Double heterozygous pathogenic germline APC and MSH2 variants in a 16-year-old female diagnosed with colorectal cancer

Abstract

Germline pathogenic variants (PVs) of the APC gene cause familial adenomatous polyposis (FAP), whereas PVs in DNA mismatch repair (MMR) genes underlie Lynch Syndrome (LS), formerly known as hereditary non polyposis colorectal cancer (HNPCC). The coexistence of both hereditary syndromes in a single individual has been reported extremely rarely and only in a few patients, and the scant literature on this topic suggests an earlier onset and a more rapidly worsening form of colorectal cancer, compared with subjects who carry a single mutation. Here we describe a 16-year-old female who presented with multiple colonic polyps as well as an invasive colorectal carcinoma, and was found to harbor heterozygous pathogenic variants (PV) both in APC and MSH2 genes. Our patient also represents the unique reported case harboring both a MSH2-EPCAM large rearrangement in association with a frameshift APC PV. This case illustrates the unique clinical phenotype that can arise from simultaneous FAP and LS pathogenic mutations, highlights the importance of comprehensive genetic testing in young patients with aggressive disease and ultimately suggests a personalized follow-up and surveillance program in this case.

Introduction

Hereditary colorectal cancer (CRC) syndromes are associated with 2–8% of all CRCs and around 20% of CRCs diagnosed before the age of 50 are related to germline pathogenic variants (PVs) in high-risk cancer genes [1, 2]. Diagnosis and accurate treatment of individuals with a hereditary form of CRC require a detailed knowledge of the primary syndrome and tumor genetics [3]. The two main representatives of such syndromes, whose clinical phenotypes differ from each other, are Familial Adenomatous Polyposis (FAP) and Lynch Syndrome (LS, formerly known as hereditary non-polyposis colorectal cancer – HNPCC).

FAP is responsible of around 1% of all colorectal cancers, and is caused by germline loss of function mutations in the APC gene, leading to the development of hundreds to thousands of adenomatous polyps, with about 100% life-time risk of colorectal cancer if untreated [1, 4]. An attenuated FAP phenotype (AFAP), with later onset of colorectal cancer and fewer polyps, is associated with a PV in exon 9 and in the 5’ or 3’ ends of the APC gene [5].

LS is caused by pathogenic variants in any of the Mismatch Repair (MMR) genes, in particular MSH2, MLH1, MSH6 and PMS2, or by a deletion in the EPCAM gene, which leads to methylation of the adjacent MSH2 promoter [1, 6]. It accounts for 2–4% of colorectal cancers and also increases the risk of developing extra-colonic neoplasms, such as endometrial, ovarian, stomach, urothelial and small intestine cancers [1, 6].

Individuals carrying both a germline APC PV and an MMR defect are extremely rare; only a handful of cases have been described [7–11], and they appear to develop malignancy more aggressively than patients with either syndrome alone [8]. Here we expand the clinical spectrum with a longitudinal description of a 16-year-old patient (later found out to have a FAP-LS overlapping genotype), who experienced invasive colon adenocarcinoma on her first colonoscopic evaluation.

Case description

The proband, a 16-year-old female, was referred to Highly Specialized Regional Reference Center for Oncological Genetics “Prof. Riccardo Cellerino” of the Azienda Ospedaliero Universitaria (AOU) delle Marche after undergoing subtotal colectomy following a colonoscopy which revealed numerous colorectal polyps and a biopsy proven adenocarcinoma at the splenic flexure. Her father was diagnosed with colon cancer at age 46 and was previously diagnosed as a carrier of a pathogenic APC variant (NM_00038.6:c.4054_4063dup; p.(Ser1355Cysfs*2)). The proband’s paternal uncle died of colorectal cancer at 42, and even her paternal grandmother was affected by the same neoplasm (the proband’s family pedigree is shown in Fig. 1).

Fig. 1.

