Frozen colonic samples collected in two different countries, Czech Republic (n = 195 specimens) and Italy (n = 80 specimens), were analyzed for the presence of HPV DNA by NGS or a multiplex Luminex genotyping assay, respectively.

Italian cohortStudy group

CRC frozen tissues (n = 40) and matched surrounding healthy tissues (n = 40) were collected at Istituto Tumori “Giovanni Paolo II” IRCCC Hospital, Bari, Italy between 2017 and 2019. The 40 patients were 26 male and 14 female, with an average age of 67.3. Tumor stage and other baseline characteristics are indicated in Table 1. Data regarding HPV status and cervical lesion history were not available.

Table 1 Characteristics of the CRC Italian patients (n = 40)Nucleic acid extraction

The simultaneous purification of DNA and RNA from frozen tissue was performed at the International Agency for Research on Cancer (IARC, Lyon) using AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Extracted DNA, RNA and proteins were stored at −80 °C until use.

HPV DNA detection assay

The prevalence of a broad spectrum of cutaneous and mucosal HPV types was determined by using type-specific multiplex genotyping (TS-MPG) assays, which is based on multiplex polymerase chain reaction (PCR) and bead-based Luminex technology (Luminex Corp., Austin, TX, USA), as described elsewhere [17]. Briefly, 10 µl of each DNA extract was subjected to a multiplex PCR with HPV type-specific primers targeting a total of 21 mucosal alpha-HPV types, namely HPV6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73 and HPV82. In addition, multiplex PCRs using type-specific primers were used for the detection of 46 beta-HPV types (HPV5, HPV8, HPV9, HPV12, HPV14, HPV15, HPV17, HPV19, HPV20, HPV21, HPV22, HPV23, HPV24, HPV25, HPV36, HPV37, HPV38, HPV47, HPV49, HPV75, HPV76, HPV80, HPV92, HPV93, HPV96, HPV98, HPV99, HPV100, HPV104, HPV105, HPV107, HPV110, HPV111, HPV113, HPV115, HPV118, HPV120, HPV122, HPV124, HPV143, HPV145, HPV150, HPV151, HPV152, HPV159 and HPV174), and 52 gamma-HPVs (HPV4, HPV48, HPV50, HPV60, HPV65, HPV88, HPV95, HPV101, HPV103, HPV108, HPV109, HPV112, HPV116, HPV119, HPV121, HPV123, HPV126, HPV127, HPV128, HPV129, HPV130, HPV131, HPV132, HPV133, HPV134, HPV148, HPV149, HPV156, HPV161, HPV162, HPV163, HPV164, HPV165, HPV166, HPV167, HPV168, HPV169, HPV170, HPV171, HPV172, HPV173, HPV175, HPV178, HPV179, HPV180, HPV184, HPV197, HPV199, HPV200, HPV201, HPV202 and SD2). Two primers for beta-globin were also used to assess the quality of the extracted DNA. After PCR amplification, 10 µl of each reaction was analysed using a multiplex Luminex-based assay as detailed previously [17].

E6*I mRNA analysis

RT-PCR was carried out using the QuantiTect Virus Kit (Qiagen, Hilden, Germany), in a total volume of 25 μl containing 5 μl of 5 × QuantiTect Virus Mastermix, 0.25 μl of 100 × QuantiTect Virus RT Mix, 0.4 μM of each oligonucleotide, and 1 μl RNA as described previously [18]. HPV specific primers and probes from a HPV type-specific E6*I mRNA assay [18] were used for the detection of viral transcripts. The assay amplifies a 65–75 base pair amplicon of HPV and an 81 base pair amplicon of ubiquitin C (ubC) cDNA. Biotinylated amplification products were hybridized to ubC and HPV type-specific probes, representing splice junction sequences, on Luminex beads, followed by staining with streptavidin–phycoerythrin, and quantified in a Luminex analyzer as previously described by Halec et al. 2013 [18].

HPV DNA detection by in situ hybridization (ISH)

The 3-µm thick sections of formalin-fixed, paraffin-embedded (FFPE) tissue were tested by in situ hybridization (ISH) using the HPV Probe (types 16, 18, 31, 33, 51) [HPV Probe Leica Biosystems, Leica, Newcastle, UK. Catalog No: PB0829] for the qualitative detection of HPV DNA, on the automated Leica BOND‐III system, according to the manufacturer’s instructions.

Briefly, slides underwent deparaffinization with the Bond Dewax solution followed by epitope retrieval using Stringency Wash solution and addition of biotinylated HPV-type-specific DNA probes followed by anti-Biotin antibody. After washings, post primary and polymer incubation, DAB staining and Hematoxylin counterstain, were performed. Slides were analyzed by using an Axio Imager A1 (Zeiss, Göttingen Germany).

Czech cohortStudy group

The CRC patients were recruited from the Department of Surgery, Teaching Hospital and Medical School, in Pilsen, Czech Republic, between January 2008 and November 2011.

Collection, processing of tissues and data acquisition of the Czech samples, was performed as previously described [19]. The clinical data, including age at diagnosis, sex, pTNM (Tumor stage, Regional lymph node involvement and distant metastasis) staging, histological grade of the tumor, and primary tumor localization were obtained from patient’s medical records (Table 2). After surgical resection, colorectal tumor and adjacent non-malignant tissues were snap frozen and stored at -80 °C.

Table 2 Clinical characteristics of the Czech patients (n = 125)DNA extraction and PCR amplification

Total DNA was extracted from frozen CRC (n = 125), and available surrounding tissues (n = 70) using the DNeasy Blood and Tissue Kit following the manufacturer’s instructions (Qiagen, Courtaboeuf, France). DNA samples were sent to IARC for HPV DNA characterization by PCRs combined with NGS. Total extracted DNA was amplified using three degenerated primer sets (i) FAP59/64 (FAP), (ii) FAPM1 and (iii) CUT protocols as previously reported [20,21,22].

Library preparation and NGS assay

PCR amplicons of the expected size (about 480 bp) were generated from 41 and 78 CRC samples using FAP and FAPM1 primers, respectively. In adjacent CRC specimens, PCR amplicons were generated from 16 and 45 specimens using FAP and FAPM1 primers, respectively. Finally, amplicons from 98 CRC and 37 adjacent samples were obtained using CUT primers and. PCR amplicons of the expected size were purified as previously described [21] and divided by PCR protocols (FAP, FAPM1, CUT) and tissue specimens (CRC and adjacent CRC) in a total of 6 different pools using the same volume of each purified amplicon. NGS analysis was performed on the pooled amplicon-based library (Nextera DNA Flex) using the Illumina MiSeq sequencer (2 X 150 paired-end reads, MiSeq reagent kit v3) (Illumina, San Diego, CA, USA) as previously reported [21]. Bioinformatic analyses were performed using PVAmpliconFinder tool [23], and all the results were based on the homology-based classification using the evolutionary placement algorithm in RAxML (Randomized Axelerated Maximum Likelihood) [24].