**Synergistic Effects of Low-Risk Variant Alleles in Cancer Predisposition**

Francesca Duraturo, Raffaella Liccardo, Angela Cavallo, Marina De Rosa and Paola Izzo

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55417

### **1. Introduction**

It has long been known that cancer can be the result of a genetic predisposition. About 5% of total cancers are associated with known Mendelian susceptibility; in these cancer types the clinical manifestations of disease are due to mutations in high-risk alleles, with a penetrance usually at least of 70%. However, there are many tumors in which the cause of hereditary predisposition can not be explained as the Mendelian syndromes. For colorectal cancer (CRC), for example, about 30% of cases are thought to be due to inherited susceptibility, which only in part can be explained by the known Mendelian inheritance, as FAP, MAP and Lynch syndrome [1]. Breast cancer has a similar gap between Mendelian and overall genetic risk. For prostate cancer, the risk is even higher, as very few cases are attributable to highrisk alleles. This gap needs to be filled by studies to identify predisposition alleles that explain the cases of hereditary tumors for which no association with gene variants has been found, so far [2].

With the advent of high-throughput technology it is now possible to analyze a great number of polymorphic variants in large cohorts of cases and controls. These studies have been used successfully by many groups leading to the identification of a large number of rare variant alleles in patients with an inherited risk of cancer [3, 4]. The simultaneous presence of rare genetic variants in the same patient might contribute in a cooperative manner to increase the risk of tumor development. Another problem is represented by variants of unknown significance (VUSs) within the cancer predisposition highly penetrant genes. These variants are usually missense or silent changes which are generally rather uncommon or rare and thus of doubtful clinical relevance, that make troublesome the genetic counseling for these cancer families. The interpretation of these variations is not easy and requires the combination of different analytical strategies to get a proper assessment of their

© 2013 Izzo et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### 4 Carcinogenesis

pathogenicity [5]. In some cases, VUSs make a more substantial overall contribution to cancer risk than the well-assessed severe Mendelian variants. It is also possible that the simultaneous presence of some polymorphisms and VUSs in cancer predisposition genes that behave as low-risk alleles, might contribute in a cooperative manner to increase the risk of hereditary cancer [6]. Therefore, current literature data suggest that a significant proportion of the inherited susceptibility to relatively common human diseases may be due to the addition of the effects of a series of low frequency variants of different genes, probably acting in a dominant and independent manner, with each of them conferring a moderate but even detectable increase in the relative cancer-risk.

Synergistic Effects of Low-Risk Variant Alleles in Cancer Predisposition 5

1/8000 100%

1/500 to 1/1000 Up to 85%

1 in 400 90%

1/100 000 90–100%

By biochemical testing

*RET* 1/30 000 70–100%c

multiple endocrine neoplasias [7]. When the benign and malignant manifestations are considered together, many of these syndromes show almost complete penetrance by age 70. An inherited cancer susceptibility is suspected in families with the following characteristics: two or more relatives with the same type of cancer on the same side of the family; several generations affected; earlier ages of cancer diagnosis than what is typically seen for that cancer type; individuals with multiple primary cancers; the occurrence of cancers in one family, which are known to be genetically related (such as breast and ovarian cancer, or colon and uterine cancer); and the occurrence of nonmalignant conditions and cancer in the same person and/or family [8]. In table 1 are reported the more frequent hereditary cancer

*Syndrome MIM#a Gene(s) Population incidence Penetranceb*

Cowden syndrome 158350 *PTEN* 1/200 000 90–95%

*APC MYH* 

*BRCA1 and BRCA2* 

*MSH2, MSH6, MLH3, PMS2*

*(SMAD4), BMPR1A* 

Li–Fraumeni syndrome 151623 *TP53* Rare 90–95%

von Hippel–Lindau (VHL) 193300 *VHL* 1/36 000 90–95% **<sup>a</sup>** MIM numbers beginning with 1 indicate autosomal dominant inheritance; those beginning with 6 are autosomal loci or phenotypes entered into the catalogue after May 1994. b Penetrance estimates are up until age 70 years, include both

malignant and benign features and with the exception of MEN2, describe clinical penetrance. c

(pentagastrin-stimulated calcitonin levels) is 95–100% by age 70.

**Table 1.** Highly penetrant cancer syndromes

137215 *CDH1* Unknown, rare 90%

131100 *MEN1* 1/100 000 95%

175200 *LKB1 (STK11)* 1/200 000 95–100%

180200 *RB* 1/13 500 to 1/25 000 90%

175100 608456

113705, 600185

171400, 162300

114500 *MLH1,* 

174900 *MADH4* 

Familial adenomatous polyposis (FAP or MAP)

Hereditary breast–ovarian

Hereditary diffuse gastric

Hereditary nonpolyposis

cancer syndrome

cancer

colon cancer

syndrome

(PJS)

(RB)

Juvenile polyposis

Multiple endocrine neoplasia type 1

Multiple endocrine neoplasia type 2

Peutz–Jeghers syndrome

Retinoblastoma, hereditary

Our studies are concerned with the molecular basis of the Lynch syndrome, which is commonly associated with mutations in mismatch repair (MMR) genes, MLH1 and MSH2. However, mutations in these genes do not account for all Lynch syndrome families. In our experience we have also identified germ-line genetic variants in the other MMR genes, called minor MMR genes: MSH6, PMS2, MLH3 and MSH3. We have shown that several patients were carriers of at least two genomic variants within the "minor" genes or a VUS in a major gene associated to a genetic variant in minor genes. We therefore speculate that the association between weak alleles in the MMR genes could determine the onset of the tumor.

### **2. Hereditary cancer syndromes**

Over 200 hereditary cancer susceptibility syndromes have been described, the majority of which are inherited in an autosomal dominant manner. Although many of these are rare syndromes, they are thought to account for at least 5–10% of all cancer, amounting to a substantial burden of morbidity and mortality in the human population (Figure 1).

**Figure 1.** The majority of most common cancers are sporadic, 5–10% are inherited and arise due to highly penetrant germ-line mutations. An additional 10–15% are referred to as 'familial' and may be caused by the interaction of low-penetrance genes, gene–environment interactions, or both.

While characterized by their markedly increased risk of malignancy, these syndromes often predispose to benign tumors and generalized disease, as in Cowden syndrome (CS) and the multiple endocrine neoplasias [7]. When the benign and malignant manifestations are considered together, many of these syndromes show almost complete penetrance by age 70. An inherited cancer susceptibility is suspected in families with the following characteristics: two or more relatives with the same type of cancer on the same side of the family; several generations affected; earlier ages of cancer diagnosis than what is typically seen for that cancer type; individuals with multiple primary cancers; the occurrence of cancers in one family, which are known to be genetically related (such as breast and ovarian cancer, or colon and uterine cancer); and the occurrence of nonmalignant conditions and cancer in the same person and/or family [8]. In table 1 are reported the more frequent hereditary cancer


**<sup>a</sup>** MIM numbers beginning with 1 indicate autosomal dominant inheritance; those beginning with 6 are autosomal loci or phenotypes entered into the catalogue after May 1994. b Penetrance estimates are up until age 70 years, include both malignant and benign features and with the exception of MEN2, describe clinical penetrance. c By biochemical testing (pentagastrin-stimulated calcitonin levels) is 95–100% by age 70.

**Table 1.** Highly penetrant cancer syndromes

4 Carcinogenesis

pathogenicity [5]. In some cases, VUSs make a more substantial overall contribution to cancer risk than the well-assessed severe Mendelian variants. It is also possible that the simultaneous presence of some polymorphisms and VUSs in cancer predisposition genes that behave as low-risk alleles, might contribute in a cooperative manner to increase the risk of hereditary cancer [6]. Therefore, current literature data suggest that a significant proportion of the inherited susceptibility to relatively common human diseases may be due to the addition of the effects of a series of low frequency variants of different genes, probably acting in a dominant and independent manner, with each of them conferring a

Our studies are concerned with the molecular basis of the Lynch syndrome, which is commonly associated with mutations in mismatch repair (MMR) genes, MLH1 and MSH2. However, mutations in these genes do not account for all Lynch syndrome families. In our experience we have also identified germ-line genetic variants in the other MMR genes, called minor MMR genes: MSH6, PMS2, MLH3 and MSH3. We have shown that several patients were carriers of at least two genomic variants within the "minor" genes or a VUS in a major gene associated to a genetic variant in minor genes. We therefore speculate that the association between weak alleles in the MMR genes could determine the onset of the tumor.

Over 200 hereditary cancer susceptibility syndromes have been described, the majority of which are inherited in an autosomal dominant manner. Although many of these are rare syndromes, they are thought to account for at least 5–10% of all cancer, amounting to a

substantial burden of morbidity and mortality in the human population (Figure 1).

**Figure 1.** The majority of most common cancers are sporadic, 5–10% are inherited and arise due to highly penetrant germ-line mutations. An additional 10–15% are referred to as 'familial' and may be caused by the interaction of low-penetrance genes, gene–environment interactions, or both.

While characterized by their markedly increased risk of malignancy, these syndromes often predispose to benign tumors and generalized disease, as in Cowden syndrome (CS) and the

moderate but even detectable increase in the relative cancer-risk.

**2. Hereditary cancer syndromes** 

syndromes that are associated with mutations in high penetrance alleles. Because of phenotypic variability, age-related penetrance, and gender-specific cancer risks, however, many families with an inherited cancer syndrome will not meet these criteria. Furthermore, because cancer is relatively common in the general population, it is possible to have a chance clusterings of the same or related cancers within a family. These familial clusterings are most likely due to low-penetrance alleles that are more common than mutations in high penetrant alleles. Thus, they will potentially account for a larger proportion of cancer in the general population than the mendelian classic syndromes. For colorectal cancer (CRC), for example, Mendelian syndrome includes FAP, MAP and Lynch syndrome.

Synergistic Effects of Low-Risk Variant Alleles in Cancer Predisposition 7

One of the fundamental elements for the success of GWAS is represented by a large collection of biospecimens in case-control and cohort studies so as to have a high degree of reliability of results. The first approaches in this regard were based on technologies such as the Denaturing High Performance Liquid Chromatography (DHPLC) and classical sequencing analysis, that provide a high degree of analytical sensitivity and specificity. However, the new challenge in the field of biotechnology has surely been to make the techniques increasingly automated in order to process multiple samples simultaneously and

The method becoming more widely used is high-throughput sequencing, which allows a massive study of DNA. This is a system able to obtain more than 400,000 different readings in a single stroke of about 8 hours. The operating principle is based on clonal amplification of DNA *in vitro* by emulsion PCR and on a protocol of pyrosequencing that, unlike the classic method of Sanger, is based on the detection of pyrophosphate released by the incorporation of a nucleotide during DNA synthesis. In high-throughput sequencing 454 instrumentation, the sample may be any DNA larger than 1500 base pairs (genomic DNA or portions, cDNAs and large amplicons). The sequences obtained are analysed, properly aligned and oriented in contigs from the sequencer software, according to the shotgun and paired-end strategy. The accuracy of the data obtained is measured in terms of "coverage", that is based on the average number of times that each is accessed (read). This technology, therefore, is able to ensure high accuracy of the results (> 99.5%), thanks also to the careful management of the enormous amount of bioinformatics sequences obtained, which minimizes the production of raw redundant data. This feature, coupled with the extraordinary speed of processing, which makes the method also more economical than the classic automated sequencer, allows the user to analyze and quantify at the same time a large amount of samples. Therefore, the sequencer ultra-massive is an extremely versatile technique for a large number of applications such as resequencing and de novo assembly of

This latter approach is now widely applied, for example, for the identification of rare variants that presumably contribute in a synergistic way and in association with other factors predisposing to the development of complex genetic diseases characterized by genetic heterogeneity. This technology therefore offers a great contribution to the studies of Genome Wide Association, because it allows quick identification of the allele frequencies of SNPs in population studies, and to analyze a given target gene in multiple genomes, or a panel of target genes in a single patient, even at the level of gene expression (transcriptome analysis) [4]. However, the high number of next generation sequencing information requires accurate statistically studies. The threshold value for discovery has been established at a high level, known as genome-wide significance, which serves two dual purposes [12]. First, it needs careful consideration of the power to detect the effect sizes expected to be observed in the study. Second, the high bar of genome wide significance protects against the probability of a false-positive finding. The latter is critical because GWAS are discovery

entire genomes, and the massive sequencing of amplicons.

especially more quickly.

However, about 30% of the variation in CRC risk is thought to be due to inherited susceptibility, which only in part can be explained by the known Mendelian inheritance [2]. Breast cancer has a similar gap between Mendelian and overall genetic risk and for prostate cancer the risk is even higher, as very few cases are attributable to high-risk alleles. It is that gap which must be filled by studies to identify cancer predisposition alleles in the general population [9]. Localization and characterization of low-penetrance alleles are the focus of much research, but the challenges are great due to the multi-factorial nature of cancer and the underlying genetic heterogeneity.

### **2.1. High-throughput technology for detection of the multiple alleles associated to cancer predisposition**

The history of human genetics has focused on mapping regions of the genome that can explain part or all of a disease or human trait.

The 'rare variant hypothesis' proposes that a significant proportion of the inherited susceptibility to relatively common human diseases may be due to the sum of the effects of a series of low frequency variants of a variety of different genes, perhaps dominantly and/or independently acting, each conferring a moderate but detectable increase in relative risk [2]. Regardless, there is good supporting evidence that rare variants will often have stronger effects on cancer risk than common variants. This evidence is based on several works whose purpose was to determine whether evaluating rare single-nucleotide polymorphism (SNPs) in case-control association studies could help to identify causal SNPs for common diseases. The sources of data of these works were generally the International HapMap Project and the SeattleSNPs project and they suggest that slightly deleterious SNPs subjected to weak purifying selection are major players in genetic control of susceptibility to common diseases, including cancer. These results suggests that studies with large sample sizes (5000 and higher) targeting SNPs will be a better strategy to identify causal disease SNPs [10]. Instead, genome wide association studies (GWAS) have emerged as an important tool for discovering regions of the genome that harbor uncommon genetic variants that confer risk for complex tumors, whose nature is probably polygenic [11]. These variants include single nucleotide variants (SNVs) or single nucleotide polymorphisms (SNPs), small insertions and deletions and structural genomic variants.

