Background
Multiple primary cancers (MPC) have been defined as two or more cancers without any subordinate relationship that occurs either simultaneously or metachronously in the same or different organs of an individual [
1]. Since Billroth proposed the concept of MPC in 1889 [
2], researchers had been attracted by the disease and emerging patients have been identified [
3‐
6] owing to advanced diagnostic technologies [
7‐
9], sustained environmental degradation [
10], and longer life expectancies of cancer survivors [
11,
12]. To date, studies on MPC were mainly descriptive with little investigating of the mechanism whereby MPC occurs [
13,
14]. Hence, it is of great urgency to learn the machinery whereby MPC occurs so as to provide prevention strategies in the future.
In clinical settings, many MPC patients had been proven to have a strong family history of cancer, while others were sporadic. Lynch syndrome is a dominant genetic disorder characterized by an increased risk of cancers of the digestive tract, gynecologic tract, and other organs [
15]. Germline mutations of DNA mismatch repair (MMR) genes including MLH1 (42%), MSH2 (33%), MSH6 (18%), and PMS2 (7%) and several less-frequent genes (PMS1, MSH3, and EPCAM) are the major causes of Lynch syndrome [
16]. Mutated MMR genes are not able to repair DNA replication errors. As cells with that specific defect continue to divide, the mistakes accumulated and usually led to cancers. Therefore, it is common that Lynch syndrome patients usually suffer more than two cancers. Strategically, Lynch syndrome patients who suffer more than two cancers provide a unique resource to study the pathogenesis of MPC.
Previously, increasing studies had focused on MPC, but most of which were descriptive with none presented with pronounced and compelling illustrations on how MPC occurs [
17‐
21]. As to Lynch syndrome, most mechanistic studies were focused on MMR genes. To the best of our knowledge, no studies had systematically investigated the mutational landscape when Lynch syndrome progressed to MPC. In the past decades, the “omics” studies had achieved many discoveries in various human malignancies, which opened a new window for understanding cancer initiation and progression. Lynch syndrome, together with genomic study strategies, provides a unique avenue to investigate the carcinogenic mechanism of MPC.
In the present study, we performed a comparative genome analysis on peripheral blood cells and two primary tumors of a patient with Lynch syndrome. We discovered a novel MSH2 mutation (G504 splicing) associating with Lynch syndrome, which segregated with disease phenotypes in a four-generational pedigree and resulted in the inactivation of MSH2 protein. Systematical comparison of somatic point mutations and copy number alterations revealed that these two cancers were evolutionarily independent. We further demonstrated that Lynch syndrome-related cancers harbored mutations in the driver genes of sporadic cancers, and that these genes might play significant roles in the carcinogenesis of Lynch syndrome. Furthermore, a model was proposed to illustrate how Lynch syndrome progressed to MPC.
Discussion
To date, multiple researches had focused on MPC with the majority of them being descriptive. Since Billroth proposed the diagnostic criteria of MPC [
2], a large amount of studies had examined in detail the incidence, origin, and classification of the disease [
13,
14,
54]. However, few studies have shown a convincing illustration on how MPC occurs. In the present study, we reported on a patient with Lynch syndrome who could also be diagnosed as MPC. After that, we preformed genome-wide sequencing on the cancer tissues of the patient, and we revealed a novel pathologic mutation on MSH2 associating with Lynch syndrome. Moreover, integrative analysis demonstrated that truncating mutations of MMR genes were significantly enriched in the patient. In addition, systematical comparison of the mutation landscape revealed that the primary cancers of the patients were evolutionarily independent. Based on the data, we proposed a model to illustrate how Lynch syndrome developed into MPC. To the best of our knowledge, this is the first study to investigate Lynch syndrome from the genomics level. Our data adds insights into the pathogenesis of MPC from the genomic level.