Proband’s family pedigree; filled symbols indicate colon cancer and further clinical/genotypical characteristics are described

It is worth underscoring that the patient’s father had not previously undergone cascade targeted testing for the APC mutation in his daughter. In fact, she was initially only followed by her local Pediatric Gastroenterology because of her celiac disease. During its instrumental follow-up, a colonoscopy was performed, which showed approximately twenty different sessile and pedunculated polyps throughout her colon and proximal rectum, as well as a 3-cm vegetating lesion at the splenic flexure: a biopsy was performed, revealing the presence of infiltrating ulcerated adenocarcinoma. Since no extra-colonic disease was confirmed, the patient was admitted to the Pediatric Surgery department at Salesi Hospital of Ancona in January 2024, where she underwent laparoscopic subtotal colectomy with ileostomy and rectal polypectomy.

Pathology report showed a well-differentiated mucinous adenocarcinoma pT2(m) that arose from an adenomatous polyp, another tubulo-villous polyp with well-differentiated mucinous adenocarcinoma (pT1, Haggitt level II), a tubular adenoma with high-grade dysplasia, other 6 tubular adenomatous polyps with low-grade dysplasia and other 6 microadenomas. Thirty-three lymph nodes resulted negative for metastases (pathological stage pT2(m) pN0 (AJCC 8th)). Immunohistochemical (IHC) analysis of the tumor demonstrated preserved nuclear expression of MLH1 and PMS2, with nuclear loss of MSH2 and MSH6 expression. Interestingly, aberrant cytoplasmatic staining of MSH2 was uniformly observed not only in adenocarcinoma tissue and adenomatous polyps, but also in dysplastic foci and colonic crypts foci. Aberrant nuclear beta-catenin staining was also observed in all histological samples. All the abnormalities were uniformly observed throughout the samples, with the difference of nuclear MSH2 and MSH6 lack of expression in cancerous tissue and adenomatous polyps compared with seemingly “normal” colonic mucosa, where both MSH2 and MSH6 were preserved. (histological and IHC findings are reported in Fig. 2). Molecular testing confirmed a high microsatellite instability (MSI-H) phenotype.

Fig. 2.

Histological and immunohistochemical specimens of the proband. and Loss of MSH6 expression (and loss of MSH2 nuclear expression with aberrant cytoplasm staining) and positive B-catenin staining (both nuclear and membrane) of adenocarcinoma samples. Loss of MSH6 expression (and loss of MSH2 nuclear expression) and positive B-catenin staining (both nuclear and membrane) of adenomatous polyp sample.: Preserved MSH6 and MSH2 and positive B-catenin staining of dysplastic foci nearby the tumor. Preserved MSH6 and MSH2 and positive B-catenin staining of colonic crypts nearby the tumor

Oncological genetic counselling was performed and a comprehensive hereditary cancer panel was proposed. Firstly, targeted cascade APC testing via Next Generation Sequencing (NGS), performed at Oncogenetics and Functional Oncogenomics Unit of Centro di Riferimento Oncologico of Aviano, identified heterozygous APC NM_00038.6:c.4054_4063dup p.(Ser1355Cysfs*2) (same mutation as the proband’s father), interpreted as pathogenic (Class 5). Then, genetic testing for hereditary tumors, using an NGS multigene panel (MLH1, MSH2, MSH6, PMS2, BRCA1, BRCA2, PALB2, CHEK2, RAD50, RAD51C, RAD51D, ATM, BARD1 and TP53) available at Highly Specialized Regional Reference Center for Oncological Genetics “Prof. Riccardo Cellerino” of the Azienda Ospedaliero Universitaria (AOU) delle Marche, identified a heterozygous large pathogenic deletion of MSH2(NM_000251.3):c.(?-125)(1386+1_1387-1)del, deleting exons 1–8, including the promoter region, and involving the exons 8 and 9 of EPCAM (NM_002354.3). This rearrangement is classified as pathogenic, as well, and leads to a diagnosis of LS [12, 13]. These results therefore confirmed concurrent FAP and LS diagnoses (a detailed proband’s clinical history timeline is shown in Fig. 3).

Fig. 3.