One of the fundamental elements for the success of GWAS is represented by a large collection of biospecimens in case-control and cohort studies so as to have a high degree of reliability of results. The first approaches in this regard were based on technologies such as the Denaturing High Performance Liquid Chromatography (DHPLC) and classical sequencing analysis, that provide a high degree of analytical sensitivity and specificity. However, the new challenge in the field of biotechnology has surely been to make the techniques increasingly automated in order to process multiple samples simultaneously and especially more quickly.

6 Carcinogenesis

syndromes that are associated with mutations in high penetrance alleles. Because of phenotypic variability, age-related penetrance, and gender-specific cancer risks, however, many families with an inherited cancer syndrome will not meet these criteria. Furthermore, because cancer is relatively common in the general population, it is possible to have a chance clusterings of the same or related cancers within a family. These familial clusterings are most likely due to low-penetrance alleles that are more common than mutations in high penetrant alleles. Thus, they will potentially account for a larger proportion of cancer in the general population than the mendelian classic syndromes. For colorectal cancer (CRC), for

However, about 30% of the variation in CRC risk is thought to be due to inherited susceptibility, which only in part can be explained by the known Mendelian inheritance [2]. Breast cancer has a similar gap between Mendelian and overall genetic risk and for prostate cancer the risk is even higher, as very few cases are attributable to high-risk alleles. It is that gap which must be filled by studies to identify cancer predisposition alleles in the general population [9]. Localization and characterization of low-penetrance alleles are the focus of much research, but the challenges are great due to the multi-factorial nature of cancer and

**2.1. High-throughput technology for detection of the multiple alleles associated** 

The history of human genetics has focused on mapping regions of the genome that can

The 'rare variant hypothesis' proposes that a significant proportion of the inherited susceptibility to relatively common human diseases may be due to the sum of the effects of a series of low frequency variants of a variety of different genes, perhaps dominantly and/or independently acting, each conferring a moderate but detectable increase in relative risk [2]. Regardless, there is good supporting evidence that rare variants will often have stronger effects on cancer risk than common variants. This evidence is based on several works whose purpose was to determine whether evaluating rare single-nucleotide polymorphism (SNPs) in case-control association studies could help to identify causal SNPs for common diseases. The sources of data of these works were generally the International HapMap Project and the SeattleSNPs project and they suggest that slightly deleterious SNPs subjected to weak purifying selection are major players in genetic control of susceptibility to common diseases, including cancer. These results suggests that studies with large sample sizes (5000 and higher) targeting SNPs will be a better strategy to identify causal disease SNPs [10]. Instead, genome wide association studies (GWAS) have emerged as an important tool for discovering regions of the genome that harbor uncommon genetic variants that confer risk for complex tumors, whose nature is probably polygenic [11]. These variants include single nucleotide variants (SNVs) or single nucleotide polymorphisms (SNPs), small insertions and

example, Mendelian syndrome includes FAP, MAP and Lynch syndrome.

the underlying genetic heterogeneity.

explain part or all of a disease or human trait.

deletions and structural genomic variants.

**to cancer predisposition** 

The method becoming more widely used is high-throughput sequencing, which allows a massive study of DNA. This is a system able to obtain more than 400,000 different readings in a single stroke of about 8 hours. The operating principle is based on clonal amplification of DNA *in vitro* by emulsion PCR and on a protocol of pyrosequencing that, unlike the classic method of Sanger, is based on the detection of pyrophosphate released by the incorporation of a nucleotide during DNA synthesis. In high-throughput sequencing 454 instrumentation, the sample may be any DNA larger than 1500 base pairs (genomic DNA or portions, cDNAs and large amplicons). The sequences obtained are analysed, properly aligned and oriented in contigs from the sequencer software, according to the shotgun and paired-end strategy. The accuracy of the data obtained is measured in terms of "coverage", that is based on the average number of times that each is accessed (read). This technology, therefore, is able to ensure high accuracy of the results (> 99.5%), thanks also to the careful management of the enormous amount of bioinformatics sequences obtained, which minimizes the production of raw redundant data. This feature, coupled with the extraordinary speed of processing, which makes the method also more economical than the classic automated sequencer, allows the user to analyze and quantify at the same time a large amount of samples. Therefore, the sequencer ultra-massive is an extremely versatile technique for a large number of applications such as resequencing and de novo assembly of entire genomes, and the massive sequencing of amplicons.

This latter approach is now widely applied, for example, for the identification of rare variants that presumably contribute in a synergistic way and in association with other factors predisposing to the development of complex genetic diseases characterized by genetic heterogeneity. This technology therefore offers a great contribution to the studies of Genome Wide Association, because it allows quick identification of the allele frequencies of SNPs in population studies, and to analyze a given target gene in multiple genomes, or a panel of target genes in a single patient, even at the level of gene expression (transcriptome analysis) [4]. However, the high number of next generation sequencing information requires accurate statistically studies. The threshold value for discovery has been established at a high level, known as genome-wide significance, which serves two dual purposes [12]. First, it needs careful consideration of the power to detect the effect sizes expected to be observed in the study. Second, the high bar of genome wide significance protects against the probability of a false-positive finding. The latter is critical because GWAS are discovery

#### 8 Carcinogenesis

tools that point investigators toward long arduous follow-up studies for unraveling the underlying biology and the pursuit of markers for risk assessment [11, 13]. However, the common cancer alleles detected by GWAS account for only 10% of the familial relative risk of disease.

Synergistic Effects of Low-Risk Variant Alleles in Cancer Predisposition 9

**2.3. The simultaneous presence of low-risk alleles increases the risk of** 

MYC oncogene and the prostate stem cell antigen gene (PSCA) [11, 28].

Genome–wide association studies in cancer based on high-throughput sequencing approaches have already identified over 150 regions associated with two dozen specific cancers, such as breast, prostate and colorectal cancer, providing new insights into common mechanisms of carcinogenesis. Since each region confers a small contribution to the cancer risk, it is daunting to consider any single nucleotide polymorphism as a clinical test, rather one should think about the synergistic action of different SNP as well as the environmental factor [11, 27]. These studies allowed researchers to identify large susceptibility chromosomal regions for many unrelated cancers. For example, the 8q24 region harbor multiple cancer susceptibility SNP loci associated with prostate cancer, colorectal cancer and precancerous colorectal adenomas, and bladder cancer risk; these loci affect genes such as

Another common cancer susceptibility chromosomal region is the 5p15.33; in this region common variants in the TERT-CLPTM1L have been identified by GWAS in association with the prostate, uterine cervix and skin cancers [11]. TERT is an attractive candidate gene, because it encodes the reverse transcriptase component of the telomerase, a gene that is critical for telomere replication and stabilization by controlling telomere length. TERT promotes epithelial proliferation and telomere maintenance has been implicated in the progression from KRAS-activated adenoma to adenocarcinoma in a murine model. There is additional evidence for its association with bladder, prostate, uterine cervix and skin cancers [11]. Moreover, phenotypic heterogeneity in the breast cancer, such as merging estrogen receptor negative and positive cases, has been need to identify other loci that might contribute to different phenotypes. Preliminary GW analysis has shown that a subset of the discovered loci may be specific to ER-pos breast cancer while select loci could be more important for ER-neg breast cancer [29]. Similar studies have identified an association between coding variants in CASP8 gene and breast cancer [30]. CASP8 belongs to many key pathways, including p53 signaling, apoptosis, and cancer [31]. The decreased risk for breast cancer with CASP8 Asp302His was revealed in an another recent association study [32]. Others proposed that rare variants within the double strand break repair genes CHEK2,

Other large studies have identified 31.7% of the novel gene-variant breast cancer significant associations between 145 variants analyzed. A large GWAS conducted with East Asian women provided convincing evidence for an association with a novel independent susceptibility locus located at 6q25.1, near the TAB2 gene (TGF-beta activated kinase 1). Furthermore this study shows that genetic variants in the ESR1 gene (estrogen receptor 1) may be related to breast cancer risk [34]. A recent study of populations conducted by Smith et al. [35] has pointed out that the simultaneous presence of mutations in the TP53 gene and single nucleotide polymorphisms (SNPs) in genes belonging to different repair systems such as complex BER, NER, MMR and DSBR (Double-Strand Break Repair) is associated with earlier age of onset of breast cancer (<50 years), thus suggesting the idea of an additive or

**hereditary cancer: review of literature data** 

BRIP1 and PALB2 predispose to breast cancer [33].

multiplicative effect.

### **2.2. Variants of unknown significance in hereditary cancer predisposition genes**

Variants of unknown significance (VUS) within the cancer predisposition genes could be responsible for cancer development, in particular when associated with another VUS or SNPs. The influence of these variants on the development of cancer is often difficult to predict [5, 14]. Several criteria have been established for the characterization of these phenotypic variants, particularly for the missense variants [15, 16]; these criteria included the co-segregation of the variant with the disease and the presence/absence of variation in the healthy population. However, these criteria are not always pursued to establish the pathogenetic significance of these variants [ 17, 18].

Segregation analysis is not always practicable, since, often the families are small or part of family members is reluctant to participate to molecular investigation. Population studies to exclude the polymorphic nature of the variant is often laborious. Recent studies have revealed new strategies to classify the VUS as pathogenic. These strategies include "in silico" analysis, using computational programs such as PolyPhen (Polymorphism Phenotiping) and SIFT (Sorting Intolerant From Tolerant) to assess whether the VUS missense type falls into a phylogenetically conserved domain and / or makes changes to the physical-chemical properties of proteins [19 -21].

The program Human Splicing Finder (HSF) [22, 23], which simultaneously uses a set of matrices already available on the network is useful to predict the effects of missense, silent and intronic variants on the signals of splicing and to identify regulators motifs associated with the processing of the mRNA. However, the results of the computational accuracy have a predictive value of about 80% and, therefore, do not always reflect the functional consequences of the variant *in vivo*. Several papers suggest to combine the results from several bioinformatics approaches especially those based on amino acid conservation status, to increase the predictive value of about 10% [19, 24].

Other studies complemented "in silico" analysis to a direct study of the mRNA, to confirm or rule out the effects of splicing variants [25, 26]. In addition, many recent literature data emphasize the importance of developing functional assays *in vitro* and *in vivo* to assess the effects of VUS on specific biological functions [18]. All studies conducted so far show that none of the above criteria, including functional assays, is an indicator of pathogenicity, if considered individually; it is necessary that most of these strategies are used in combination with each other so that they can lead to a correct evaluation pathogenicity of numerous variant data.

### **2.3. The simultaneous presence of low-risk alleles increases the risk of hereditary cancer: review of literature data**

8 Carcinogenesis

of disease.

variant data.

tools that point investigators toward long arduous follow-up studies for unraveling the underlying biology and the pursuit of markers for risk assessment [11, 13]. However, the common cancer alleles detected by GWAS account for only 10% of the familial relative risk

**2.2. Variants of unknown significance in hereditary cancer predisposition genes** 

Variants of unknown significance (VUS) within the cancer predisposition genes could be responsible for cancer development, in particular when associated with another VUS or SNPs. The influence of these variants on the development of cancer is often difficult to predict [5, 14]. Several criteria have been established for the characterization of these phenotypic variants, particularly for the missense variants [15, 16]; these criteria included the co-segregation of the variant with the disease and the presence/absence of variation in the healthy population. However, these criteria are not always pursued to establish the

Segregation analysis is not always practicable, since, often the families are small or part of family members is reluctant to participate to molecular investigation. Population studies to exclude the polymorphic nature of the variant is often laborious. Recent studies have revealed new strategies to classify the VUS as pathogenic. These strategies include "in silico" analysis, using computational programs such as PolyPhen (Polymorphism Phenotiping) and SIFT (Sorting Intolerant From Tolerant) to assess whether the VUS missense type falls into a phylogenetically conserved domain and / or makes changes to the

The program Human Splicing Finder (HSF) [22, 23], which simultaneously uses a set of matrices already available on the network is useful to predict the effects of missense, silent and intronic variants on the signals of splicing and to identify regulators motifs associated with the processing of the mRNA. However, the results of the computational accuracy have a predictive value of about 80% and, therefore, do not always reflect the functional consequences of the variant *in vivo*. Several papers suggest to combine the results from several bioinformatics approaches especially those based on amino acid conservation status,

Other studies complemented "in silico" analysis to a direct study of the mRNA, to confirm or rule out the effects of splicing variants [25, 26]. In addition, many recent literature data emphasize the importance of developing functional assays *in vitro* and *in vivo* to assess the effects of VUS on specific biological functions [18]. All studies conducted so far show that none of the above criteria, including functional assays, is an indicator of pathogenicity, if considered individually; it is necessary that most of these strategies are used in combination with each other so that they can lead to a correct evaluation pathogenicity of numerous

pathogenetic significance of these variants [ 17, 18].

physical-chemical properties of proteins [19 -21].

to increase the predictive value of about 10% [19, 24].

Genome–wide association studies in cancer based on high-throughput sequencing approaches have already identified over 150 regions associated with two dozen specific cancers, such as breast, prostate and colorectal cancer, providing new insights into common mechanisms of carcinogenesis. Since each region confers a small contribution to the cancer risk, it is daunting to consider any single nucleotide polymorphism as a clinical test, rather one should think about the synergistic action of different SNP as well as the environmental factor [11, 27]. These studies allowed researchers to identify large susceptibility chromosomal regions for many unrelated cancers. For example, the 8q24 region harbor multiple cancer susceptibility SNP loci associated with prostate cancer, colorectal cancer and precancerous colorectal adenomas, and bladder cancer risk; these loci affect genes such as MYC oncogene and the prostate stem cell antigen gene (PSCA) [11, 28].

