Hereditary Nonpolyposis Colorectal Cancer

Hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome), which affects about 1 in 200 individuals, is the most common inherited cancer syndrome. It behaves as an autosomal dominant trait. The phenotype is characterized by few colonic polyps (<100) and early onset of multiple tumors in the from the human genetics files

Philadelphia Chromosome, Two-Hit Hypothesis, and Comparative Genomic Hybridization

In the early 1970s, when chromosome banding was a newly developed technique, Janet Rowley (b.1925) demonstrated the first consistent association between a chromosome translocation [t(8;21)(q22;q22)] and a cancer (acute myeloblastic leukemia, AML). Shortly afterward, she solved the mystery of the Philadelphia chromosome (Ph'). Since 1960, the Philadelphia chromosome, named after the city where it was first discovered, had been observed consistently in cases of chronic myeloid leukemia (CML). On the basis of whole chromosome staining, the Philadelphia chromosome appeared to consist of half of either chromosome 21 or 22. At this level of resolution, there was no evidence that it was part of a translocation. Rowley found that the Philadelphia chromosome was actually a translocation between chromosomes 9 and 22 [t(9;22)(q34;q11)]. After finding a third example of a specific association of a translocation [t(15;17)(q22;q11.2)] with a cancer (acute promyelocytic leukemia, APL), Rowley was convinced that these chromosome abnormalities were not coincidental and that chromosomal changes in leukemias, and probably other cancers, did not arise after the cancer was formed but instead contributed to its onset. As she has said, ". . . Chromosome changes were an essential component of the leuke-mogenic process, and I soon became a 'missionary,' attending hematology meetings in the 1970s and early 1980s carrying the 'gospel' that chromosome abnormalities were an essential component of hema-tologic malignant diseases to which the clinical community should pay attention."

As more correlations between chromosome abnormalities and specific cancers accumulated, it became clear that Rowley was right. Her work focused the attention of cancer researchers on chromosomes and raised provocative questions about the genes that were disrupted by translocations in the various cancers. By 1982, with the development of recombinant DNA technology, the t(8;14)(q24;q32) breakpoint was cloned. This translocation is associated with both acute lymphocyte leukemia and Burkitt lymphoma. Within a few years, the genes involved in the t(9;22) translocation and other cancer-specific chromosome abnormalities were isolated. Once these genes were available for study, it became clear that the translocations in leukemias and other cancers activate genes by either putting them under the control of the expression elements of other genes or creating fusion proteins as a result of the joining of two reading frames from different genes. Not only did this work establish the molecular basis of cancer, but it facilitated the development of new diagnostic and management protocols.

As Rowley was painstakingly matching chromosome bands, Alfred Knudson (b. 1922) was delving into the relationship, if any, between hereditary and nonhereditary forms of the same cancer. Specifically, he examined the features, including onset, of bilateral (hereditary) and unilateral (non-hereditary) retinoblastoma (RB). He concluded that both forms of RB were related and postulated that both were caused by two independent, successive mutation events, that is, two hits. In cases of hereditary RB, one of the mutations preexists in the germline cells and is passed on to all somatic cells during embryogenesis. The second hit occurs, by chance, in a somatic cell. Under these circumstances, one would expect that cells in both retinas likely would have two hits and bilateral tumors would develop. By contrast, in nonhereditary RB, the two mutations occur independently in the same somatic cell. In this case, it is highly unlikely that cells in both retinas would accumulate two hits, and, therefore, unilateral tumor formation is expected. Although there were other possibilities, Knudson opted for the two-hit hypothesis, because it was the simplest interpretation of his observations. The loss of activity in two alleles of a gene raised the possibility that a wild-type gene product could prevent tumor formation. Knudson called such a gene an "anti-oncogene."This term has been replaced by "tumor suppressor gene."

The two-hit hypothesis was supported when chromosome G-banding analyses showed a deletion at 13q14 in the tumor cells of many patients with hereditary RB. After the retinoblastoma gene (RB1) was isolated and characterized, mutational screening established conclusively that both RB1 alleles were mutated in retinoblastoma tumor DNA. Knudson's insights formed the conceptual framework for understanding tumor suppressor genes.