The proband’s clinical history timeline (CT: computerized tomography; EGD: esophagogastroduodenoscopy; FAP: familial adenomatous polyposis; GE: gastroenterology; LS: Lynch syndrome; MRI: magnetic resonance imaging; MSI-H: high microsatellite instability; PET: positron emission tomography; SUV: Standardized Uptake Value)

Because of these simultaneous diagnoses, the aforementioned hereditary cancer detection NGS panel and cascade APC testing were also used for Our patient’s parents, brother and sister: her mother was found to be a carrier of an MSH2 PV and, as of this case report’s publication date, she is still undergoing active surveillance for LS, always resulting negative; her brother resulted negative for both FAP and LS; her sister was diagnosed with FAP alone and underwent a subtotal colectomy in July 2025, due to the endoscopic finding of many polyps with low-grade dysplasia. Our proband’s maternal uncle also tested positive for LS, therefore excluding a de novo MSH2 PV of the probands’ mother.

The demonstration of the APC variant segregating on the paternal side (FAP) and the MSH2 variant segregating on the maternal side (LS) confirms that the proband represents a genuine double heterozygote, resulting from the chance convergence of two separate hereditary cancer lineages.

Discussion

Our patient represents one of the few well-documented cases of germline APC and MMR gene double heterozygosity worldwide [7–11] and, so far, only the third reported case in literature describing a simultaneous germline APC and MSH2 mutation [7, 8]. Moreover, Our patient also represents the unique reported case harboring both a MSH2-EPCAM large rearrangement in association with a frameshift APC PV.

The clinical presentation is notable for several reasons. First, the age at CRC onset (16 years) is strikingly early, preceding the typical median age of CRC presentation in both FAP (about 40 years [14]) and LS (ranging 44–52 years [15]) alone. Secondly, the combination of a high polyp burden (characteristic of FAP-spectrum APC mutations) with an MSI-H tumor (typical of MMR deficiency) within the same individual suggests that both genetic defects contributed independently to the pathogenesis of this malignancy.

The most informative aspect of this case is the direct phenotypic comparison with Our proband's sister, who carries the identical APC PV, but lacks the MSH2 deletion. Our proband’s sister, in fact, developed the expected FAP phenotype (extensive polyposis leading to prophylactic surgery) by adolescence, undergoing subtotal colectomy due to low-grade dysplasia but not adenocarcinoma. The proband, on the other hand, carrying the same high burden of APC-driven polyps plus the MSH2 PV, as well as confirmed MSI-H status, progressed directly to invasive adenocarcinoma by around the same age. This within-family comparison provides compelling evidence that the additional loss of MMR function accelerates the adenoma-to-carcinoma transition, likely through the accumulation of secondary somatic mutations in critical driver genes that would otherwise manifest later in the setting of proficient MMR [16–18].

Aberrant MSH2 cytoplasmatic expression was uniformly observed in all histological samples: this feature has been previously described [19] and it has been associated with specific MSH2 variants where large genomic deletions also occurring in EPCAM gene were found. This is exactly what was found in Our patient, as germline testing confirmed the presence of a large deletion of exons 1–8 of MSH2, also including the promoter region, and deletion of exons 8–9 of EPCAM gene.

We believe that, similarly to what was described previously, the result of the aberrant fusion transcript of EPCAM and MSH2 is the leading cause of the observed cytoplasmic MSH2 staining. All samples that were assessed for this case, comprising of both tumoral, adenomatous polyp, dysplastic foci and colonic crypts samples had aberrant cytoplasmic staining, thus confirming the presence of one mutated germline allele in all specimens.

On the other hand, complete loss of nuclear expression of MSH2 was only evident in cancer tissue and in adenomatous polyp, whereas in dysplastic foci and in colonic crypts, even if aberrant cytoplasmic staining of MSH2 was observed, standard nuclear expression of MSH2 was also found. Coupled with that, MSH6 nuclear staining was only found in dysplastic foci and in colonic crypts foci, while, as expected, was lost in cancerous tissue and in adenomatous polyps.