Another common cancer susceptibility chromosomal region is the 5p15.33; in this region common variants in the TERT-CLPTM1L have been identified by GWAS in association with the prostate, uterine cervix and skin cancers [11]. TERT is an attractive candidate gene, because it encodes the reverse transcriptase component of the telomerase, a gene that is critical for telomere replication and stabilization by controlling telomere length. TERT promotes epithelial proliferation and telomere maintenance has been implicated in the progression from KRAS-activated adenoma to adenocarcinoma in a murine model. There is additional evidence for its association with bladder, prostate, uterine cervix and skin cancers [11]. Moreover, phenotypic heterogeneity in the breast cancer, such as merging estrogen receptor negative and positive cases, has been need to identify other loci that might contribute to different phenotypes. Preliminary GW analysis has shown that a subset of the discovered loci may be specific to ER-pos breast cancer while select loci could be more important for ER-neg breast cancer [29]. Similar studies have identified an association between coding variants in CASP8 gene and breast cancer [30]. CASP8 belongs to many key pathways, including p53 signaling, apoptosis, and cancer [31]. The decreased risk for breast cancer with CASP8 Asp302His was revealed in an another recent association study [32]. Others proposed that rare variants within the double strand break repair genes CHEK2, BRIP1 and PALB2 predispose to breast cancer [33].

Other large studies have identified 31.7% of the novel gene-variant breast cancer significant associations between 145 variants analyzed. A large GWAS conducted with East Asian women provided convincing evidence for an association with a novel independent susceptibility locus located at 6q25.1, near the TAB2 gene (TGF-beta activated kinase 1). Furthermore this study shows that genetic variants in the ESR1 gene (estrogen receptor 1) may be related to breast cancer risk [34]. A recent study of populations conducted by Smith et al. [35] has pointed out that the simultaneous presence of mutations in the TP53 gene and single nucleotide polymorphisms (SNPs) in genes belonging to different repair systems such as complex BER, NER, MMR and DSBR (Double-Strand Break Repair) is associated with earlier age of onset of breast cancer (<50 years), thus suggesting the idea of an additive or multiplicative effect.

In prostate cancer, there are at least 35 distinct loci harboring common susceptibility alleles identified by GWAS that could distinguish between aggressive and non-aggressive disease, but other studies are required [36]. These analyses were conducted in both European and Asian populations [37]. Moreover, a fine mapping of a region of chromosome 11q13 showed a complex genomic architecture characterized by multiple independent signals contributing to prostate cancer risk. This study further annotates common and uncommon variants across this region. In particular, a variant in the promoter of the MSMB gene on chromosome 10q13, is known to have influence in the gene expression, and in the protein PSP94 (prostate secretory protein 94) levels, showing significant association with prostate cancer. This chromosomal region was extensively resequenced and it is possible that a neighboring gene, the androgen receptor coactivator (NCOA4), could also be a candidate gene for analysis [38]. Moreover, GWAS for chromosomal 19q13.33 region, that harbors the gene responsible for the prostate serum antigene (PSA), suggested that variants in this gene, including a nonsynonymous SNP, could contribute to both prostate carcinogenesis and PSA levels [39].

Synergistic Effects of Low-Risk Variant Alleles in Cancer Predisposition 11

CHEK2 (I157T and possibly del1100C) [45]. Numerous GWAS data for susceptibility cancer specially for colorectal cancer have been the subject of several functional studies to demonstrate the effective association and to test the hypothesis of a synergistic effect

In a recent study on the genome of yeast, it has been shown that the weak alleles of MMR complex cause a weak mutator phenotype, but when these interact with each other cause a strong mutator phenotype. In this work, 11 SNPs and 14 missense variants of doubt pathogenetic meaning, previously identified in these genes, have been studied. The mutator effect of these variants both individually and in combination with each other was assayed by testing complementation, in selective media for the amino acids lysine and tyrosine, and for resistance to canavanine [46]. Finally, Demogines et al. [47] have used yeast strains, that differed in terms of geographic and environmental factors, to demonstrate that the association of polymorphic variants, identified in the MMR genes MLH1 and PMS1, affecting the same or different genetic loci, may act as modifiers intra - or inter-gene and this phenomenon may play a role in both the penetrance of the colorectal disease (mutator

phenotype) and in the process of evolutionary adaptation (genomic compatibility).

In this chapter we report the results of our studies on detection of mutations in MisMatch Repair (MMR) genes as responsible for Lynch syndrome. Because many patients with hereditary cancer syndrome did not show mutations in high penetrance genes, we speculate that association of several low penetrance alleles could determine a genetic predisposition to

Colon cancer is a multifactorial disease. It's caused by enviromental factors, nutritional factors and genetic predisposition. Our studies are related to the genetic susceptibility of colon cancer, in particular the molecular basis of Lynch syndrome (Hereditary Non Polyposis colorectal cancer, HNPCC). The Lynch Syndrome is one of the syndromes of hereditary cancer with higher incidence in the population [48]. It has an autosomal dominant transmission and occurs in two forms: as Lynch I with an early age of occurrence (25% at 50 years and 70–80% within 70 years), predilection for the proximal colon (60–80%), and high rates of metachronous colorectal cancer (30% at 10 years and 50% at 15 years from the first tumor); and Lynch II, has the same characteristics but also extracolonic tumors involving the uterus (25–60%), ovaries (8–14%), stomach (13%), and urinary tract (4%)

This syndrome accounts for 5–15% of all colorectal cancers, although the true incidence is unknown, confounded by incomplete penetrance (<80%), rapid progression of adenoma to carcinoma (<5 years), development of extracolonic neoplasms, and the inter- and, occasionally, intra-familiar heterogeneity of the lesions [49]. In Lynch syndrome, the adenomas have the same frequency as in sporadic cases, but a more rapid progression to carcinoma. Due to the deficiency in DNA-repair genes, adenomas accumulate mutations

between low risk allelic variants.

**3. The Lynch syndrome** 

cancer development.

(Figure 2).

A large GWAS conducted in several populations (European Americans and African Americans) showed that genetic associations by race are modified by interactions between individual SNPs and prostate cancer and that significance of particular GWAS "hits" is not the same between racial groups. This study highlights the need to conduct GWAS and GWAS replication studies in a variety of racial groups in order to gain a more complete understanding of differences in risk alleles by race and in order to study gene-gene and gene-environment interactions [40]. A similar study conducted in two European populations suggested a list of SNP–SNP interactions that can be followed in other confirmation studies. to explore the etiology of prostate cancer [41].

Finally several papers report numerous GWAS for colorectal cancer, identifying a total of 16 new susceptibility loci for colorectal cancer. SNPs both in common genes as MMR genes and in other novel loci as SMAD7 and MYC seem to associate with different clinical outcomes [42], or different pharmacological responses [43]. Moreover, GWAS for chromosomal 20p12.3 region, a site bereft of genes or predicted protein-encoding transcripts, suggested that particular SNP in this region could contribute to colorectal cancer progression. Interestingly, the bone morphogenetic protein 2 (BMP2) maps 342 kb telomeric to this locus, which is an initiator of BMP signaling by binding to its corresponding receptors. BMP signaling can suppress the Wnt pathway to ensure a balanced control of intestinal stem cell self-renewal. As reflected by earlier studies, mutations of BMP pathway have been described in juvenile polyposis, an inherited syndrome that predisposes to CRC. Considering all this information, it has been speculated that this locus might alter the BMP signaling transduction by the effect on BMP2 and thus affect CRC incidence [44].

A different GWAS study assessed a set of single-nucleotide polymorphisms (SNPs) near 157 DNA repair genes in three studies on colorectal cancer (CRC). Although no individual SNP showed evidence of association, the set of SNPs as a whole was associated with colorectal cancer risk, in particular the MLH1 promoter SNP -93G>A (rs1800734) and rare variants in CHEK2 (I157T and possibly del1100C) [45]. Numerous GWAS data for susceptibility cancer specially for colorectal cancer have been the subject of several functional studies to demonstrate the effective association and to test the hypothesis of a synergistic effect between low risk allelic variants.

In a recent study on the genome of yeast, it has been shown that the weak alleles of MMR complex cause a weak mutator phenotype, but when these interact with each other cause a strong mutator phenotype. In this work, 11 SNPs and 14 missense variants of doubt pathogenetic meaning, previously identified in these genes, have been studied. The mutator effect of these variants both individually and in combination with each other was assayed by testing complementation, in selective media for the amino acids lysine and tyrosine, and for resistance to canavanine [46]. Finally, Demogines et al. [47] have used yeast strains, that differed in terms of geographic and environmental factors, to demonstrate that the association of polymorphic variants, identified in the MMR genes MLH1 and PMS1, affecting the same or different genetic loci, may act as modifiers intra - or inter-gene and this phenomenon may play a role in both the penetrance of the colorectal disease (mutator phenotype) and in the process of evolutionary adaptation (genomic compatibility).

### **3. The Lynch syndrome**

10 Carcinogenesis

levels [39].

In prostate cancer, there are at least 35 distinct loci harboring common susceptibility alleles identified by GWAS that could distinguish between aggressive and non-aggressive disease, but other studies are required [36]. These analyses were conducted in both European and Asian populations [37]. Moreover, a fine mapping of a region of chromosome 11q13 showed a complex genomic architecture characterized by multiple independent signals contributing to prostate cancer risk. This study further annotates common and uncommon variants across this region. In particular, a variant in the promoter of the MSMB gene on chromosome 10q13, is known to have influence in the gene expression, and in the protein PSP94 (prostate secretory protein 94) levels, showing significant association with prostate cancer. This chromosomal region was extensively resequenced and it is possible that a neighboring gene, the androgen receptor coactivator (NCOA4), could also be a candidate gene for analysis [38]. Moreover, GWAS for chromosomal 19q13.33 region, that harbors the gene responsible for the prostate serum antigene (PSA), suggested that variants in this gene, including a nonsynonymous SNP, could contribute to both prostate carcinogenesis and PSA

A large GWAS conducted in several populations (European Americans and African Americans) showed that genetic associations by race are modified by interactions between individual SNPs and prostate cancer and that significance of particular GWAS "hits" is not the same between racial groups. This study highlights the need to conduct GWAS and GWAS replication studies in a variety of racial groups in order to gain a more complete understanding of differences in risk alleles by race and in order to study gene-gene and gene-environment interactions [40]. A similar study conducted in two European populations suggested a list of SNP–SNP interactions that can be followed in other

Finally several papers report numerous GWAS for colorectal cancer, identifying a total of 16 new susceptibility loci for colorectal cancer. SNPs both in common genes as MMR genes and in other novel loci as SMAD7 and MYC seem to associate with different clinical outcomes [42], or different pharmacological responses [43]. Moreover, GWAS for chromosomal 20p12.3 region, a site bereft of genes or predicted protein-encoding transcripts, suggested that particular SNP in this region could contribute to colorectal cancer progression. Interestingly, the bone morphogenetic protein 2 (BMP2) maps 342 kb telomeric to this locus, which is an initiator of BMP signaling by binding to its corresponding receptors. BMP signaling can suppress the Wnt pathway to ensure a balanced control of intestinal stem cell self-renewal. As reflected by earlier studies, mutations of BMP pathway have been described in juvenile polyposis, an inherited syndrome that predisposes to CRC. Considering all this information, it has been speculated that this locus might alter the BMP

signaling transduction by the effect on BMP2 and thus affect CRC incidence [44].

A different GWAS study assessed a set of single-nucleotide polymorphisms (SNPs) near 157 DNA repair genes in three studies on colorectal cancer (CRC). Although no individual SNP showed evidence of association, the set of SNPs as a whole was associated with colorectal cancer risk, in particular the MLH1 promoter SNP -93G>A (rs1800734) and rare variants in

confirmation studies. to explore the etiology of prostate cancer [41].

In this chapter we report the results of our studies on detection of mutations in MisMatch Repair (MMR) genes as responsible for Lynch syndrome. Because many patients with hereditary cancer syndrome did not show mutations in high penetrance genes, we speculate that association of several low penetrance alleles could determine a genetic predisposition to cancer development.

Colon cancer is a multifactorial disease. It's caused by enviromental factors, nutritional factors and genetic predisposition. Our studies are related to the genetic susceptibility of colon cancer, in particular the molecular basis of Lynch syndrome (Hereditary Non Polyposis colorectal cancer, HNPCC). The Lynch Syndrome is one of the syndromes of hereditary cancer with higher incidence in the population [48]. It has an autosomal dominant transmission and occurs in two forms: as Lynch I with an early age of occurrence (25% at 50 years and 70–80% within 70 years), predilection for the proximal colon (60–80%), and high rates of metachronous colorectal cancer (30% at 10 years and 50% at 15 years from the first tumor); and Lynch II, has the same characteristics but also extracolonic tumors involving the uterus (25–60%), ovaries (8–14%), stomach (13%), and urinary tract (4%) (Figure 2).

This syndrome accounts for 5–15% of all colorectal cancers, although the true incidence is unknown, confounded by incomplete penetrance (<80%), rapid progression of adenoma to carcinoma (<5 years), development of extracolonic neoplasms, and the inter- and, occasionally, intra-familiar heterogeneity of the lesions [49]. In Lynch syndrome, the adenomas have the same frequency as in sporadic cases, but a more rapid progression to carcinoma. Due to the deficiency in DNA-repair genes, adenomas accumulate mutations

#### 12 Carcinogenesis

about three times faster than in sporadic disease. These mutations occur predominantly in microsatellite DNA sequences, a condition defined as microsatellite instability (MSI), which are more susceptible to errors in these genes replication because of their repetitive nature. The microsatellite sequences are also present in very important colorectal cancer tumorigenesis genes, thus the accumulation of errors in these genes determine rapid cellular proliferation. MSI is present in over 90% Lynch cases [50]. The clinical diagnosis of Lynch syndrome is performed upon the Amsterdam Criteria (Tab. 2). However, the Amsterdam Criteria do not identify up to 30% of potential Lynch syndrome carriers [51].

