Abstract IA02: The discovery of the hereditary colorectal cancer genes: A historical perspective
Authors: Boland C. Richard 
Published: March 2017

When I was first introduced to the concept of carcinogenesis as a medical student in 1969, the problem was conceptually a “black box”. Certain stimuli were carcinogenic, something happened to a cell, and cancer resulted. It had been noted in the beginning of the 20th Century that cancer cells had abnormal chromosomes (Boveri); it had been noted that chicken sarcomas could be transmitted by a “filterable agent” (Rous sarcoma virus); a variety of compounds could cause cancers on the skin of mice (chemical carcinogenesis), as well other physical agents (UV light, Xrays). There were other empirical observations, but there were no unifying concepts of what was inside the black box of any cancer, let alone the hereditary forms.

The mechanisms of inheritance were known to be related to the nucleus, but it was not until 1953 that the structure of DNA was deduced, and actually a few years later that the number of human chromosomes was accurately determined to be 46. Concepts of inheritance were entirely descriptive. Progress was excruciatingly slow. It had been a century since the initial appreciation of dominant and recessive inheritance. However, only a few diseases with very distinctive phenotypes were clearly identified as familial, and only a few of these had any biochemical explanation. There was no obvious place to begin the search for the genetic basis of familial diseases. Worse, for the more common diseases such as cancer, diabetes and hypertension, it was thought that environmental influences were much more important than genetic ones.

The discovery process accelerated through technical advances that permitted deeper dives into the genetic processes underlying these diseases. Chromosomal banding was only useful when there were grossly detectable defects in a chromosome. DNA sequencing was a slow, laborious process. However, in the 1970s, the pace of discovery heated up (automated sequencing, oncogenes), and this increased ever faster over the next 3-4 decades. The 1980s represented the “inflection point” of discovery into the causes of cancer. By the end of the 1980s, the concept of multistep carcinogenesis driven by “alterations” in the genome was advanced by Vogelstein's lab at Johns Hopkins.

Alfred Knudson in 1971, based upon studies of familial and sporadic retinoblastoma (RB) that two copies of a gene related to this disease had to be disabled in the evolution of RB, and that individuals with the familial form of RB had a germline mutation in that gene, which accounted for the increased risk and early onset. This set the stage for the appearance of techniques to prove the concept.

In the 1970s, familial adenomatous polyposis (FAP), Peutz-Jeghers Syndrome (PJS) and juvenile polyposis syndrome (JPS) were recognized as distinctive clinical phenotypes, and occurred on a familial basis. Actually, this first required the standardized pathological interpretation of colonic and intestinal polyps. Then, in 1986 Lemuel Herrera, a surgeon at Roswell Park in Buffalo, identified a patient with multiple congenital abnormalities and “Gardner's Syndrome” (FAP), which his family did not have. He asked his colleague Avery Sandberg to do chromosomal analysis, which led to the discovery of a microscopically visible interstitial deletion on 5q. They accurately proposed that this represented the loss of multiple genes on 5q, leading to a complex (and unique) phenotype, and that one of those genes deleted was connected to FAP. In 1987, Walter Bodmer's lab in London used restriction fragment length polymorphism (RFLP) analysis and linked families with FAP to the 5q locus. Moreover, they showed losses on 5q in 20% of sporadic colorectal cancers (CRCs).

This resonated with the Knudson hypothesis and suggested that a gene responsible for FAP would be on chr 5q21-22, but it took four years and the collaboration of several labs to finally clone the APC gene in 1991. The function of the gene was a complete surprise, as it played a central role in the WNT growth-regulatory pathway. The APC gene was also the 5q event that Vogelstein placed at the beginning of the multistep carcinogenesis model of 1990. These early days were largely driven by techniques that could identify genetic losses in tumor DNA.

The discovery of microsatellite instability (MSI) and its link to Lynch Syndrome (LS) came as lightning strike in 1993. Many labs were looking for the losses of tumor suppressor genes (TSGs) in cancers using RFLPs, and then extended the analyses by using variable number tandem repeats (VNTRs) and microsatellites (simple VNTRs with cassettes of 1-4 nucleotides), both of which are widespread throughout the human genome and valuable for genomic mapping. In 1992, Perucho (in San Diego) used “arbitrarily-primed PCR” to generate a large number of PCR products from DNA, separated them on PAGE gels, and compared results from CRCs with the patient's normal DNA to look for allelic losses (or gains), which would then lead to TSGs (or oncogenes) involved in carcinogenesis. However, his careful eye noted that some of the differences between the AP-PCR products from cancers and normal DNA represented band shifts, due to a small change in the length of the PCR product in cancer. He sequenced the altered bands, and discovered deletions (or insertions) in microsatellite sequences that were very widespread in a subset of the cancers, but not present in most other CRCs. He proposed that this was a unique “pathway” to carcinogenesis for ~15% of CRCs, but had a hard time convincing reviewers of this novel concept. While he was trying to get his findings published, Steve Thibodeau discovered the same thing at the Mayo Clinic. At the same time, Lauri Aaltonen (Finland; part of the Vogelstein-de la Chapelle collaboration) used a microsatellite marker (D2S123) located on chr 2p and found significant linkage between this locus and Lynch Syndrome (LS). Microsatellites markers were then used to look for TSG losses on 2p21 and elsewhere. Instead, MSI was recognized, and suddenly there were 3 groups with the same discovery published in May and June of 1993.

