A COMPARISON OF CANCER HISTORIES AND MANAGEMENT OF WOMEN CARRIERS OF CHEK2 TRUNCATING AND MISSENSE MUTATIONS

Transcription

1 A COMPARISON OF CANCER HISTORIES AND MANAGEMENT OF WOMEN CARRIERS OF CHEK2 TRUNCATING AND MISSENSE MUTATIONS A Thesis Presented to the Faculty of California State University, Stanislaus In Partial Fulfillment of the Requirements for the Degree of Master of Genetic Counseling By Sojin Durrant April 2018

2 CERTIFICATION OF APPROVAL A COMPARISON OF CANCER HISTORIES AND MANAGEMENT OF WOMEN CARRIERS OF CHEK2 TRUNCATING AND MISSENSE MUTATIONS by Sojin Durrant Signed Certification of Approval page is on file with the University Library Dr. Catherine Bethan Powell, MD Gynecological Oncologist Kaiser Permanente San Francisco Medical Center Date Christine Kobelka, MS, LCGC Genetic Counselor, Genetics Department Kaiser Permanente San Francisco Medical Center Date Dr. Janey Youngblom, PhD, MS Professor of Genetics, Biology Department CSU Stanislaus Date

3 2018 Sojin Durrant ALL RIGHTS RESERVED

4 DEDICATION To Appa for pushing me and inspiring me to go further, overcome challenges and achieve more. To Umma for your selfless support and always being on my side. iv

5 ACKNOWLEDGEMENTS I would like to thank my committee members, Dr. Bethan Powell, Christine Kobelka, and Janey Youngblom, for guiding me with this project and providing me with thoughtful feedback throughout. I really appreciated the support of Laurie Nemzer and the Kaiser Permanente genetics group; thank you for answering my many questions and for the enlightening discussions. v

6 TABLE OF CONTENTS PAGE Dedication... Acknowledgements... List of Tables... iv v vii List of Figures... viii Abstract... ix Introduction... 1 Multi-gene Panel Testing... 1 Checkpoint Kinase The Founder Mutations... 4 c.1100delc Truncating Mutation... 5 p.i157t Missense Mutation... 6 Purpose of Study... 7 Methods... 9 Study Population... 9 Genetic Testing Pedigree Data Statistical Data Analysis Results Personal History of Cancer Breast Cancer Characteristic Family History of Breast Cancer Family History of All Cancer Breast Cancer Management Colon Cancer Management Other Cancer Management Discussion Personal History of Cancer Family History of Cancer vi

7 Observations of CHEK2 truncating and missense mutation carriers Management Study Limitation Conclusion Future studies References vii

8 LIST OF TABLES TABLE PAGE 1. Personal history of cancer comparison Breast cancer clinical characteristic comparison Family history of breast cancer comparison Family history all cancer comparison Breast cancer management comparison Colon cancer management comparison Other cancer management comparison viii

9 LIST OF FIGURES FIGURE PAGE 1. Distribution of cancer in relatives Management of breast cancer in CHEK2 mutation positive patients ix

10 ABSTRACT Through Next Generation Sequencing (NGS) technology, multi-gene panel testing is now more clinically accessible. Multi-gene panels allow testing of high-risk genes, as well as other lower cancer risk genes such CHEK2, where the significance of the mutations is not fully understood. Prior research on CHEK2 has focused primarily on Northern and Eastern European founder mutations, and suggests a difference in cancer risk and phenotype between CHEK2 truncating mutation c.1100delc and missense mutation p.i157t. Our study tries to determine if there is a need for separate management guidelines for CHEK2 truncating mutation and missense mutation carriers. We explored whether the difference in risk and phenotype between the two founder mutations c.1100delc and p.i157t can be generalized to all CHEK2 truncating and missense mutations. We also examined if there were differences in the uptake of management options and utilization of surveillance or risk reducing surgery between CHEK2 truncating and missense mutation carriers. We retrospectively reviewed the Electronic Health Record (EHR) of 72 individuals with CHEK2 pathogenic, or likely pathogenic mutations. We examined both founder and nonfounder CHEK2 mutations from an ethnically diverse population. No significant difference in personal and family history of cancer was observed between truncating and missense CHEK2 mutation carriers. Also, there were no significant differences between management of breast cancer, colon cancer, and other cancers of CHEK2 truncating and missense mutation carriers. These findings suggest the difference in x