Synergistic Effects of Low-Risk Variant Alleles in Cancer Predisposition 13

*All of the following must apply for a putative diagnosis of HNPCC to be made in a family*  There are at least three relatives with an HNPCC-associated cancer (large bowel,

One affected person is a first-degree relative of the other two

At least one person was diagnosed before the age of 50 years

instability, there is a higher likelihood for a Lynch syndrome diagnosis.

Individuals with cancer in families that meet the Amsterdam Criteria

Individuals with signet-ring–cell-type CRC diagnosed at age < 45 years

metachronous CRC or associated extracolonic cancers

Individuals with adenomas diagnosed at age < 40 years

histopathology diagnosed at age < 45 years

**Table 3.** Bethesda Guidelines for MSI Testing

Individuals with two HNPCC-associated cancers, including synchronous and

Individuals with CRC and a first-degree relative with CRC and/or HNPCC-related extracolonic cancer and/or a colorectal adenoma diagnosed at age < 40 years Individuals with CRC or endometrial cancer diagnosed at age < 45 years

Individuals with right-sided CRC with an undifferentiated pattern (solid or cribriform) on

Germ-line mutations in the MLH1 and MSH2 genes account for a majority of families with Lynch Syndrome. The majority of research into mutations has focused on MLH1 and MSH2, however mutations in these two gene are not present in many patients. So far, 10% of mutations in MMR genes have been identified in the MSH6 gene and a total of 5% in MLH3 and PMS2 and very recently germ-line mutations in the MSH3 gene [53]. These genes are defined as "minor MMR genes" because they have redundant functions in mismatch repair in replication. It is known that as well as being involved in mismatch repair in replication,

At least two successive generations are affected

Familial adenomatous polyposis has been excluded Tumors have been verified by pathologic examination

brain, bladder, or skin)

**Table 2.** Amsterdam Criteria I and II

*continue for MMR testing* 

endometrium, small bowel, ureter, or renal pelvis, although not including stomach, ovary,

For this reason, in some patients with colon cancer, as suggested by the Bethesda guidelines (Table 3) [52], it is possible to analyse microsatellite instability in colon tumor specimens, to identify the inefficiency of DNA mismatch repair complex. If there is microsatellite

*Tumors from any of the following should be tested for MSI and then positive patients should* 

**Figure 2.** Lifetime Risk of development of cancer associated with Lynch Syndrome.

*All of the following must apply for a putative diagnosis of HNPCC to be made in a family* 

There are at least three relatives with an HNPCC-associated cancer (large bowel, endometrium, small bowel, ureter, or renal pelvis, although not including stomach, ovary, brain, bladder, or skin)

One affected person is a first-degree relative of the other two

At least two successive generations are affected

At least one person was diagnosed before the age of 50 years

Familial adenomatous polyposis has been excluded

Tumors have been verified by pathologic examination

**Table 2.** Amsterdam Criteria I and II

12 Carcinogenesis

about three times faster than in sporadic disease. These mutations occur predominantly in microsatellite DNA sequences, a condition defined as microsatellite instability (MSI), which are more susceptible to errors in these genes replication because of their repetitive nature. The microsatellite sequences are also present in very important colorectal cancer tumorigenesis genes, thus the accumulation of errors in these genes determine rapid cellular proliferation. MSI is present in over 90% Lynch cases [50]. The clinical diagnosis of Lynch syndrome is performed upon the Amsterdam Criteria (Tab. 2). However, the Amsterdam

Criteria do not identify up to 30% of potential Lynch syndrome carriers [51].

**Figure 2.** Lifetime Risk of development of cancer associated with Lynch Syndrome.

For this reason, in some patients with colon cancer, as suggested by the Bethesda guidelines (Table 3) [52], it is possible to analyse microsatellite instability in colon tumor specimens, to identify the inefficiency of DNA mismatch repair complex. If there is microsatellite instability, there is a higher likelihood for a Lynch syndrome diagnosis.

*Tumors from any of the following should be tested for MSI and then positive patients should continue for MMR testing* 

Individuals with cancer in families that meet the Amsterdam Criteria

Individuals with two HNPCC-associated cancers, including synchronous and metachronous CRC or associated extracolonic cancers

Individuals with CRC and a first-degree relative with CRC and/or HNPCC-related extracolonic cancer and/or a colorectal adenoma diagnosed at age < 40 years

Individuals with CRC or endometrial cancer diagnosed at age < 45 years

Individuals with right-sided CRC with an undifferentiated pattern (solid or cribriform) on histopathology diagnosed at age < 45 years

Individuals with signet-ring–cell-type CRC diagnosed at age < 45 years

Individuals with adenomas diagnosed at age < 40 years

**Table 3.** Bethesda Guidelines for MSI Testing

Germ-line mutations in the MLH1 and MSH2 genes account for a majority of families with Lynch Syndrome. The majority of research into mutations has focused on MLH1 and MSH2, however mutations in these two gene are not present in many patients. So far, 10% of mutations in MMR genes have been identified in the MSH6 gene and a total of 5% in MLH3 and PMS2 and very recently germ-line mutations in the MSH3 gene [53]. These genes are defined as "minor MMR genes" because they have redundant functions in mismatch repair in replication. It is known that as well as being involved in mismatch repair in replication, the MMR system also has other functions [54], such as: DNA damage response, diversification of antibody, promotion of meiotic crossover. In these functions the "minor" MMR genes play an important role.

Synergistic Effects of Low-Risk Variant Alleles in Cancer Predisposition 15

IVS7 -9 T>C AM+

ex12 c.1860G>A (Asp>Asn)

(Pro>Ser) IVS6-64 C>T AM+

ex4 c.693G>A (Pro) ex20 c.2732 T>G (Leu>Trp)

IVS6-64 C>T AM+

NO AM MSI-H

NO AM later onset MSI-H

AM+ later onset MSI-L

> AM+ MSI-H

> MSI-H

AM+ MSI-H

capable of polygenic interactions with other MMR gene alleles that might lead to tumour

*PATIENTS MSH6 PMS2 MLH3 MSH3 PHENOTYPE* 

ex1 c.2530 C>T (Pro>Ser) c.2533 T>C (Ser>Pro)

ex1 c.2533 T>C (Ser>Pro)

ex1 c.2533 T>C (Ser>Pro)

ex1 c.2530 C>T (Pro>Ser) c.2533 T>C (Ser>Pro)

ex1 c.666 G>A (Lys) c.2191 G>T (Val>Phe) c.2533A>G (Ser>Gly)

ex1 c.2530 C>T

ex 1 c.2027G>A (Arg>Lys)

A>G (Ile>Val) IVS12-4 G>A IVS6-64 C>T AM+

ex14 c.2324 A>G (Asn>Ser)

ex6 c.665G>C (Ser>Thr) IVS6 +16A>G

IVS6+16A>G ex13 c.2324 T>C (Phe)

(Thr>Lys)

**Table 4.** Patients carrying variants in several MMR genes: MSH6, PMS2, MSH3, MLH3; \*the patient

In detail, we report the case of a Lynch family with mutations in several MMR genes. The index case of family 504 (II-5 in Figure 4), who had developed an adenocarcinoma of the left colon at the age of 34 years, an adenocarcinoma of the right colon at the age of 53 years and

progression in Lynch syndrome.

<sup>9525</sup>ex4 c.2633

<sup>103</sup>ex5 c.3261\_62insC (Phe>stop)

013

015

504\*

T>C (Val>Ala)

423 IVS12-4G>A

<sup>416</sup>ex11 c.1714C>A

shows also the UV in MSH2 gene (c.984 C>T)

ex5 c.3295\_97delTT (Ile>stop)

<sup>210</sup>ex4 c.2941 A>G (Ile>Val)

<sup>211</sup>ex4 c.2941

### **3.1. Results of mutation detection analysis in MMR genes**

Recently, several studies have shown that association of low penetrance alleles could determine a genetic predisposition to cancer development [46,47]. For this reason, we studied 63 Lynch families recruited from various health centres in Campania (Southern Italy). Of these, forty families met the Amsterdam criteria and twenty-three patients with high microsatellite instability (MSI-H) met the Bethesda guidelines, in which no pathogenetic germline mutations were identified in MLH1 and MSH2 genes. We performed detection mutation analysis in each minor MMR gene (MSH6, MLH3, PMS2 and MSH3) by DHPLC. All samples exbiting abnormal DHPLC profiles were analyzed by directed sequencing (Figure 3). In our studies we have identified overall 65 genetic variants in these "minor" MMR genes.

**Figure 3.** A) Chromatogram and B) electropherogram of the missense mutation c.2732 T>G (Leu>Trp) in MSH3 gene.

The analysis of the damaged point mutations at the structural level is considered to be very important to understand the functional activity of the protein concerned. For this purpose we used the server PolyPhen (bibl), which is available at http://coot.embl.de/PolyPhen/, for missense mutations identified in this study. Moreover, we also used the bioinformatic analysis for the silent and intronic variants.

These variants were analyzed by the software "Human Splicing Finder", a tool to predict the effects of mutations on splicing signals or to identify splicing motifs in any human sequence. Most of these variants result in a polymorphism, which, however, can cause phenotypic variability, affecting the accuracy and efficiency of the protein function [24]. Interestingly, several patients were carriers of at least two genomic variants within the "minor" genes or a VUS in a major gene associated with a genetic variant in minor genes (Table 4 ).

Recently, the effect of polymorphisms and missense mutations in human MMR genes was studied in a *Saccharomyces cerevisiae*-based system. A number of weak alleles of MMR genes and MMR gene polymorphisms that are capable of interacting with other weak alleles of MMR genes to produce strong polygenic MMR defects, have been identified [46]. A similar situation found in our studies might support the hypothesis that weak MMR gene alleles are capable of polygenic interactions with other MMR gene alleles that might lead to tumour progression in Lynch syndrome.

14 Carcinogenesis

MMR genes play an important role.

"minor" MMR genes.

in MSH3 gene.

analysis for the silent and intronic variants.

the MMR system also has other functions [54], such as: DNA damage response, diversification of antibody, promotion of meiotic crossover. In these functions the "minor"

Recently, several studies have shown that association of low penetrance alleles could determine a genetic predisposition to cancer development [46,47]. For this reason, we studied 63 Lynch families recruited from various health centres in Campania (Southern Italy). Of these, forty families met the Amsterdam criteria and twenty-three patients with high microsatellite instability (MSI-H) met the Bethesda guidelines, in which no pathogenetic germline mutations were identified in MLH1 and MSH2 genes. We performed detection mutation analysis in each minor MMR gene (MSH6, MLH3, PMS2 and MSH3) by DHPLC. All samples exbiting abnormal DHPLC profiles were analyzed by directed sequencing (Figure 3). In our studies we have identified overall 65 genetic variants in these

**Figure 3.** A) Chromatogram and B) electropherogram of the missense mutation c.2732 T>G (Leu>Trp)

The analysis of the damaged point mutations at the structural level is considered to be very important to understand the functional activity of the protein concerned. For this purpose we used the server PolyPhen (bibl), which is available at http://coot.embl.de/PolyPhen/, for missense mutations identified in this study. Moreover, we also used the bioinformatic

These variants were analyzed by the software "Human Splicing Finder", a tool to predict the effects of mutations on splicing signals or to identify splicing motifs in any human sequence. Most of these variants result in a polymorphism, which, however, can cause phenotypic variability, affecting the accuracy and efficiency of the protein function [24]. Interestingly, several patients were carriers of at least two genomic variants within the "minor" genes or a

Recently, the effect of polymorphisms and missense mutations in human MMR genes was studied in a *Saccharomyces cerevisiae*-based system. A number of weak alleles of MMR genes and MMR gene polymorphisms that are capable of interacting with other weak alleles of MMR genes to produce strong polygenic MMR defects, have been identified [46]. A similar situation found in our studies might support the hypothesis that weak MMR gene alleles are

VUS in a major gene associated with a genetic variant in minor genes (Table 4 ).

**3.1. Results of mutation detection analysis in MMR genes** 


**Table 4.** Patients carrying variants in several MMR genes: MSH6, PMS2, MSH3, MLH3; \*the patient shows also the UV in MSH2 gene (c.984 C>T)

In detail, we report the case of a Lynch family with mutations in several MMR genes. The index case of family 504 (II-5 in Figure 4), who had developed an adenocarcinoma of the left colon at the age of 34 years, an adenocarcinoma of the right colon at the age of 53 years and a new malignancy of the colon at 59 years of age, show two mutations in MSH3 gene, the c.2732 T>G in exon 20 and c.693 G>A in exon 4, and an UV within the MSH2 gene, the c.984 C>T in exon 6. The PolyPhen in silico analysis showed that the missense variant in MSH3 might alter the function of the protein, because it falls into a highly conserved region in different species, while the silent variant, analyzed by HSF could affect the splicing process.

Synergistic Effects of Low-Risk Variant Alleles in Cancer Predisposition 17

**Figure 4.** Pedegree of 504 family [53]. Symbols and abbreviations used are denoted as fellow: Arrows, analysed members of family; black symbol, colorectal cancer or cancer associate with HNPCC; gray symbols, adenomas or cancer not associated with HNPCC; CRC, colorectal cancer; Br, brain cancer; GU, gastric ulcer; BL, bladder cancer; Bre, breast cancer; TA, tubular adenoma. Number next to diagnosis

**Table 5.** Genotypes of analysed patients; the patients are identified with number of pedigree (Fig.4).

denote age at oneset; l not detected.

