous squamous cell carcinoma induced by the somatic inactivation of P121/00208 and co-funded by FEDER and the European Union; and retinoblastoma and Trp53 tumor suppressors. Cancer Res 2008;68:683–92. grants from the Spanish Fundacion Anemia de Fanconi and Fanconi [23] Moral M, Segrelles C, Lara MF, Martinez-Cruz AB, Lorz C, Santos M, et al. Akt Anemia Research Fund USA. J.P. was supported by a FEDER co-funded activation synergizes with Trp53 loss in oral epithelium to produce a novel mouse grant (ref PEJ2018-002040-A) from the Ministerio de Ciencia, model for head and neck squamous cell carcinoma. Cancer Res 2009;69:1099–108. [24] Page A, Bravo A, Suarez-Cabrera C, Sanchez-Baltasar R, Oteo M, Morcillo MA, et al. Innovación y Universidades. J.O. was supported by a FEDER co-funded IKKbeta overexpression together with a lack of tumour suppressor genes causes grant (ref PEJ-2019-TL_BMD-12905) from the Comunidad de Madrid. ameloblastic odontomas in mice. Int J Oral Sci 2020;12:1. The funding sources were not involved in study design; in the collection, [25] Cheng NC, van de Vrugt HJ, van der Valk MA, Oostra AB, Krimpenfort P, de Vries Y, et al. Mice with a targeted disruption of the Fanconi anemia homolog analysis and interpretation of data; in the writing of the report; and in Fanca. Hum Mol Genet 2000;9:1805–11. the decision to submit the article for publication. [26] Jonkers J, Meuwissen R, van der Gulden H, Peterse H, van der Valk M, Berns A. Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer. Nat Genet 2001;29(4):418–25. Appendix A. Supplementary material [27] Bogliolo M, Pujol R, Aza-Carmona M, Muñoz-Subirana N, Rodriguez-Santiago B, Casado JA, et al. Optimised molecular genetic diagnostics of Fanconi anaemia by Supplementary data to this article can be found online at https://doi. whole exome sequencing and functional studies. J Med Genet 2020;57(4):258–68. 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