As also previously described by Dong et al. [19] this specific pattern of expression might be associated with the “double hit” phenomenon where the paternal allele (that was not harboring germline MSH2 variants), upon receiving a secondary mutation leading to its inactivation, would result in the loss of MSH2 (and MSH6) nuclear expression while aberrant cytoplasmic staining of MSH2 would still be present due to the aberrant localization of the fusion transcript of the germline variant inherited from the patient’s maternal line. Beta-catenin aberrant expression, a typical hallmark of APC function loss, was instead observed in all histological samples, even including those with “normal” colonic mucosa and in aberrant dysplastic foci. Due to the different pattern of nuclear expression of beta-catenin, that was present in all samples, and the loss of nuclear expression of MSH2, that was present only in cancerous tissue and adenomatous polyps even when aberrant cytoplasmic MSH2 expression was still evident in all samples, we made Our hypothesis that APC variants might “initiate” the process, leading to an increased number of polyps and only when polyps reach a critical point (related to their size or their number or both) secondary mutations in MSH2 might hasten the development of cancer.

Table 1 summarizes all reported cases of germline APC and MMR gene double heterozygotes in the literature. Most of these patients presented with CRC in their first or second decade of life, a pattern that is notably earlier than the expected presentation for either FAP or LS alone. The variation in polyp burden across cases likely reflects the specific APC variant (classic FAP versus AFAP) and may also be modulated by the identity of the affected MMR gene and the level of residual repair capacity.

Table 1.

Comparison between Our proband and the other double-mutated APC/MMR genes cases reported in literature (AFAP, attenuated familial adenomatous polyposis; CRC, colorectal cancer; FAP, familial adenomatous polyposis; HGD, high-grade dysplasia; MMR, Mismatch Repair; PV, Pathogenic Variant)

Case	Age at CRC	APC PV (*)	MMR Gene (*)	Polyp Burden	Extra-colonic Lesions	Surgical Approach
Scheenstra (2003) [10]	10	Exon 15 (NM_000038.2**, Frameshift)	MLH1 NM_000249.4:c.677G>A (Splice site)	>100 adenomas, rapidly progressive to HGD	Not reported	Proctocolectomy with ileal pouch-anal anastomosis
Okkels (2006) [11]	18	NM_000038.6:c.7504G>A, (Missense)	MSH6 NM_000179.3:c.1836C>A, NM_000179.3:c.3226C>T, (Compound het: nonsense + missense)	Multiple adenomas with synchronous CRC	Not reported	Not specified
Uhrhammer (2008) [7]	16	NM_000038.6:c.3183_3187del (Frameshift)	MSH2 NM_000251.3:c.258_259del (Frameshift)	Polyposis noted (limited details)	Family: uterus, kidney, bladder, small intestine cancers	Not specified
Soravia (2005) [8]	25	NM_000038.6:c.3471_3474del (Frameshift)	MSH2 NM_000251.3:c.1191_1192dup (Frameshift)	<10 polyps (AFAP phenotype)	Gastric fundic + antral + duodenal adenomas; intra-abdominal desmoid tumor	Prophylactic colectomy (pT1 N0)
Yuan (1998) [9]	Not reported	NM_000038.6:c.3920 T>A (I1307K, Missense risk factor)	MLH1 c.1784delT§ (Frameshift)	Not reported	Not reported	Not specified
PRESENT CASE	16	NM_000038.6:c.4054_4063dup (Frameshift)	Large MSH2-EPCAM deletion (NM_000251.3: exons 1–8, NM_002354.3: ex8-9)	Around 20 polyps at colonoscopy, confirmed 15 polyps at histologic specimen	Gastric fundic pseudopolyps, esophagitis	Subtotal colectomy with ileostomy + rectal polypectomy

^*The RefSeq sequences were not cited in the publications; Variants have been annotated using the current MANE Select RefSeq whenever possible

^**The reference sequences have been inferred based on the publication year and exon numbering

^§The variant is not straightforward to annotate according to current nomenclature

Albeit various LS and FAP guidelines delineate comprehensive follow-up and management programs for their patients, there are currently no reported strategies regarding intensified surveillance when both pathways are disrupted. Therefore, here we suggest a possible practical integrated FAP/LS follow up recommendations for this extremely rare and specific category of patients:

Colorectal management

In FAP patients, prophylactic colectomy is commonly recommended once polyposis be-comes unmanageable endoscopically or when high grade dysplasia/cancer is identified; subtotal colectomy with ileo-rectal anastomosis or proctocolectomy with ileal pouch may be chosen based on rectal polyposis and patient factors [20]. Since LS also confers early CRC risk, surveillance intervals for residual rectum (if preserved) should be short (every 6–12 months, hypothetically) with subsequent proctectomy if rectal disease becomes uncontrolled. Our patient has already undergone subtotal colectomy and ileostomy, so a careful endoscopic surveillance of the preserved rectum per FAP protocols is appropriate. LS lifetime CRC risk is high (50–80%), but colonoscopic surveillance markedly reduces CRC incidence and mortality in MMR carriers [21]. Moreover, in well surveilled cohorts, more deaths now occur from extracolonic LS cancers than from CRC [22].

Upper gastrointestinal (GI) surveillance

FAP upper GI protocols with periodic EGDs integrated with Spigelman staging for duodenal polyposis [22] are to be followed, because APC pathogenic variants noticeably increase duodenal and gastric cancer risk. Our proband’s gastric polyps should therefore be adequately monitored and biopsied.

Extra colonic screening for LS associated risks

Since MSH2 pathogenic variants carry higher risks for endometrial, ovarian, urinary tract and other LS associated tumors [23], gynecological surveillance and counseling (annual endometrial sampling or transvaginal ultrasound, discussion of risk reducing hysterectomy and bilateral salpingo-oophorectomy after childbearing), as well as urinary cytology are to be recommended.

Potential immunotherapy administration

If these patients’ neoplasms were to evolve into metastatic diseases, it is worth noticing that MSI-H CRCs are strong candidates for anti-Programmed-Death (PD) 1 immunotherapy, which has regular approval in numerous countries [24].

Multidisciplinary care and timing of interventions

Because this patient combines risks from both syndromes, multidisciplinary coordination among CRC surgery, medical oncology, gastroenterology, gynecology and clinical genetics is essential to orchestrate surveillance and surgical timing, optimize quality of life and plan fertility preservation when relevant.

Genetic counseling and cascade testing

Each parent can transmit each pathogenic allele independently: first degree relatives should consequently be offered targeted testing both for the familial APC and the MSH2 vari-ant, because inheritance follows autosomal dominant transmission for each gene. Relatives who carry only APC mutation need FAP surveillance; relatives who carry only MSH2 mutation need LS surveillance; relatives who inherit both (or children of two carriers) may be at risk for earlier and/or more aggressive disease and should be counseled accordingly.

Conclusion

In conclusion, this case represents one of the extremely rare documented instances of simultaneous germline APC and MSH2 pathogenic variants, stressing the need for broad genetic testing in young patients with aggressive CRC disease.

In recent years, a widely increasing, progressive and exponential use of genetic tests has been reported, both for prevention and therapeutic purposes, due to the widespread use of biological drugs targeting specific molecular path-ways. This process, called "mainstreaming genetic testing", probably will increase the number of probands carrying simultaneous pathogenic variants in hereditary-cancer associated genes.

Reporting additional cases, ideally with prospective monitoring, is warranted, in order to steadily outline a comprehensive follow-up for these peculiar but clinically crucial genotypes.

Authors contributions

Conceptualization: E.M, R.B (Riccardo Bacalini), A.L, C.C, R.G and R.B (Rossana Berardi) Methodology: E.M and R.G Validation: E.M, R.B (Riccardo Bacalini) Investigation and data collection: E.M, R.B, A.L, C.C, F.B, C.B, L.B, E.A, N.C, N.E.S, G.R, V.T, M.S, F.M, M.A, S.V, V.A, G.M, A.M, G.G and P.C Data Curation: E.M, R.B (Riccardo Bacalini), R.G and R.B (Rossana Berardi) Writing and original draft preparation: R.B (Riccardo Bacalini), E.M, A.L, C.C Writing and review and editing: R.G and R.B (Rossana Berardi).

Data availability

No datasets were generated or analysed during the current study.