To elucidate whether the mutation was associated with the disease in this family, we analysed another eight members. These variants was found in a brother of the index case, with the same phenotype. Instead, another brother (II-8 in Fig. 4) showed only a variant in the MSH2 gene and no genetic variants in the MSH3 gene. This patient had developed a polyp of the colon at 47 years of age. Today he is 59 years old, undergoes regular colonoscopy and so far has not presented other polyps. In the third generation (Fig. 4), we analysed four affected family members. Subjects III-1 and III-2, in Figure 4, showed a silent variant in MSH3 and a variant in MSH2; both subjects showed an early-onset right colon tumour. Subjects III-3 and III-4, in Figure 4, the sons of our proband, developed colon cancer at 36 years of age and a tubular adenoma of the colon at 34 years of age, respectively. Both subjects showed a silent variant in MSH2 and a missense variant and a silent variant in MSH3. The MSI analysis performed on DNA extracted from tumour tissues of patients II-5 and III-3 showed an MSI-H status. Thus, both subjects presented a strong mutator phenotype, probably due to an additive effect by several variants that leads to inefficiency of the MMR complex. The other family members analysed showed only one mutation in the MSH3 gene and they do not present a typical phenotype of Lynch syndrome (Tab.4). Therefore, it is clear that all subjects in this family with the Lynch phenotype showed the c.984T allele of MSH2 and a germ-line variant in the MSH3 gene (a missense and/or silent variant).

Patients belonging to other families showed mutations in several MMR genes; however, for these families it wasn't possible to perform segregation analysis of mutations with disease because no other family members were available for the analysis. In conclusion, several germ-line variants have been identified in several MMR genes using a DHPLC procedure; a method robust, automated, highly sensitive, fast, feasible and particularly useful for highthroughput analyses.

On the basis of this study, it is conceivable to hypothesize a model in which these genetic variants behave as low-risk alleles that contribute to the risk of colon cancer in Lynch families, mostly together with other low-risk alleles of other MMR genes. Therefore, if our assumptions are correct, these studies may indicate a novel inheritance model in the Lynch syndrome, and might suggest that the risk alleles identified to date represent just the tip of an iceberg of risk variants likely to include hundreds of modest effects and possibly thousands of very small effects. This could pave the way toward new diagnostic perspectives. Moreover, The same situation could occur in other forms of hereditary cancer and it may explain the large number of cases remained unresolved as well as the phenotypic heterogeneity that characterizes all hereditary cancer syndromes.

16 Carcinogenesis

variant).

throughput analyses.

a new malignancy of the colon at 59 years of age, show two mutations in MSH3 gene, the c.2732 T>G in exon 20 and c.693 G>A in exon 4, and an UV within the MSH2 gene, the c.984 C>T in exon 6. The PolyPhen in silico analysis showed that the missense variant in MSH3 might alter the function of the protein, because it falls into a highly conserved region in different species, while the silent variant, analyzed by HSF could affect the splicing process. To elucidate whether the mutation was associated with the disease in this family, we analysed another eight members. These variants was found in a brother of the index case, with the same phenotype. Instead, another brother (II-8 in Fig. 4) showed only a variant in the MSH2 gene and no genetic variants in the MSH3 gene. This patient had developed a polyp of the colon at 47 years of age. Today he is 59 years old, undergoes regular colonoscopy and so far has not presented other polyps. In the third generation (Fig. 4), we analysed four affected family members. Subjects III-1 and III-2, in Figure 4, showed a silent variant in MSH3 and a variant in MSH2; both subjects showed an early-onset right colon tumour. Subjects III-3 and III-4, in Figure 4, the sons of our proband, developed colon cancer at 36 years of age and a tubular adenoma of the colon at 34 years of age, respectively. Both subjects showed a silent variant in MSH2 and a missense variant and a silent variant in MSH3. The MSI analysis performed on DNA extracted from tumour tissues of patients II-5 and III-3 showed an MSI-H status. Thus, both subjects presented a strong mutator phenotype, probably due to an additive effect by several variants that leads to inefficiency of the MMR complex. The other family members analysed showed only one mutation in the MSH3 gene and they do not present a typical phenotype of Lynch syndrome (Tab.4). Therefore, it is clear that all subjects in this family with the Lynch phenotype showed the c.984T allele of MSH2 and a germ-line variant in the MSH3 gene (a missense and/or silent

Patients belonging to other families showed mutations in several MMR genes; however, for these families it wasn't possible to perform segregation analysis of mutations with disease because no other family members were available for the analysis. In conclusion, several germ-line variants have been identified in several MMR genes using a DHPLC procedure; a method robust, automated, highly sensitive, fast, feasible and particularly useful for high-

On the basis of this study, it is conceivable to hypothesize a model in which these genetic variants behave as low-risk alleles that contribute to the risk of colon cancer in Lynch families, mostly together with other low-risk alleles of other MMR genes. Therefore, if our assumptions are correct, these studies may indicate a novel inheritance model in the Lynch syndrome, and might suggest that the risk alleles identified to date represent just the tip of an iceberg of risk variants likely to include hundreds of modest effects and possibly thousands of very small effects. This could pave the way toward new diagnostic perspectives. Moreover, The same situation could occur in other forms of hereditary cancer and it may explain the large number of cases remained unresolved as well as the phenotypic

heterogeneity that characterizes all hereditary cancer syndromes.

**Figure 4.** Pedegree of 504 family [53]. Symbols and abbreviations used are denoted as fellow: Arrows, analysed members of family; black symbol, colorectal cancer or cancer associate with HNPCC; gray symbols, adenomas or cancer not associated with HNPCC; CRC, colorectal cancer; Br, brain cancer; GU, gastric ulcer; BL, bladder cancer; Bre, breast cancer; TA, tubular adenoma. Number next to diagnosis denote age at oneset; l not detected.


**Table 5.** Genotypes of analysed patients; the patients are identified with number of pedigree (Fig.4).

### **4. Further research**

The Lynch syndrome is associated mainly with germ-line mutations in MSH2 and MLH1 genes. However, mutational analysis of these two genes do not always provide informative results for genetic counseling of patients with a clinical diagnosis strongly predisposing to cancer development. Therefore, these subjects are considered candidates with simultaneous molecular analysis of all MMR genes. For this reason, high-throughput sequencing could be considered as an analytical approach that adapts better to clarify the molecular basis for each subject with a significant colorectal cancer history. In the future, these new technologies will enable faster identification of the molecular basis of cancer; it will improve the genotype-phenotype correlations the purpose of implementing a clinical treatment more personalized.

Synergistic Effects of Low-Risk Variant Alleles in Cancer Predisposition 19

Ministero Salute - Ricerca Oncologica - RECAM-2006-353005; PRIN 2007-prot. 2007EN8F7T-

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[6] Chung CC., Chanock SJ. Current status of genome-wide association studies in cancer.

[7] Galatola M, Paparo L, Duraturo F, Turano M, Rossi GB, Izzo P, De Rosa M. Beta catenin and cytokine pathway dysregulation in patients with manifestations of the "PTEN

[8] Frank TS, Critchfield GC. Hereditary risk of women's cancers. Best Pract Res Clin

[9] Bodmer W., Bonilla C. Common and rare variants in multifactorial susceptibility to

[10] Gorlov IP, Gorlova OY, Sunyaev SR, Spitz MR, Amos CI.. Shifting paradigm of association studies: value of rare single-nucleotide polymorphisms. Am J Hum Genet.

[11] Chung CC., Magalhaes W., Gonzalez-Bosquet J., Chanock SJ. Genome-wide association studies in cancer—current and future directions. Carcinogenesis 2010;

[12] Barrett JC, Cardon LR. Evaluating coverage of genome-wide association studies. Nat

[13] Erichsen HC, Chanock SJ. SNPs in cancer research and treatment. Br J Cancer. 2004 Feb

[14] Syngal S., Fox EA., Li C, Dovidio M, Eng C, Kolodner RD, Garber JE.Interpretation of genetic test results for hereditary nonpolyposis colorectal cancer: implications for

**Acknowledgement** 

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31(1): 111–120.

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unclassified genetic variants. Hum Mutat. 2008;29(11):1314-26.

hamartoma tumor syndrome". BMC Med Genet. 2012; 13:28.

clinical predisposition testing. JAMA. 1999; 282(3):247-53.

**6. References** 

004

### **5. Conclusions**

A field of biology where the "high-throughput technologies" is now widely applied is certainly the genetics of cancer for identification of constitutive and somatic mutations of putative genes associated with hereditary predisposition to cancer, particularly for those diseases characterized by genetic heterogeneity. Nowadays, we are witnessing a revolution in oncologic medicine, and the hope is that an increasing understanding of genetics will one day unlock the potential of personalized medicine. Clinical cancer genetics has traditionally been associated with risk estimation. Genome-wide germ-line mutation analysis will result in the identification of new cancer-associated alleles across the spectrum of risk. This may in time permit more precise estimation of development cancer risk. The new genetics will bridge the gap between germ-line and somatic genetics; prior analysis of the genetic makeup of the person and their tumour at time of diagnosis will be needed in order to tailor therapy. Central to this endeavour will be the increasing use of next-generation sequencers as whole cancer genomes become unravelled, revealing critical pathways that drive tumour progression and resistance. In the future these new technologies will enable faster identification of the molecular basis of cancer and thus improve the genotypephenotype correlations, in order to implement more personalized monitoring and clinical treatment.

### **Nomenclature**

den Dunnen JT, Antonarakis SE. "*Nomenclature for the description of human sequence variations".* Hum Genet. *2001 Jul;109(1):121-4.*

### **Author details**

Francesca Duraturo, Raffaella Liccardo, Angela Cavallo, Marina De Rosa and Paola Izzo *Department of Molecular Medicine and Medical Biotechnologie, University of Naples Federico II, Italy* 

### **Acknowledgement**

Ministero Salute - Ricerca Oncologica - RECAM-2006-353005; PRIN 2007-prot. 2007EN8F7T-004

### **6. References**

18 Carcinogenesis

personalized.

treatment.

**Nomenclature** 

**Author details** 

*Italy* 

*variations".* Hum Genet. *2001 Jul;109(1):121-4.*

**5. Conclusions** 

**4. Further research** 

The Lynch syndrome is associated mainly with germ-line mutations in MSH2 and MLH1 genes. However, mutational analysis of these two genes do not always provide informative results for genetic counseling of patients with a clinical diagnosis strongly predisposing to cancer development. Therefore, these subjects are considered candidates with simultaneous molecular analysis of all MMR genes. For this reason, high-throughput sequencing could be considered as an analytical approach that adapts better to clarify the molecular basis for each subject with a significant colorectal cancer history. In the future, these new technologies will enable faster identification of the molecular basis of cancer; it will improve the genotype-phenotype correlations the purpose of implementing a clinical treatment more

A field of biology where the "high-throughput technologies" is now widely applied is certainly the genetics of cancer for identification of constitutive and somatic mutations of putative genes associated with hereditary predisposition to cancer, particularly for those diseases characterized by genetic heterogeneity. Nowadays, we are witnessing a revolution in oncologic medicine, and the hope is that an increasing understanding of genetics will one day unlock the potential of personalized medicine. Clinical cancer genetics has traditionally been associated with risk estimation. Genome-wide germ-line mutation analysis will result in the identification of new cancer-associated alleles across the spectrum of risk. This may in time permit more precise estimation of development cancer risk. The new genetics will bridge the gap between germ-line and somatic genetics; prior analysis of the genetic makeup of the person and their tumour at time of diagnosis will be needed in order to tailor therapy. Central to this endeavour will be the increasing use of next-generation sequencers as whole cancer genomes become unravelled, revealing critical pathways that drive tumour progression and resistance. In the future these new technologies will enable faster identification of the molecular basis of cancer and thus improve the genotypephenotype correlations, in order to implement more personalized monitoring and clinical

den Dunnen JT, Antonarakis SE. "*Nomenclature for the description of human sequence* 

Francesca Duraturo, Raffaella Liccardo, Angela Cavallo, Marina De Rosa and Paola Izzo *Department of Molecular Medicine and Medical Biotechnologie, University of Naples Federico II,* 


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#### 24 Carcinogenesis

[54] Jun S.-H., Kim TG., Ban C. DNA mismatch repair system. Classical and fresh roles. FEBS Journal 2006; 273: 1609-19.

**Chapter 2** 

© 2013 Carmo and Caldeira, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Binary System of Grading Epithelial Dysplasia in** 

Cancers of the oral cavity and oropharynx account for approximatelly 3% of all malignancies among men and 2% among women in the United States, and oral squamous cell carcinoma represents 90% of these tumors. Despite great achievements concerning surgery, radiation and chemotherapy, survival rates in 5 years remain near 50 to 55%. As this survival time is directly related to the time of diagnosis of the lesion, prevention and early diagnosis remain important aspects to reduce incidence of the disease, as well as to

Oral squamous cell carcinoma can be preceded by potentially malignant alterations [1,3]. Such alterations are classified as potentially malignant due to the following evidence: 1) it was observed that these lesions evolved to malignant ones during follow-up; 2) typical alterations of potentially malignant lesions are seen co-existing in the margins of squamous cell carcinoma; 3) a proportion of these lesions show cytological and morphological alterations that are observed in malignant lesions; 4) some chromosomal, genomic, and molecular alterations are found in both, potentially malignant and malignant lesions [4].

In a recently published paper, leukoplakia has been defined as ''a white plaque of questionable risk having excluded (other) known diseases or disorders that carry no increased risk for cancer" [5]. Nevertheless, the most used definition of leukoplakia is still the one proposed by the World Health Organization (WHO) in 1978, which states that "leukoplakia is a predominantly white patch that cannot be characterized clinically or

Oral leukoplakia (OL) is the most common potentially malignant lesion of the oral mucosa [1,3]. In a published systematic review [8], the author estimated a global prevalence of OL of

and reproduction in any medium, provided the original work is properly cited.

**1.1. Definition, epidemiology, and etiology of oral leukoplakias** 

histopathologically as any other definable lesion" [6,7].

Maria Auxiliadora Vieira do Carmo and Patrícia Carlos Caldeira

**Oral Leukoplakias** 

http://dx.doi.org/10.5772/54466

**1. Introduction** 

Additional information is available at the end of the chapter

enhance the survival rate of patients [1,2].

Maria Auxiliadora Vieira do Carmo and Patrícia Carlos Caldeira

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54466

### **1. Introduction**

24 Carcinogenesis

FEBS Journal 2006; 273: 1609-19.

[54] Jun S.-H., Kim TG., Ban C. DNA mismatch repair system. Classical and fresh roles.