Obviously, our data suggested that MSH2 gene was critical during Lynch syndrome progression to MPC. This promoted us to investigate whether MSH2 mutation alone was strong enough to induce the occurrence of Lynch syndrome-related cancers. However, systematical literature review indicated that genetic disorder and dietary and/or environment factors had synergistic effect in promoting cancer initiation in MSH2-defective individuals. For instance, Belcheva A and colleagues indicated that interaction between microbiota and dietary factors tends to reduce the occurrence of colorectal cancer and other cancers in APC (Min/+)MSH2(−/−) mice [
55]. Moreover, there was also report that germline ablation of SMUG1 DNA glycosylase causes loss of 5-hydroxymethyluracil- and UNG-backup uracil-excision activities and increases cancer predisposition of Ung−/−Msh2−/− mice [
56]. This suggested that other genetic instabilities were also effective in MSH2-defective resultant cancers. These data in combination reminded that appropriate dietary and lifestyle intervention might also be effective in preventing Lynch syndrome progression.
Over the past decades, “omics” studies had achieved many discoveries in various human malignancies. For instance, scientists had obtained the genomic mutation landscape of the major human cancers by genome-wide sequencing. Bioinformatics analysis suggested that a typical tumor has two to eight “drive gene” mutations, which manifest selective growth advantage, while others are passenger mutations [
37]. The mutation rate varies from one cancer to another, with an average of 1/Mb, which increases to 10/Mb in MSI tumors [
35]. Additionally, single-cell sequencing and multi-region sequencing were used to infer tumor progression, which largely extends our knowledge of carcinogenesis. Unfortunately, these technologies had rarely been used to explore the initiation and progression of MPC. Genetic testing of MMR genes has been widely applied to aid the diagnosis of Lynch syndrome. As a major member of MMR genes, MSH2 exhibits a novel mutation in our analysis. MSH2 functions to repair DNA replication errors, whose dysfunction results in accumulated mutation of the cells and, finally, cancer. We collected known pathologic mutations of hereditary tumors from public databases and analyzed the association between MSH2 mutation spectrum and Lynch syndrome. We showed that mutations within the entire length of the coding sequence of MSH2 were positively correlated with Lynch syndrome, which suggested that the mutation of MSH in our study is a novel pathogenic factor. The mutation pattern of MSH2 was further studied by comparing germline mutations in inherited tumors with somatic mutations in sporadic tumors. We found that truncating mutations were more likely to be causal in hereditary Lynch syndrome than missense mutations. This will assist in the annotation of pathogenicity of MMR genetic variants.
Patients with Lynch syndrome tend to develop cancers in multiple tissues, such as colorectal, pancreas, stomach, and so on. However, it is still unclear whether they are related and share the similar mutational landscape. Moreover, the mutation landscapes of the cancers of the proband indicated that they developed independently. This supported the fact that multiple cancers in Lynch syndrome are primary but not metastasis.
Of note, we proposed a double-hits theory during Lynch syndrome progression to MPC in our study. As put, the first hit was the genetic mutation of MSH2, and the second hit was caused by somatic mutations. The second hit, including the loss of heterozygosity at the MSH2 mutation site in the renal pelvic and a new MSH3 somatic mutation in the small intestine might be distinct among tumor tissues. A previous study reported the loss of the wild type MLH1 gene in hereditary nonpolyposis colorectal cancer [
48]. These results suggested that double hits of DNA MMR genes might be a common event in the development of other malignancies in Lynch syndrome patients.
Even more interesting is that some SMGs of sporadic tumors also had high-frequency mutations in Lynch syndrome-related cancers. Higher alternative allele frequency indicates that SMG mutations were not a random event, and they might occur earlier than other passenger mutations. This finding highlights a potential role of SMGs in the carcinogenesis of Lynch syndrome. Based on these data, we proposed a mutation progress model of MPC in Lynch syndrome, which include germline mutations of MMR genes, double hits of MMR system, mutations in tissue-specific driver genes, and rapid accumulation of additional passenger mutations. This model may advance the elucidation of carcinogenic theory. Although this model was established based only on a single patient, it was consistent with our prior knowledge of Lynch syndrome.