We searched our pathology reports databases for patients with rSCC, using the keywords “squamous cell carcinoma” and “rectum” and selected those who meet the inclusion criteria. We also used our SNOMED (Systematized Nomenclature of Medicine) International Code coding system, looking for the codes “squamous cell carcinoma” and “rectum” associated with our reports. Only tissue from patients with rSCC that meets the following 4 strict diagnostic criteria has been used: (1) No continuity between the tumor and the anal squamous epithelium or the gynecological tract, (2) Absence of a SCC in another primary site, (3) Absence of squamous-lined fistula in the context of inflammatory bowel disease, (4) Finally histological confirmation of rSCC. All the cases of rSCC biopsy, in whom the complementary work-up (pelvic MRI, ano-rectoscopy, colonoscopy) shows an anal origin (or extension) of the gynecological tract, or another primary tumor location (non-rectal) have been excluded. The study is authorized by the competent Ethics Committee of Geneva (Project-ID: 2021-00,149).

We identified nine patients at HUG (Geneva University Hospitals) and another three cases were selected from the cantonal hospital of Sion (2 institutions study). These two institutions count about an average of 70 new rectal cancer cases per year, meaning approximately 2100 patients in the last 30 years (period during which pathology reports databases have been searched in our study). In other words, our 10 rSCC cases correspond to 0, 4% of all rectal cancers of our 2 centers, a percentage that is in absolute adequacy with rSCC’s prevalence in the literature.

Tissue sample selection was performed during the first half of 2021. After careful histological review, two cases were dropped—one had mixed adeno-squamous histology and the other was a poorly differentiated adenocarcinoma. One patient (clinical case 1) was initially diagnosed with a concomitant squamous esophageal carcinoma in addition to his rSCC. To understand if both tumors were related (exclusion criteria), we carried out an HPV profiling, which showed positivity for the rectal tumor and negativity for the esophageal tumor, speaking in favor of 2 unrelated tumors.

The 10 selected patients (Table 1) were profiled by NGS sequencing, CNV analysis and HPV typing. Both tumor and non-tumor tissues from the 10 selected patients are used in our assays. In 3 out of 10 patient cases (clinical cases 5, 6, and 10), molecular analyses were carried out on post-CRT samples, because of their higher percentage of tumor cells compared to tissue biopsies taken on pre-CRT.

Genomic DNA extraction and purification using the QIAamp DNA FFPE (Fixed-Formalin, Paraffin-embedded) tumor tissue Kit (cat. 56,404; QIAGEN, Hilden, Germany) and copy number profiling and quantification, using the OncoScan Assay kit (cat. 902,695; ThermoFisher Scientific) were performed following manufacturer’s instructions, as previously described [21]. For NGS sequencing, libraries of a custom 462-gene panel (SureSelect-HS library, Agilent) were built from genomic DNA. Paired-end sequencing, 2 × 150 nt, has been performed on a NextSeq500 sequencer (Illumina) as previously described [22]. The size of our custom NGS panel is > 1Mbp.

Copy number variation was performed, using the OncoScan Assay kit (cat. 902,695; ThermoFisher Scientific) following manufacturer’s instructions, as previously described [21]. Data were analyzed using OncoScan Console and Chromosome Analysis Suite (CHAS) software.

CNV segments were classified into four categories: “gain’’, when there are one or two extra copies with respect to the diploid state; “amplification’’, in case of a gain of five or more copies; “loss’’, when the number of copies is lower than the normal number (two in a human genome); and loss of heterozygosity (LOH), when there is a loss of the maternal or paternal allele without any loss of copies.

The Cancer Gene Census, COSMIC (Catalogue of Somatic Mutations In Cancer), CIViC (Clinical Interpretations of Variants in Cancer), OncoKb (PMID: 28,890,946) were used for variant interpretation and classification according to international guidelines (PMID: 25,741,868, PMID: 27,993,330).

DNA extracted from fixed material was of sufficient quality to perform PCR-Blot analysis (DNA control and HPV positivity). Analysis for HPV virus DNA was realized by polymerase chain reaction (PCR) amplification of the region conserved L1 and hybridization of the PCR product on blot, making it possible to identify high-risk HPV types (16,18,31,33,35,39,45,51,52,56,58,59,68a), probably high risk (26,53,66,70,73,82), low risk (6,11,40,42,43,44,54,61), and HPV of uncharacterized pathogenicity (62,67,83,89).

For HPV genotyping, the Inno-LiPA HPV Genotyping Extra II (cat.81534, Fujirebio) was used according to the manufacturer’s instructions. We proceeded to a PCR analysis using a kit that can detect the presence of HPV and then hybridization to determine the HPV subtype.

We hypothesized that comprehensive mutation profiling of a cohort of rSCC tumors, could assist in defining the genomic landscape of this rare cancer. We compared our data with public rADC databases from TCGA (The Cancer Genome Atlas Program) [22,23,24,25,26]. Concerning aSCC, there is no public database including a complete molecular characterization of this tumoral entity. In this context, we used genomic profile published in the scientific literature for aSCC in pre- and post-CRT for local and metastatic disease [25, 27, 28].