Cancers of the oral cavity and oropharynx account for approximatelly 3% of all malignancies among men and 2% among women in the United States, and oral squamous cell carcinoma represents 90% of these tumors. Despite great achievements concerning surgery, radiation and chemotherapy, survival rates in 5 years remain near 50 to 55%. As this survival time is directly related to the time of diagnosis of the lesion, prevention and early diagnosis remain important aspects to reduce incidence of the disease, as well as to enhance the survival rate of patients [1,2].

Oral squamous cell carcinoma can be preceded by potentially malignant alterations [1,3]. Such alterations are classified as potentially malignant due to the following evidence: 1) it was observed that these lesions evolved to malignant ones during follow-up; 2) typical alterations of potentially malignant lesions are seen co-existing in the margins of squamous cell carcinoma; 3) a proportion of these lesions show cytological and morphological alterations that are observed in malignant lesions; 4) some chromosomal, genomic, and molecular alterations are found in both, potentially malignant and malignant lesions [4].

### **1.1. Definition, epidemiology, and etiology of oral leukoplakias**

In a recently published paper, leukoplakia has been defined as ''a white plaque of questionable risk having excluded (other) known diseases or disorders that carry no increased risk for cancer" [5]. Nevertheless, the most used definition of leukoplakia is still the one proposed by the World Health Organization (WHO) in 1978, which states that "leukoplakia is a predominantly white patch that cannot be characterized clinically or histopathologically as any other definable lesion" [6,7].

Oral leukoplakia (OL) is the most common potentially malignant lesion of the oral mucosa [1,3]. In a published systematic review [8], the author estimated a global prevalence of OL of

#### 26 Carcinogenesis

2.6%, which is in accordance with the consensus that OL prevalence is between 1% and 5% [9,10]. However, isolated reports show variable rates from 0.5% to 26.92% [8].

Binary System of Grading Epithelial Dysplasia in Oral Leukoplakias 27

**Figure 3.** Homogeneous thick leukoplakia in the tongue.

transformation [4,10].

**Figure 4.** Non-homogeneous (speckled) leukoplakia in the upper alveolar ridge.

There is also the proliferative verrucous leukoplakia, characterized by multifocal evolvement, mainly in elderly female patients that do not present known risk factors (Figure 5 and 6). These lesions are usually resistant to treatment and show a high risk for malignant

**Figure 5.** Proliferative verrucous leukoplakia. Notice the multifocal involvement in the lower gingiva.

OL is more frequent in middle-aged and elderly men, with higher indexes correlated with increased age. The most common sites are cheek, alveolar mucosa, and lower lip [1]. Nonetheless, lesions affecting the floor of the mouth, lateral border of tongue, and lower lip seem to present displastic or malignant alterations more frequently [1,9].

The main risk factor associated with OL is the use of tobacco. OL is six times more frquent among smokers than non-smokers [10]. The effects of alcohol, betel, human papiloma virus, and diet are associated as well, but their exact role is yet to be established [1,9-11]. In addition, there are some OL for which no obvious aetiological factor can be identified, and these lesions are named idiopathic leukoplakias. It is believed that such lesions are significantly more prone to develop into cancer than those OL with known causative factors [9].

### **1.2. Clinical and histological features**

Clinically, OL can be classified as homogeneous and non-homogeneous lesions. Homogeneous OL arises as a white patch slightly elevated, thin, white to gray, uniform, and can present well defined borders or may gradually mix with normal adjacent mucosa (Figure 1 to 3). Non-homogeneous OL can be nodular, verrucous, or speckled (erythroplastic) (Figure 4) [4,10].

**Figure 1.** Homogeneous thin leukoplakia in the tongue.

**Figure 2.** Homogeneous leukoplakia in the lower lip.

**Figure 3.** Homogeneous thick leukoplakia in the tongue.

26 Carcinogenesis

2.6%, which is in accordance with the consensus that OL prevalence is between 1% and 5%

OL is more frequent in middle-aged and elderly men, with higher indexes correlated with increased age. The most common sites are cheek, alveolar mucosa, and lower lip [1]. Nonetheless, lesions affecting the floor of the mouth, lateral border of tongue, and lower lip

The main risk factor associated with OL is the use of tobacco. OL is six times more frquent among smokers than non-smokers [10]. The effects of alcohol, betel, human papiloma virus, and diet are associated as well, but their exact role is yet to be established [1,9-11]. In addition, there are some OL for which no obvious aetiological factor can be identified, and these lesions are named idiopathic leukoplakias. It is believed that such lesions are significantly more prone

Clinically, OL can be classified as homogeneous and non-homogeneous lesions. Homogeneous OL arises as a white patch slightly elevated, thin, white to gray, uniform, and can present well defined borders or may gradually mix with normal adjacent mucosa (Figure 1 to 3). Non-homogeneous OL can be nodular, verrucous, or speckled

[9,10]. However, isolated reports show variable rates from 0.5% to 26.92% [8].

seem to present displastic or malignant alterations more frequently [1,9].

to develop into cancer than those OL with known causative factors [9].

**1.2. Clinical and histological features** 

**Figure 1.** Homogeneous thin leukoplakia in the tongue.

**Figure 2.** Homogeneous leukoplakia in the lower lip.

(erythroplastic) (Figure 4) [4,10].

**Figure 4.** Non-homogeneous (speckled) leukoplakia in the upper alveolar ridge.

There is also the proliferative verrucous leukoplakia, characterized by multifocal evolvement, mainly in elderly female patients that do not present known risk factors (Figure 5 and 6). These lesions are usually resistant to treatment and show a high risk for malignant transformation [4,10].

**Figure 5.** Proliferative verrucous leukoplakia. Notice the multifocal involvement in the lower gingiva.

#### 28 Carcinogenesis

Binary System of Grading Epithelial Dysplasia in Oral Leukoplakias 29

OL may persist unchanged, progress, regress, or even disappear [9]. The malignant transformation risk varies from 3.6% to 36.0%, and some features such as the presence and degree of dysplasia, female gender, time of duration, non-smoker patient, location at floor of the mouth or tongue, size higher than 200mm², and non-homogeneous type, seem to be associated with a worse prognosis [10,19-22]. Surgical excision, cryosurgery, laser surgery, topical or systemic retinoids, therapy with mouth rinses with attenuated adenovirus, and photodynamic therapy are possible therapeutics [10,13,23]. Recurrence rates are highly

Many efforts have attemped to identify molecular markers to predict cancer development in OL. However, the presence and degree of epithelial dysplasia in OL is yet regarded as the most relevant indicator of progression and prognosis, influencing the management of the

The term "dysplasia" is generally employed in the sense of a disordered development [24]. In a stratified squamous epithelium, architectural disturbances affecting normal maturation and stratification may occur. When such alterations are accompanied by cytological atypia, which can be detected as variations in the size and shape of the keratinocytes, the term

Despite many efforts towards new evaluative methods, the histological analysis is still the

The concept of a sequential developmental process from a normal epithelium through a dysplasia, ending in a carcinoma, was introduced from studying pathological changes in the uterine cervix [24]. It is believed that through this process there is an accumulation of genetic and epigenetic alterations and more and more layers of the epithelium are progressively involved, until it is replaced by atypical cells in full length. It is considered that the more severe the degree of dysplasia, the greater the likelihood of malignant transformation. Despite the imperfection of currently available systems, they remain essential, and the diagnosis is a prerequisite for the establishment of the treatment that

The elaboration of a classifying system is not a simple issue as the system may be, above all, an indicator of prognosis, guiding or at least helping in the establishment of the best treatment. Moreover, it should be reproducible, reliable, and as simple and objective as possible. Many classification schemes have been proposed over time, with variable

**1.3. Evolution and prognosis** 

patients [9,10,12].

**2.1. Relevance** 

"dysplasia" is applied [7,12].

provides the best prognosis [12,24].

**2.2. Proposed systems** 

variable among studies, from 0 to 30.0% [10].

**2. Grading oral epithelial dysplasia** 

most useful method for grading epithelial dysplasia in OL [12].

**Figure 6.** Proliferative verrucous leukoplakia. This elderly woman presented multiple lesions affecting different sites of the oral mucosa.

Many lesions must be excluded before formulating a diagnostic hypothesis of OL, such as chemical injuries, candidiasis, frictional lesion, hairy leukoplakia, leukoedema, linea alba, nicotinic stomatitis, among others [4,10]. Because of variable clinical presentation of the potentially malignant lesions, when a provisional clinical diagnosis of OL is made, a biopsy must be performed to obtain the histopathological diagnosis [12].

The microscopic presentation of OL can vary from slightly hyperkeratotic epithelium to lesions with severe dysplasia [13]. The frequencies of dysplastic or malignant alterations in OL vary from 15.6% to 39.2%, and a rate of 19.9% was found in a retrospective study of 3,300 white lesions of the oral cavity [14]. Epithelial dysplasia is characterized by the presence of architectural alteration and cytological atypia, and can be graded as mild, moderate, severe, and carcinoma *in situ* [10]. Nevertheless, there is a notable inter- and intra-observer variation in the interpretation and classification of dysplasia, which makes this method subjective with low reproducibility [12,15]. Thus, many different grading systems have been suggested to enhance the reproducibility and the predictive value for malignant transformation of OL.

It has been suggested a possible correlation between clinical and histopathological features of OL [16]. Following this proposal, thin and flat OL would show hyperkeratosis, acanthosis, and occasional lymphocytes. Thick fissured OL lesions would present, besides these microscopic alterations, mild to moderate dysplasia. The verrucous or granular OL would show irregular hyperkeratosis, drop-shaped rete ridges, a moderate amount of lymphocytes, and moderate to severe dysplasia. Finally, speckled OL and erythroplakia could show irregular hyperkeratosis, epithelial atrophy, numerous lymphocytes, and severe dysplasia or carcinoma *in situ*.

A research group published a proposal of a staging system for OL, in which a clinical feature of the lesion would be taken into account [17,18]. The lesion would be classified into one of the four stages (I, II, III, or IV), according to the association between two parameters. The first characteristic to be evaluated would be the size of the lesion, with four possible categories (L1, L2, L3, and Lx). The second item concerned the histopathological presentation, focused on the presence of dysplasia, with three possible categories (P0, P1, and Px). Therefore, a somehow similar strategy to that of TNM (extent of the tumor (T), spread to regional lymph nodes (N), and distant metastasis (M)) for oral cancer would be used to stage OL, and the authors intended to promote a uniform reporting of treatment or management of OL lesions.

### **1.3. Evolution and prognosis**

28 Carcinogenesis

different sites of the oral mucosa.

dysplasia or carcinoma *in situ*.

management of OL lesions.

must be performed to obtain the histopathological diagnosis [12].

**Figure 6.** Proliferative verrucous leukoplakia. This elderly woman presented multiple lesions affecting

Many lesions must be excluded before formulating a diagnostic hypothesis of OL, such as chemical injuries, candidiasis, frictional lesion, hairy leukoplakia, leukoedema, linea alba, nicotinic stomatitis, among others [4,10]. Because of variable clinical presentation of the potentially malignant lesions, when a provisional clinical diagnosis of OL is made, a biopsy

The microscopic presentation of OL can vary from slightly hyperkeratotic epithelium to lesions with severe dysplasia [13]. The frequencies of dysplastic or malignant alterations in OL vary from 15.6% to 39.2%, and a rate of 19.9% was found in a retrospective study of 3,300 white lesions of the oral cavity [14]. Epithelial dysplasia is characterized by the presence of architectural alteration and cytological atypia, and can be graded as mild, moderate, severe, and carcinoma *in situ* [10]. Nevertheless, there is a notable inter- and intra-observer variation in the interpretation and classification of dysplasia, which makes this method subjective with low reproducibility [12,15]. Thus, many different grading systems have been suggested to enhance the reproducibility and the predictive value for malignant transformation of OL. It has been suggested a possible correlation between clinical and histopathological features of OL [16]. Following this proposal, thin and flat OL would show hyperkeratosis, acanthosis, and occasional lymphocytes. Thick fissured OL lesions would present, besides these microscopic alterations, mild to moderate dysplasia. The verrucous or granular OL would show irregular hyperkeratosis, drop-shaped rete ridges, a moderate amount of lymphocytes, and moderate to severe dysplasia. Finally, speckled OL and erythroplakia could show irregular hyperkeratosis, epithelial atrophy, numerous lymphocytes, and severe

A research group published a proposal of a staging system for OL, in which a clinical feature of the lesion would be taken into account [17,18]. The lesion would be classified into one of the four stages (I, II, III, or IV), according to the association between two parameters. The first characteristic to be evaluated would be the size of the lesion, with four possible categories (L1, L2, L3, and Lx). The second item concerned the histopathological presentation, focused on the presence of dysplasia, with three possible categories (P0, P1, and Px). Therefore, a somehow similar strategy to that of TNM (extent of the tumor (T), spread to regional lymph nodes (N), and distant metastasis (M)) for oral cancer would be used to stage OL, and the authors intended to promote a uniform reporting of treatment or OL may persist unchanged, progress, regress, or even disappear [9]. The malignant transformation risk varies from 3.6% to 36.0%, and some features such as the presence and degree of dysplasia, female gender, time of duration, non-smoker patient, location at floor of the mouth or tongue, size higher than 200mm², and non-homogeneous type, seem to be associated with a worse prognosis [10,19-22]. Surgical excision, cryosurgery, laser surgery, topical or systemic retinoids, therapy with mouth rinses with attenuated adenovirus, and photodynamic therapy are possible therapeutics [10,13,23]. Recurrence rates are highly variable among studies, from 0 to 30.0% [10].

Many efforts have attemped to identify molecular markers to predict cancer development in OL. However, the presence and degree of epithelial dysplasia in OL is yet regarded as the most relevant indicator of progression and prognosis, influencing the management of the patients [9,10,12].

### **2. Grading oral epithelial dysplasia**

The term "dysplasia" is generally employed in the sense of a disordered development [24]. In a stratified squamous epithelium, architectural disturbances affecting normal maturation and stratification may occur. When such alterations are accompanied by cytological atypia, which can be detected as variations in the size and shape of the keratinocytes, the term "dysplasia" is applied [7,12].

