With respect to oncologic outcomes, MMR-deficient CRC is associated with a better prognosis and therapeutic responses because the MMR pathway is involved in triggering cell death after chemotherapy-induced DNA damage [14]. The prognosis in patients with MMR-deficient CRC tends to be better, with regard to stage-for-stage comparison, than in those with MMR-proficient cancer [14]. Patients with early-stage MMR-deficient CRC do not appear to benefit from adjuvant 5-Fluorouracil monotherapy [15]; however, in some patients with metastatic MMR-deficient CRC, treatment with immune checkpoint inhibitors has been associated with an excellent response [16].
However, a considerable number of MMR-deficient CRC tumors have an unknown etiology, other than confirmed LS and methylation of MLH1. In our study, a high proportion of patients with MMR-deficient CRC were diagnosed as having LLS, which was consistent with the data of a previous study [9]. Therefore, multigene panel testing should be recommended for all MMR-deficient patients to distinguish LS and LLS.
While management of LS has been well described, the inability to define the molecular basis of the LLS entity not only hampers the appropriate clinical management of probands, but also the cancer screening recommendations for affected families. Comparison of clinical and molecular characteristics of patients with LLS and features of their CRC tumors with those of confirmed patients with LS can contribute to the development of appropriate management recommendations for patients with LLS and their affected family members.
The genetic causes of LLS are still unknown, although advanced NGS approaches have facilitated the discovery of novel genetic events that may allow the definition of clinical and molecular phenotypes of LLS. In our study, variants were unidentified in nearly half of the LLS cohort. Current techniques of analysis may be missing complex or cryptic variants in MMR genes, and some deep intronic variants may be overlooked [17, 18]. Furthermore, there may be some unidentified variants in the regulatory regions of MMR genes, which are hardly screened [19]. Thus, we suggest that this subset may have been a mixture of patients with LS, whose germline variants were not detected, and those with sporadic CRC. Future advances in NGS techniques may allow obtaining more accurate genetic information for discriminating between patients with LS and LLS.
Among the variants identified in this study, the largest category was VUS in MMR genes. The classification of these patients is still uncertain, and they were grouped as patients with LLS in the current study. Some of the patients carrying VUS in MMR genes may have been true patients with LS, which was supported by a high frequency of metachronous CRC. The pathogenicity of these VUS should be confirmed in functional experiments. The high frequency of metachronous CRC observed in our study suggests that patients with LLS should be considered high-risk cases, and strategies for cancer prevention must be implemented for this group of patients and their relatives. In clinical practice, aggressive surgical protocols, such as extended colectomy and subtotal colectomy, should be recommended. Platinum-based chemotherapy should be routinely applied, and treatment with immune checkpoint inhibitors may provide a considerable benefit to patients with metastatic MMR-deficient CRC. Meanwhile, stringent colonoscopy surveillance should be performed in patients with LLS, who carry both PVs and VUS in MMR genes.
In addition to MMR genes, most of the other PVs and likely PVs were detected in the MUTYH and GJB2 genes. Biallelic MUTYH variants have been detected in 1.8–3.1% of patients with LLS [20, 21]. MUTYH-associated polyposis is extremely variable, ranging from severe polyposis coli to attenuated forms with a late age of onset or few adenomas, or CRC, which creates a phenotypic overlap with LS [20, 22]. GJB2 encodes a gap junction protein, also known as connexin 26. Variants in this gene are responsible for as much as 50% of prelingual, recessive deafness [23]. The cytoplasmic Cx26 protein has been associated with the tumor progression and a poor prognosis in patients with breast cancer and esophageal squamous cell carcinoma [24, 25]. To the best of our knowledge, this is the first study to demonstrate the involvement of GJB2, as a novel candidate gene, in LLS-linked CRC. The pathogenicity of the frameshift variant in GJB2 is being evaluated by functional analysis, and the results will be reported separately. Variants in the exonuclease domain of the polymerase proofreading genes POLE and POLD1 cause polymerase proofreading-associated polyposis, which is a dominant-inheritance and high-penetrance hereditary syndrome conferring a predisposition to attenuated colorectal polyposis and early-onset CRC [26]. The association between variants of polymerase proofreading genes and MMR deficiency has been reported previously [27]. In our study, VUS in the POLE and POLD1 genes were predicted to be deleterious and were among the most frequently detected variants. Some other variants were identified in BRCA1, BRCA2, and RAD50, which are involved in the homologous recombination pathway. Defects in the BRCA genes are known to be pathogenic causes of hereditary breast and ovarian cancers [28], in addition to conferring a high risk of developing CRC [29].
Therefore, it is possible that some cases of LLS can be due to the pleiotropism of certain gene variants, manifesting as genetic overlaps with other hereditary cancer syndromes. Because of the mixture, a higher prevalence of extra-CRCs and a lower prevalence of CRCs were revealed in the LLS families. The high risk of extra-CRCs found in our study suggests that stringent surveillance of other organs should be recommended for probands and their affected family members. The surveillance regimen can be based on the gene variants and family history. For example, gastroduodenoscopy should be regularly performed in patients carrying MUTYH variants, while gynecological and breast examinations would be recommended for patients carrying BRCA variants. Furthermore, functional analysis of the undefined variants found in patients with LLS should be performed to elucidate the underlying molecular etiology of LLS.
The difference in the age at onset of CRC between patients with LS and LLS remains controversial; some studies demonstrated similar proportions of early-onset patients in the LS and LLS groups [13], whereas one report showed that the population of patients with LLS was older [30]. Our results supported the latter findings, with age differences being manifested in both probands and related family members. Variants in genes such as POLE and BRCA, which were found in patients with LLS, may confer a higher risk of CRC; however, these variants show moderate penetrance [31]. Because sporadic CRC is combined with moderate penetrance of other variants, a delayed onset of CRC was demonstrated in probands with LLS. It is noteworthy that more than half of the patients in the LLS group were early-onset cases, which is significantly higher than the reported rate of sporadic CRC [32]. Therefore, MSI and multigene panel testing should be recommended for the early-onset subset, and screening colonoscopy at an early age should be performed in affected family members.
In terms of the CRC localization, our study showed a striking clustering of tumors in the rectum of probands with LLS, indicating that the rectum as the preferred organ can be described as a clinical feature of LLS-associated CRC. A higher frequency of left colon cancer was consistent with the findings of our previous study, which investigated clinical features of LS in an Asian population [33]. While LS-associated CRC is characterized by mucinous differentiation [34], a reasonably lower proportion of mucinous tumors was observed in the LLS cohort in this study.
Another interesting finding was a larger number of male patients in LLS families. A higher prevalence of male patients in LS families was reported in a previous review [35], but has not been previously described in LLS families. This discovery of the sex-dependent tendency of disease in LLS families indicates that more attention should be paid to the screening and surveillance of male members.
There are some limitations of our study. First, because this was a retrospective study, the potential bias in the selection of subjects could not be eliminated. Second, MSI testing was not performed, which may have resulted in an incorrect interpretation of the molecular evidence. Lastly, the sample size needs to be increased, and a long-term follow-up is required.