The three groups who found MSI published their odd-looking autoradiographs in Science and Nature, but none of the human geneticists immediately recognized the implications. However, those who had been working in basic yeast genetics for years (Richard Kolodner, Rick Fishel, Paul Modrich, Thomas Petes, Michael Liskay and others) recognized that this probably represented loss of the DNA mismatch repair (MMR) system in the tumors, which would represent a totally unique mechanistic basis for this subset of about 15% of CRCs. In May, 1993, by coincidence, Kolodner's group at Dana Farber in Boston had just cloned the hMSH2 gene based upon its similarity to the yeast MSH2 gene, but they had to identify pedigrees with LS and find a germline mutation to make their point. The human geneticists had the families and familiarity with the human genome (organized differently than in yeast), and soon suspected DNA MMR genes themselves. The race was on, and by December 1993, both groups linked LS to germline mutations in hMSH2, and reported this in Cell; Kolodner won the race by 2 weeks, although he mistook a simple SNP for the mutation The Vogelstein-de la Chapelle collaboration cloned the hMSH2 gene, found mutations in families, and found a cell line with MSI (which happened not to have an MSH2 mutation; rather it was mutated at the hMLH1 locus, reported in a few months). What is notable is how quickly this story went from the empirical observation of MSI to the cloning of the familial CRC genes.

The pace of discovery continued to be astonishing, and both Kolodner's and the Vogelstein-de la Chapelle groups found germline mutations in the hMLH1 gene in other LS families—published on consecutive days in Nature and Science on March 17 and 18, 1994. Kolodner used hybridization techniques to find hMLH1 based upon the yeast MLH sequence, but Vogelstein's group also used bioinformatics approaches to find not only hMLH1, but 2 more yeast MLH homologs, hPMS1 and hPMS2. hPMS2 turned out to be a legitimate LS gene; hPMS1 did not. The LS story had a long tail after this initial cluster of reports; hMSH6 was initially cloned by Vogelstein and Jiricny in June 1995, but not linked to LS families until Miyaki did so in November 1997. EPCAM was linked to some MSH2-like LS families in 2006. It would be an understatement to say that this led to a subsequent series of insights into how the DNA MMR system works, and what happens when this system is disabled.

The 21st Century has provided ongoing broad insight into gut development and carcinogenesis in the context of both WNT signaling and DNA MMR. Most colorectal neoplasms begin by disabling the WNT pathway that regulates epithelial proliferation. It is now appreciated that about 15-20% of CRCs are hypermutated though the inactivation of both MMR genes and (less often) DNA polymerase proofreading subunits, and develop through a unique pathway of “target” genes, that they grow and evolve differently, have different clinical behaviors, and respond differently to oncological treatment. Tumors with MSI do not respond to classical cytotoxic chemotherapy (unlike non-MSI CRCs), but do respond to immune checkpoint therapies—to which non-MSI tumors are non-responsive. Moreover, we now have panel that test for multiple genes for any of the hereditary cancer syndromes.

Finally the genes for PJS, JPS, Cowden's Disease (CD) and Bannayan-Riley Ruvalcaba Syndrome (BRRS) were all discovered by variations on positional cloning, but with less notoriety. Whereas PJS has a classic phenotype that has been linked to just one gene (LKB1/STK11), JPS has been a much more problematic disease because of the clinical heterogeneity of the phenotype generally and clinical overlap with CD and BRRS. Several genes have been linked to JPS, but this disease is still confusing. There are probably several diseases that we call JPS, which confuses the genetics. SMAD4, BMPR1A, SMAD9, and other genes have all been linked to JPS. Even Cowden's Disease, linked to the PTEN gene, can present with hyperplastic or hamartomatous polyps. BRRS may be a pediatric variant of CD.

At this time, there are candidate genes for almost all of the familial polyposis genes, from APC to POLD1 and POLE (Polymerase Proofreading Associated Polyposis), to the recessive polyposis genes, MutYH and NTHL1. However, there are families with these phenotypes in which no genetic diagnosis can be made. Biallelic MMR gene mutations, or constitutional mismatch repair deficiency syndrome—CMMRDS—can present with colonic polyposis in childhood. Finally, the serrated polyposis syndrome with multiple polyps that look a bit like hyperplastic polyps is rarely familial, is linked to another CRC phenotype (CpG island methylator phenotype or CIMP), and is the final outlier without any consensus germline mutations causing the syndrome.

Citation Format: C. Richard Boland. The discovery of the hereditary colorectal cancer genes: A historical perspective. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr IA02.