Cancer cytogenetic studies have shown that chromosome abnormalities are among the hallmarks of cancer cells. In addition to losses or gains of whole chromosomes (aneuploidy) and translocations, more subtle changes can lead to the loss and/or gain of chromosome subregions. A number of methods have been developed to detect gain and loss of interstitial chromosome regions. Of these, a two-color FISH procedure called comparative genomic hybridization (CGH) has been quite effective. Briefly, for CGH, total genomic DNA is extracted from a tumor and labeled with a green fluorescent dye. DNA from normal cells is labeled with a red fluorescent dye. The two DNA samples are combined and hybridized, along with an unlabeled sample of highly repetitive DNA, to normal human metaphase spreads from noncancerous cells. The unlabeled repetitive DNA is included in the hybridization mixture to prevent labeled repetitive sequences in the DNA samples from binding to chromosome DNA. Consequently, only labeled single-copy DNA sequences hybridize to chromosome DNA.

If a chromosome region has the same copy number in both of the labeled DNA samples, that is, in cancer and noncancer cells, then after hybridization the fluorescence will be a blend of green and red fluorescent dyes. If DNA is lost from a chromosome subregion in tumor cells, then the comparable regions of the hybridized chromosomes will fluoresce with more red than green dye. Finally, if a chromosome region is amplified in tumor cells, then after hybridization the corresponding chromosome regions will fluoresce with more green than red dye. Comparative genomic hybridization also detects the loss or gain of whole chromosomes. Computer-assisted image analysis distinguishes differences in fluorescence and calculates the relative amount of each fluorescent dye in defined segments of each chromosome. As part of the analysis, a green-to-red intensity ratio profile is calculated for sections of each chromosome; significant deviations from 1.0 are localized to specific chromosome regions; and, depending on whether the green-to-red ratio is greater or less than 1.0, the gain or loss, respectively, of chromosome material is noted. For example, when pancreatic carcinomas were screened by CGH, gains of chromosome DNA were consistently found on 3q, 5p, 7q, 8q, 12p, and 20q and losses in 8p, 9p, 17p, 18q, 19p, and 21.

Comparative genomic hybridization has been used to track chromosome copy number changes during the progression of a cancer. Generally, CGH overcomes the problems of poor metaphase chromosome spreads from tightly bound cancer cells and the relatively low frequency of cells in metaphase in many tumors. As well, the level of resolution detects amplifications as small as 1 Mb. On the other hand, CGH is blind to translocations. Thus, for a complete cytogenetic picture of a cancer cell, a combination of techniques, including spectral karyotyping, G-banding, and FISH is often used. For the most part, cancer cytogeneticists are interested in identifying consistent chromosome abnormalities that can be used as diagnostic indicators and demarcating chromosome regions that carry cancer-causing genes.

transverse and ascending portions of the colon (Figure 16.13). In a number of HNPCC families, tumors of the ovary, kidney, brain, pancreas, stomach, and the innermost layer of the uterus (endometrium) also occur, but to a lesser extent than colon cancer. Generally, HNPCC tumors appear in patients before they are 45 years old.

Stringent criteria were established for the clinical designation of a family with HNPCC to minimize heterogeneity among data sets to be used for linkage and other studies. These requirements, which have been dubbed the Amsterdam criteria, are (a) at least three relatives must have colon cancer, with at least one of them being a first-degree relative to the other two, (b) at least two successive generations must have affected individuals, (c) at least one family member should have colorectal cancer before the age of 50, and (d) FAP should be excluded as the basis of the disorder. As knowledge about a disease accumulates, diagnostic guidelines are reworked. Currently, the revised Bethesda guidelines (2003) are often used to define cases of HNPCC (Table 16.6).

Linkage studies with large HNPCC kindreds indicated that the disorder was genetically heterogeneous. Sites for HNPCC were mapped to chromo-

Table 16.6 Revised Bethesda (2003) guidelines for diagnosing HNPCC.

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