Despite many efforts towards new evaluative methods, the histological analysis is still the most useful method for grading epithelial dysplasia in OL [12].

### **2.1. Relevance**

The concept of a sequential developmental process from a normal epithelium through a dysplasia, ending in a carcinoma, was introduced from studying pathological changes in the uterine cervix [24]. It is believed that through this process there is an accumulation of genetic and epigenetic alterations and more and more layers of the epithelium are progressively involved, until it is replaced by atypical cells in full length. It is considered that the more severe the degree of dysplasia, the greater the likelihood of malignant transformation. Despite the imperfection of currently available systems, they remain essential, and the diagnosis is a prerequisite for the establishment of the treatment that provides the best prognosis [12,24].

### **2.2. Proposed systems**

The elaboration of a classifying system is not a simple issue as the system may be, above all, an indicator of prognosis, guiding or at least helping in the establishment of the best treatment. Moreover, it should be reproducible, reliable, and as simple and objective as possible. Many classification schemes have been proposed over time, with variable

#### 30 Carcinogenesis

acceptance and employment. Herein, three of the most mentioned systems will first be discussed, followed by the recently suggested binary system.

Binary System of Grading Epithelial Dysplasia in Oral Leukoplakias 31

In 1997, the WHO published the "Histopathological Typing of Cancer and Precancer of the Oral Mucosa" and in the latest WHO's classification of Head and Neck Tumours, a grading system based on "thirds" was described [7,29]. This resembled the system described since

The WHO's classification system is truly widely accepted among pathologists. However, it is not able to reflect the clinical behaviour of every single lesion and does not provide a clear therapeutic guideline to clinicians [24]. Moreover, in spite of its wide acceptance, this system presents great variability and low reproducibility [10,12]. According to it, lesions are allocated into categories considering firstly the architectural features, followed by

**Figure 7.** Left: specimen of an oral leukoplakia showing irregular epithelial stratification. Right: Normal

*2.2.3. World health organization* 

cytological alterations [7].

Drop-shaped rete ridges

the 1970's for lesions of the uterine cervix [24].

The architectural features that should be addressed are:

Premature keratinisation in single cells (dyskeratosis) (Figure 9)

 Irregular epithelial stratification (Figure 7) Loss of polarity of basal cells (Figure 8)

oral mucosa. Hematoxylin and eosin, 200X magnification.

 Increased number of mitotic figures Abnormally superficial mitoses

Keratin pearls within rete pegs

### *2.2.1. Squamous intraepithelial neoplasia (SIN)*

This classification is a modification of a previously suggested system for cervical premalignant lesions, named cervical intraepithelial neoplasia [25]. After that, this concept has been adopted and extended to other sites, including oral mucosa, named "oral intraepithelial neoplasia" [12,26]. The term squamous intraepithelial neoplasia is also used to encompass all sites of the upper aerodigestive tract [26]. However, there is no evidence that many of the potentially malignant lesions of the oral mucosa are committed on a path to malignancy. Moreover, the SIN terminology would not clarify the knowledge concerning this issue, which would not justify replacing the widely accepted concept of dysplasia. Additionally, the WHO consensus group did not favour this system [12].

According to this system, lesions are classified as:


### *2.2.2. Ljubljana classification of squamous intraepithelial lesions*

This classification system was proposed by laryngeal pathologists in 1971 and additionally formulated in 1997 by a Working Group of the European Society of Pathology [27,28]. Very detailed criteria have been published and this system is more complex than the concept of dysplasia, so that even experienced pathologists would require time to adapt to it. Moreover, despite some publications, the usefulness of this grading system for oral lesions is doubtful [12,24].

Briefly, lesions are classified into four groups according to this system:


The first two degrees are considered mainly benign lesions, showing minimum risk for malignant transformation. The third degree would be a potentially malignant lesion, and the last one is actually considered a malignant lesion already. Additionally, the "atypical hyperplasia" and "carcinoma *in situ*" degrees are divided into basal cell type and spinous cell type [12,24].

### *2.2.3. World health organization*

30 Carcinogenesis

acceptance and employment. Herein, three of the most mentioned systems will first be

This classification is a modification of a previously suggested system for cervical premalignant lesions, named cervical intraepithelial neoplasia [25]. After that, this concept has been adopted and extended to other sites, including oral mucosa, named "oral intraepithelial neoplasia" [12,26]. The term squamous intraepithelial neoplasia is also used to encompass all sites of the upper aerodigestive tract [26]. However, there is no evidence that many of the potentially malignant lesions of the oral mucosa are committed on a path to malignancy. Moreover, the SIN terminology would not clarify the knowledge concerning this issue, which would not justify replacing the widely accepted concept of dysplasia.

This classification system was proposed by laryngeal pathologists in 1971 and additionally formulated in 1997 by a Working Group of the European Society of Pathology [27,28]. Very detailed criteria have been published and this system is more complex than the concept of dysplasia, so that even experienced pathologists would require time to adapt to it. Moreover, despite some publications, the usefulness of this grading system for oral lesions

Basal/parabasal cell hyperplasia or abnormal hyperplasia, considered essentially a basal

Atypical hyperplasia, also named risky hyperplasia, shows epithelial stratification, but

 Carcinoma *in situ*, characterized by loss of stratification throughout epithelium, but three to five layers of compressed cells may be present on the surface. Also, there is

The first two degrees are considered mainly benign lesions, showing minimum risk for malignant transformation. The third degree would be a potentially malignant lesion, and the last one is actually considered a malignant lesion already. Additionally, the "atypical hyperplasia" and "carcinoma *in situ*" degrees are divided into basal cell type and spinous

discussed, followed by the recently suggested binary system.

Additionally, the WHO consensus group did not favour this system [12].

SIN 3, would combine severe dysplasia and carcinoma *in situ*

*2.2.2. Ljubljana classification of squamous intraepithelial lesions* 

Briefly, lesions are classified into four groups according to this system: Simple hyperplasia, which is an increase in the stratum spinosum

*2.2.1. Squamous intraepithelial neoplasia (SIN)* 

According to this system, lesions are classified as:

marked atypia and mitotic abnormalities

 SIN 1, would be similar to mild dysplasia SIN 2, would be similar to moderate dysplasia

is doubtful [12,24].

cell hyperplasia

with atypia

cell type [12,24].

In 1997, the WHO published the "Histopathological Typing of Cancer and Precancer of the Oral Mucosa" and in the latest WHO's classification of Head and Neck Tumours, a grading system based on "thirds" was described [7,29]. This resembled the system described since the 1970's for lesions of the uterine cervix [24].

The WHO's classification system is truly widely accepted among pathologists. However, it is not able to reflect the clinical behaviour of every single lesion and does not provide a clear therapeutic guideline to clinicians [24]. Moreover, in spite of its wide acceptance, this system presents great variability and low reproducibility [10,12]. According to it, lesions are allocated into categories considering firstly the architectural features, followed by cytological alterations [7].

The architectural features that should be addressed are:


**Figure 7.** Left: specimen of an oral leukoplakia showing irregular epithelial stratification. Right: Normal oral mucosa. Hematoxylin and eosin, 200X magnification.

 **Figure 10.** Left: in this specimen, it can be noticed anisonucleosis, anisocytosis, nuclear and cellular pleomorphism, increased nuclear size, increased nuclear-cytoplasm ratio, and increased number and size of nucleoli of keratinocytes in an oral leukoplakia. Right: normal keratinocytes. Hematoxylin and

**Figure 11.** Keratinocytes exhibiting atypical mitotic figures (arrows). Hematoxylin and eosin, 400X

The observation of these alterations should be done considering the epithelium divided into "thirds". Accordingly, lesions should be classified into five categories, as described below:

1. Hyperplasia (Figure 13): describes a lesion showing an increase in cell number in the spinous layer and/or in the basal/parabasal cell layers. There is regular stratification and

2. Mild dysplasia (Figure 14): architectural disturbance only in the lower third of the

eosin, 1000X magnification.

magnification.

no cellular atypia.

epithelium with cytological atypia.

**Figure 8.** Loss of polarity of basal cells in a photomicrograph of an oral leukoplakia specimen (arrows). Hematoxylin and eosin, 400X magnification.

**Figure 9.** A keratinocyte showing dyskeratosis. Hematoxylin and eosin, 1000X magnification.

The cytological alterations to be observed are as follows:


32 Carcinogenesis

Hematoxylin and eosin, 400X magnification.

**Figure 8.** Loss of polarity of basal cells in a photomicrograph of an oral leukoplakia specimen (arrows).

**Figure 9.** A keratinocyte showing dyskeratosis. Hematoxylin and eosin, 1000X magnification.

 Nuclear pleomorphism: abnormal variation in nuclear shape (Figure 10) Cellular pleomorphism: abnormal variation in cell shape (Figure 10) Anisonucleosis: abnormal variation in nuclear size (Figure 10) Anisocytosis: abnormal variation in cell size (Figure 10)

The cytological alterations to be observed are as follows:

Increased nuclear size (Figure 10)

Atypical mitotic figures (Figure 11)

Increased nuclear-cytoplasm ratio (Figure 10)

Increased number and size of nucleoli (Figure 10 and 12)

**Figure 10.** Left: in this specimen, it can be noticed anisonucleosis, anisocytosis, nuclear and cellular pleomorphism, increased nuclear size, increased nuclear-cytoplasm ratio, and increased number and size of nucleoli of keratinocytes in an oral leukoplakia. Right: normal keratinocytes. Hematoxylin and eosin, 1000X magnification.

**Figure 11.** Keratinocytes exhibiting atypical mitotic figures (arrows). Hematoxylin and eosin, 400X magnification.

The observation of these alterations should be done considering the epithelium divided into "thirds". Accordingly, lesions should be classified into five categories, as described below:


#### 34 Carcinogenesis

3. Moderate dysplasia (Figure 15): architectural disturbance extending into the middle third of the epithelium is the initial criteria, but the degree of cytological atypia may require upgrading it to "severe".

Binary System of Grading Epithelial Dysplasia in Oral Leukoplakias 35

**Figure 13.** Sample of an oral leukoplakia showing hyperplasia. Note an increased number of basal / parabasal cells and a hyperkeratotic surface. Regular stratification is observed, as well as no cytological

**Figure 14.** These specimens of oral leukoplakia exhibited mild dysplasia. Observe architectural disturbances affecting the lower third of the epithelium and cytological atypia. Hematoxylin and eosin,

atypia. Hematoxylin and eosin, 200X magnification.

200X magnification (left), 400X magnification (right).


**Figure 12.** Oral leukoplakia specimen exhibiting increased number and size of nucleoli (arrows). AgNOR staining, 200X magnification.

34 Carcinogenesis

require upgrading it to "severe".

of the epithelium, with cytological atypia.

abnormal superficial mitoses are common.

3. Moderate dysplasia (Figure 15): architectural disturbance extending into the middle third of the epithelium is the initial criteria, but the degree of cytological atypia may

4. Severe dysplasia (Figure 16): architectural disturbance affecting greater than two thirds

5. Carcinoma *in situ* (Figure 17): theorically, indicates that malignant transformation has occurred but invasion has not. Full or almost full thickness architectural disturbance in viable cellular layers with pronounced cellular atypia. Atypical mitotic figures and

**Figure 12.** Oral leukoplakia specimen exhibiting increased number and size of nucleoli (arrows).

AgNOR staining, 200X magnification.

**Figure 13.** Sample of an oral leukoplakia showing hyperplasia. Note an increased number of basal / parabasal cells and a hyperkeratotic surface. Regular stratification is observed, as well as no cytological atypia. Hematoxylin and eosin, 200X magnification.

**Figure 14.** These specimens of oral leukoplakia exhibited mild dysplasia. Observe architectural disturbances affecting the lower third of the epithelium and cytological atypia. Hematoxylin and eosin, 200X magnification (left), 400X magnification (right).

**Figure 17.** An oral leukoplakia that showed microscopic features of carcinoma *in situ*: architectural disturbances are observed in the full thickness of epithelium with pronounced cellular atypia. No

As mentioned above, the proposed systems to grade epithelial dysplasia published so far, including the WHO's proposal, showed some shortcomings, such as great variability and low reproducibility. Thus, studies concerning classification criteria are being performed, looking for an enhancement for grading epithelial dysplasia in OL. In 2006, a new binary system was proposed, which could be a more feasible and reliable tool for grading epithelial dysplasia in OL [15]. According to this system, pathologists would observe the same morphological criteria used in the WHO classification, but lesions would be classified as low-risk OL (former "no/ mild / questionable" dysplasia) or as high-risk OL (former "moderate/ severe" dysplasia) [15,10,12]. This would provide more reliable criteria upon

Interestingly, in 1988 the "Bethesda classification" for cervical cytopathology, already included only two grades [30]. According to this, lesions would be classified as low-grade squamous epithelial lesions, corresponding to former cervical intraepithelial neoplasia grade 1, and high-grade squamous epithelial lesions, corresponding to grades 2 and 3. This

After the publication of those papers on the binary system for grading epithelial dysplasia in OL, a study was performed with 218 patients with OL, from which 39 (17.9%) developed into cancer [31]. The authors reported that high-risk OL was associated with a 4.57-fold

superficial keratinisation can be observed. Hematoxylin and eosin, 200X magnification.

which to rely for the selection of patient treatment.

system has also been mentioned in some reports for oral lesions [26].

*2.2.4. Binary system* 

**Figure 15.** Microscopic presentation of an oral leukoplakia showing moderate dysplasia. Architectural disturbances extending into the middle third of epithelium, along with cytological atypia. Hematoxylin and eosin, 100X magnification.

**Figure 16.** Histological section of oral leukoplakia exhibiting severe dysplasia. Architectural disturbances affecting greater than two thirds of the epithelium. Pronounced cytological atypia is evident. Hematoxylin and eosin, 100X magnification.