11 cancer risk penetrance and phenotype between the two founder mutations c.1100delc and p.i157t cannot be generalized to all CHEK2 truncating and missense mutations. Also, despite the knowledge of p.i157t missense mutation carriers having lower risk, clinical recommendations were not adjusted accordingly, indicating a potential for overtreatment. Chart review identified cases of potential overtreatment for CHEK2 mutation carriers with respect to other cancers such as ovarian, kidney and thyroid cancers. xi

12 CHAPTER I INTRODUCTION Multi-gene Panel Testing The introduction of Next Generation Sequencing (NGS) technology allows for rapid simultaneous screening of a large number of genes at significantly lower cost. This has drastically increased clinical availability of multi-gene panel testing for hereditary breast and ovarian cancer (HBOC). Through the multi-gene panel testing, individuals have the potential to discover the underlying etiology for the hereditary cancer risk in their family. When a pathogenic mutation in such high risk genes as BRCA1 or BRCA2 is identified on multiplex panel testing, clear hereditary cancer risk, cancer screening and risk-reducing guidelines can be provided with confidence (Ellisen, et al., 2015). However, many of the other genes that are included in multigene panels are recent discoveries, and thought to be associated with moderate-risk or lower-risk of breast or ovarian cancer. When a pathogenic mutation is identified in one of these moderate to lower-risk genes, the significance of the result is not always clear since information on hereditary cancer risk, penetrance, and clinical utility can be more limited, and national guidelines may not exist or may be vague (Young, et al., 2016). Currently, the National Comprehensive Cancer Network (NCCN) Genetic/Familial High-Risk Assessment Guideline recommends screening with yearly breast MRI, in addition to mammogram, when lifetime breast cancer risk exceeds 20% (National Comprehensive Cancer Network, 2015). Moderate-risk genes 1

13 including ATM, CHEK2, PALB2, and NF1 are thought to have lifetime breast cancer risk close to or slightly exceeding 20%. Although the risk is significantly lower than that of high-risk genes, it is potentially still high enough to influence medical management of individuals (Young, et al., 2016). More research on cancer risk, penetrance, personal and family history is necessary to understand the moderate to lower risk genes and to help guide individuals make better clinical decisions based on these multi-gene panel testing results. Checkpoint Kinase 2 CHEK2 is one of the moderate-risk genes that is commonly included on multigene panels. Its susceptibility to hereditary cancer was first identified when several CHEK2 germline mutations were detected among patients and families meeting clinical criteria for Li-Fraumeni syndrome. Although evidence from different studies suggests that CHEK2 is not a predisposition gene to Li-Fraumeni syndrome, certain mutations in CHEK2 are associated with an increased risk of developing breast cancer (Nevanlinna and Bartek, 2006). According to the NCCN Genetic/Familial High-Risk Assessment Guideline, the cumulative lifetime risk for breast cancer in women with pathogenic mutations in the CHEK2 gene has been estimated to range from approximately 28% to 37%. Studies have shown that this risk is higher in CHEK2 mutation carriers with a strong family history of breast cancer than those who do not have family history of breast cancer (Cybulski, et al., 2011). In addition to breast cancer, pathogenic mutations of CHEK2 have been associated with other cancer risk including prostate cancer, and colorectal cancer (Cybulski, et al., 2011). A few 2

