Aim: High-resolution HLA-G typing in selected patients with locally advanced rectal cancer (LARC) enrolled at the National Cancer Institute "Centro di Riferimento Oncologico di Aviano (CRO) IRCCS" (Italy) (CRO-2023-77).
Methods: HLA-G genotyping was conducted in 405 LARC patients with allele identification at the genomic (4-field), exonic (3-field), and protein (2-field) levels. Genomic DNA was extracted from buffy coat samples, and full-length HLA-G amplicons were obtained by long-range PCR. DNA libraries were prepared with the QIAseq FX DNA Library kit (Qiagen) and sequenced on the Illumina MiSeq platform (2x250bp). High-coverage sequencing data (>50x) were analyzed using an updated version (v5) of the hla-mapper workflow.
Results: This LARC cohort predominantly included patients from the North-East regions of Italy. Variants in the HLA-G gene were mostly located in intronic (50, 41.0%) and regulatory regions, including promoter, 5'UTR, and 3'UTR (50, 41%), compared to exonic regions (22, 18.0%). Most variants were bi-allelic single nucleotide variants, although insertions/deletions were also detected. Almost half of patients (186, 45.9%) showed at least one missense (rs41551813, rs770412396, rs12722477, or rs12722482), start-lost (rs143732275), or frameshift (rs41557518) variants. Moreover, in 60 (14.8%) patients, 39 different rare or putative novel variants were found, all in heterozygosity. One patient carried the G*01:01:11 rare allele, previously reported only in two Brazilian relatives of Italian ancestry. The most prevalent allotypes were HLA-G*01:01, HLA-G*01:04, HLA-G*01:06, HLA-G*01:03, and the HLA-G*01:05N null allele, consistent with European reference data. Our LARC cohort shows frequencies similar to those reported for the Italian population. The differences observed comparing HLA-G 4-field allele frequencies between this LARC cohort and the European population for G*01:01:01:01 (p = 0.0004, q = 0.0048), G*01:01:02:01 (p = 0.0033, q = 0.0132), G*01:01:01:05 (p = 0.0012, q = 0.0072), G*01:01:01:04 (p = 0.0093, q = 0.0279), G*01:03:01:02 (p = 0.0354, q = 0.0708), and rare 4-field alleles (p = 0.0117, q = 0.0281) reflected the known allele differences across European countries.
Conclusions: This study represents the largest Italian cohort to date with comprehensive high-resolution HLA-G genotyping in LARC patients. Ongoing analyses aim to correlate these genetic findings with clinical parameters and HLA-G expression levels in liquid biopsies. This integrative approach may identify HLA-G-related genetic factors influencing prognosis, therapeutic response, and treatment-related adverse events in patients with LARC.
Keywords: Germline variants; HLA-G; Haplotypes; Immune checkpoint; Rectal cancer.
Copyright © 2026. Published by Elsevier Inc.