**Figure 17.** An oral leukoplakia that showed microscopic features of carcinoma *in situ*: architectural disturbances are observed in the full thickness of epithelium with pronounced cellular atypia. No superficial keratinisation can be observed. Hematoxylin and eosin, 200X magnification.

### *2.2.4. Binary system*

36 Carcinogenesis

and eosin, 100X magnification.

**Figure 15.** Microscopic presentation of an oral leukoplakia showing moderate dysplasia. Architectural disturbances extending into the middle third of epithelium, along with cytological atypia. Hematoxylin

**Figure 16.** Histological section of oral leukoplakia exhibiting severe dysplasia. Architectural disturbances affecting greater than two thirds of the epithelium. Pronounced cytological atypia is

evident. Hematoxylin and eosin, 100X magnification.

As mentioned above, the proposed systems to grade epithelial dysplasia published so far, including the WHO's proposal, showed some shortcomings, such as great variability and low reproducibility. Thus, studies concerning classification criteria are being performed, looking for an enhancement for grading epithelial dysplasia in OL. In 2006, a new binary system was proposed, which could be a more feasible and reliable tool for grading epithelial dysplasia in OL [15]. According to this system, pathologists would observe the same morphological criteria used in the WHO classification, but lesions would be classified as low-risk OL (former "no/ mild / questionable" dysplasia) or as high-risk OL (former "moderate/ severe" dysplasia) [15,10,12]. This would provide more reliable criteria upon which to rely for the selection of patient treatment.

Interestingly, in 1988 the "Bethesda classification" for cervical cytopathology, already included only two grades [30]. According to this, lesions would be classified as low-grade squamous epithelial lesions, corresponding to former cervical intraepithelial neoplasia grade 1, and high-grade squamous epithelial lesions, corresponding to grades 2 and 3. This system has also been mentioned in some reports for oral lesions [26].

After the publication of those papers on the binary system for grading epithelial dysplasia in OL, a study was performed with 218 patients with OL, from which 39 (17.9%) developed into cancer [31]. The authors reported that high-risk OL was associated with a 4.57-fold

#### 38 Carcinogenesis

increased risk for malignant transformation, compared with low-risk OL. Those authors suggested that high-risk dysplasia would be a significant indicator for evaluating malignant transformation risk in OL.

Binary System of Grading Epithelial Dysplasia in Oral Leukoplakias 39

After that, we conducted a comparative immunohistochemical and histochemical study encompassing those same samples of OL [34]. At that time, the hMLH1 immunoexpression was compared to p53 immunoexpression (Figure 19) and AgNOR counting. Thus, we could assess the possible association between a protein of DNA repair, a tumor suppressor protein, and the cellular proliferation in OL with different degrees of dysplasia, *i.e.* no, mild, moderate, and severe. We concluded that it seemed reasonable that other molecular alterations may take place in early phases of carcinogenesis, related to tumor suppressor

 **Figure 19.** Immunoexpression of p53 in oral leukoplakia with mild dysplasia (left) and severe dysplasia

Recently, we decided to reevaluate those previous results in the light of the binary system to grade epithelial dysplasia in OL [35]. Therefore, we grouped OL formerly classified as showing no and mild dysplasia into low-risk lesions. Accordingly, OL previously classified as having moderate and severe dysplasia were defined as high-risk lesions. After that, we performed the statistical analyses again. Our findings showed statistically significant differences for hMLH1, p53, and AgNOR indexes between low- and high-risk OL. This suggests that the biological processes linked to the impairment of those proteins remain enhancing from low-risk OL to high-risk OL. Thus, the use of the binary system would give support to a more reliable clinical approach involving the removal of high-risk OL. Moreover, we could speculate that OL classified as low-risk may be reasonably named this way, since comparisons between hMLH1 and AgNOR indexes of this group and normal oral mucosa did not reach statistical significance, despite their different median values.

Apart from those investigations pointing towards an adaptation of the WHO classification to a binary system, there are also other recently published papers on different proposals to

As reviewed before, the Japanese Society for Oral Pathology reported a definition of carcinoma *in situ* and proposes the term "oral intraepithelial neoplasia", which in turn could

genes, like p53, as well as modifications in proliferation rates.

(right). Streptoavidin-biotin, 100X magnification.

*2.2.5. Other proposals* 

evaluate epithelial dysplasia.

Subsequently, the same research group published a study in which they identified significant risk factors for malignant transformation in a long-term follow-up cohort of patients with oral epithelial dysplasia [32]. Of the 138 patients with histologically confirmed oral dysplasia, 115 had OL and 23 had lichen planus. From these 138 lesions, 37 (26.8%) developed into cancer and the "high-risk" degree of dysplasia was an independent risk factor for transformation. Moreover, high-risk degree of dysplasia was associated with a 2.78-fold increased risk of transformation compared with low-risk degree. The authors then suggest the utilization of high-risk dysplasia as a significant indicator for evaluating malignant transformation risk in patients with potentially malignant lesions. According to them, this would also help guiding treatment in clinical practice. In spite of these great achievements, it must be mentioned that malignancy also developed in some patients previously presenting low-risk potentially malignant lesions [32].

In our first paper [33], we investigated the immunoexpression of hMLH1 (a protein of the mismatch repair system) (Figure 18) in OL with different degrees of dysplasia, according to the WHO grading system. We evaluated lesions showing no, mild, moderate, and severe dysplasia, and we found that the greater difference in the hMLH1 immunoexpression was detected comparing OL with mild and moderate dysplasia, with decreasing indexes. Therefore, we suggested that this result would be in accordance with the proposed binary system of grading dysplasia in OL, as the morphological dysplastic alterations observed in routinely stained slides may be related to molecular changes.

**Figure 18.** Immunoexpression of hMLH1 in oral leukoplakia showing no dysplasia (left) and severe dysplasia (right). Advance HRP, 200X magnification.

After that, we conducted a comparative immunohistochemical and histochemical study encompassing those same samples of OL [34]. At that time, the hMLH1 immunoexpression was compared to p53 immunoexpression (Figure 19) and AgNOR counting. Thus, we could assess the possible association between a protein of DNA repair, a tumor suppressor protein, and the cellular proliferation in OL with different degrees of dysplasia, *i.e.* no, mild, moderate, and severe. We concluded that it seemed reasonable that other molecular alterations may take place in early phases of carcinogenesis, related to tumor suppressor genes, like p53, as well as modifications in proliferation rates.

**Figure 19.** Immunoexpression of p53 in oral leukoplakia with mild dysplasia (left) and severe dysplasia (right). Streptoavidin-biotin, 100X magnification.

Recently, we decided to reevaluate those previous results in the light of the binary system to grade epithelial dysplasia in OL [35]. Therefore, we grouped OL formerly classified as showing no and mild dysplasia into low-risk lesions. Accordingly, OL previously classified as having moderate and severe dysplasia were defined as high-risk lesions. After that, we performed the statistical analyses again. Our findings showed statistically significant differences for hMLH1, p53, and AgNOR indexes between low- and high-risk OL. This suggests that the biological processes linked to the impairment of those proteins remain enhancing from low-risk OL to high-risk OL. Thus, the use of the binary system would give support to a more reliable clinical approach involving the removal of high-risk OL. Moreover, we could speculate that OL classified as low-risk may be reasonably named this way, since comparisons between hMLH1 and AgNOR indexes of this group and normal oral mucosa did not reach statistical significance, despite their different median values.

### *2.2.5. Other proposals*

38 Carcinogenesis

transformation risk in OL.

increased risk for malignant transformation, compared with low-risk OL. Those authors suggested that high-risk dysplasia would be a significant indicator for evaluating malignant

Subsequently, the same research group published a study in which they identified significant risk factors for malignant transformation in a long-term follow-up cohort of patients with oral epithelial dysplasia [32]. Of the 138 patients with histologically confirmed oral dysplasia, 115 had OL and 23 had lichen planus. From these 138 lesions, 37 (26.8%) developed into cancer and the "high-risk" degree of dysplasia was an independent risk factor for transformation. Moreover, high-risk degree of dysplasia was associated with a 2.78-fold increased risk of transformation compared with low-risk degree. The authors then suggest the utilization of high-risk dysplasia as a significant indicator for evaluating malignant transformation risk in patients with potentially malignant lesions. According to them, this would also help guiding treatment in clinical practice. In spite of these great achievements, it must be mentioned that malignancy also developed in some patients

In our first paper [33], we investigated the immunoexpression of hMLH1 (a protein of the mismatch repair system) (Figure 18) in OL with different degrees of dysplasia, according to the WHO grading system. We evaluated lesions showing no, mild, moderate, and severe dysplasia, and we found that the greater difference in the hMLH1 immunoexpression was detected comparing OL with mild and moderate dysplasia, with decreasing indexes. Therefore, we suggested that this result would be in accordance with the proposed binary system of grading dysplasia in OL, as the morphological dysplastic alterations observed in

**Figure 18.** Immunoexpression of hMLH1 in oral leukoplakia showing no dysplasia (left) and severe

previously presenting low-risk potentially malignant lesions [32].

routinely stained slides may be related to molecular changes.

dysplasia (right). Advance HRP, 200X magnification.

Apart from those investigations pointing towards an adaptation of the WHO classification to a binary system, there are also other recently published papers on different proposals to evaluate epithelial dysplasia.

As reviewed before, the Japanese Society for Oral Pathology reported a definition of carcinoma *in situ* and proposes the term "oral intraepithelial neoplasia", which in turn could

#### 40 Carcinogenesis

be classified as differentiated and basaloid type [24]. The main difference between them would be the presence of keratinisation in the epithelium surface in the differentiated type. Additionally, some authors analyzed individual features of dysplasia in oral lesions and determined the reproducibility of scoring each one [36]. They suggested that those data might be used to improve or to develop simpler routine diagnostic methods.

Binary System of Grading Epithelial Dysplasia in Oral Leukoplakias 41

[6] Kramer IR, Lucas RB, Pindborg JJ, Sobin LH. Definition of leukoplakia and related lesions: an aid to studies on oral precancer. Oral Surgery, Oral Medicine, Oral

[7] World Health Organization. Tumours of the Oral Cavity and Oropharynx. In: Barnes L, Eveson JW, Reichart P, Sidransky D, editors. Pathology & genetics. Head neck tumors.

[8] Petti S. Pooled estimate of world leukoplakia prevalence: a systematic review. Oral

[9] Napier SS, Speight PM. Natural history of potentially malignant oral lesions and conditions: an overview of the literature. Journal of Oral Pathology & Medicine

[10] van der Waal I. Potentially malignant disorders of the oral and oropharyngeal mucosa; terminology, classification and present concepts of management. Oral Oncology

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### **3. Conclusion**

To date, no system is free of presenting failures in identifying those OL prone to evolve to oral squamous cell carcinoma. Furthermore, the reproducibility and subjectivity are still key points to be addressed. Therefore, robust research on the predictive value, relevance, applicability, and feasibility of the binary system for grading epithelial dysplasia are clearly warranted. Such research should aim to establish of a reliable and reproducible method that, above all, could provide a better and less empiric clinical management of the patient.

### **Author details**

Maria Auxiliadora Vieira do Carmo and Patrícia Carlos Caldeira *School of Dentistry, Universidade Federal de Minas Gerais, Brazil* 

### **Acknowledgement**

The authors acknowledge National Council of Technological and Scientific Development (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for the received grants and research funding.

### **4. References**


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40 Carcinogenesis

**3. Conclusion** 

**Author details** 

**Acknowledgement** 

grants and research funding.

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Medicine 2007;36(10):575-80.

**4. References** 

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To date, no system is free of presenting failures in identifying those OL prone to evolve to oral squamous cell carcinoma. Furthermore, the reproducibility and subjectivity are still key points to be addressed. Therefore, robust research on the predictive value, relevance, applicability, and feasibility of the binary system for grading epithelial dysplasia are clearly warranted. Such research should aim to establish of a reliable and reproducible method that,

The authors acknowledge National Council of Technological and Scientific Development (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for the received

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Maria Auxiliadora Vieira do Carmo and Patrícia Carlos Caldeira *School of Dentistry, Universidade Federal de Minas Gerais, Brazil* 

might be used to improve or to develop simpler routine diagnostic methods.


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**Section 2** 

**Cancer Development and Progression** 


**Cancer Development and Progression** 

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**Chapter 3** 

© 2013 Castillo, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Human Papillomavirus and Carcinogenesis** 

Infectious agents are suspected to play causal roles in a variety of human malignancies. The public health impact of the oncogenic effects of these infections is considerable. Infection is estimated to be responsible for about 17.8% of all incident cases of cancer worldwide, accounting for 26.3% of all malignancies in economically developing countries and 7.7% in

The evaluation of causality for these infectious agents as human carcinogens is difficult given their ubiquitous nature, the substantial length of time between infection and the cancer event, the nature of cofactors, and the rarity of malignancy among those infected. Thus, a central problem for the epidemiologist is to define the natural history of infection and to identify those factors that are related to the development of cancer. Hence, informative biomarkers of the agent (such as viral load), of the host (such as abnormal antibody pattern), and of other oncogenic exposures (such as tobacco use) are required for

Case-control studies have now recognized that human papilloma virus (HPV) infection in the oral cavity is a strong risk factor for head and neck squamous cell carcinoma (HNSCC), and mostly for oropharyngeal cancer. The risk is increased for high-risk HPV-16 infection [3]. Therefore, HPV infection in the oral cavity has important health consequences, requiring more studies about these aspects to clarify the implications of a diagnosis of HPV in the oral

HPVs are nonenveloped icosahedral viruses with a diameter of 55 nm, belonging to the *papillomaviridae* family. This epitheliotropic virus has 72 capsomers enclosing an 8 kbp-long

and reproduction in any medium, provided the original work is properly cited.

understanding the viral-human interactions and for developing interventions [2].

**in the Upper Aero-Digestive Tract** 

Additional information is available at the end of the chapter

Andrés Castillo

**1. Introduction** 

developed countries [1].

cavity and HNSCC.

**2. Human papillomavirus (HPV)** 

http://dx.doi.org/10.5772/54800