14 studies have suggested thyroid cancer, kidney cancer, and gastrointestinal cancer are also associated with CHEK2 pathogenic mutations. Results from these small studies have not been replicated by others, therefore more comprehensive studies are needed to confirm or disprove these results (Cybulski, et al., 2004), (Teodorczyk, et al., 2013). CHEK2 is likely a tumor suppressor gene. In response to DNA damage, the protein product, checkpoint kinase (CHEK2, Chk2) aids pathways including: DNA repair, cell cycle regulation and apoptosis in order to protect genomic integrity. The CHEK2 protein structure shows three characteristic domains; 1) an N-terminal SQ/TQ cluster regulatory domain containing ataxia telangiectasia-mutated (ATM) protein kinase phosphorylation sites for activation, 2) a fork head-associated (FHA) domain which participates in dynamic protein phosphoprotein interaction of CHEK2 during activation and signaling of DNA damage, and 3) a serine/threonine kinase domain (Nevanlinna & Bartek, 2006). CHEK2 gene is activated by the ATM protein and then reacts with other proteins such as BRCA1 or BRCA2, CDC25A, p53 protein, NEK6, and transcription factor FOXM1 to regulate cell division. CHEK2 is essential for cell cycle regulation and its abnormal expression could thus lead to abnormal cell growth and ultimately cancer (Apostulou & Papasotiriou, 2017). Mutations found in the germline CHEK2 gene can cause abnormal functioning of the Chk2 protein. However, depending on the type of mutation, the effects of the mutation on the protein function may vary. Truncating and missense mutations are two classifications of mutations of CHEK2 associated with an increased risk of 3

15 cancer. Protein truncating mutations are a type of DNA variation that disrupts normal protein synthesis. Typically, the proteins are not expressed, or incompletely expressed, which leads to loss of the protein function. With missense mutations, a single nucleotide change results in substitution of a different amino acid. The substitution can happen anywhere in the three domains, and the effect of the mutation varies substantially depending on the location of the substitution or the type of amino acid that is replaced. The mutation may be silent with no effect, may disable ATM activation, or disrupt the interaction between other proteins in chk2. Typically, it is difficult to accurately predict the effects of a missense mutation on its protein function, and the effects could range anywhere from benign to dominant negative in which the gene products are altered to interfere with the function of normal gene products. The Founder Mutations The studies investigating the association between breast cancer risk and CHEK2 genes are primarily based on four common founder mutations (c.1100delc, c G > A, EX8_9del, and p.i157t) which were identified in Eastern and Northern European population studies. The c.1100delc carrier frequency is highest (over 1%) in the Netherlands and Finland (Muranen, et al., 2016), c G > A and EX8_9del are found mainly in the Slavic populations of Poland (Cybulski, et al., 2011), and p.i157t is most frequent in Finland and Poland (about 5%) (Nevanlinna & Bartek, 2006). It has been estimated that the three founder truncating mutations c.1100delc, c G > A and EX8_9del in combination on average confers about 4

16 a 2.3 fold increased risk of breast cancer. Whereas, p.i157t, the missense mutation has been shown to have less impact on breast cancer risk conferring about a 1.4 fold increased risk (Tavtigian & Chenevix-Trench, 2014). Non-founder mutations are rarer, and incorporating larger numbers of mutation carriers in studies to further evaluate cancer risks and phenotypic presentation can be challenging. To this point, there is a significant lack of data on penetrance and phenotypic presentation in nonfounder mutation carriers. c.1100delc Truncating Mutation Of the four founder mutations, the most extensively studied mutation is the c.1100delc protein-truncating mutation. CHEK2 c.1100delc mutation carriers are at a two to three-fold increased risk for breast cancer compared to the general population (Weischer, et al., 2007). On average C.1100delC carriers develop breast cancer at an earlier age than non-carriers (Oldenburg, et al., 2003). Penetrance is known to be dependent on the family history of cancer. The risk for c.1100delc carriers with a strong family history of breast cancer is greater than c.1100delc carriers without family history of breast cancer (Gronwald, et al., 2009). Sisters and mothers of c.1100delc carriers with breast cancer, who are also c.1100delc carriers have cumulative incidence of 85% for breast cancer at an average age of 70 years (Adank, et al., 2013). c.1100delc carriers have an elevated risk for bilateral breast cancer (Nevanlinna and Bartek, 2006), and are also found to be associated with the more aggressive luminal B subtype tumors (Domagala, et al., 2012). Estrogen receptor (ER) positive, progesterone receptor (PR) positive tumors tend to be more 5

17 common among c.1100delc carriers, however they seem to have a worse prognosis of breast cancer (De Bock, et al., 2004). Male c.1100delc carriers have increased risk for prostate cancer (Cybulski, et al., 2004A) (Hale, et al., 2014). C.1100delC carriers may confer a lower risk or no increase in risk of colon cancer (Cybulski, et al., 2007). Gastric and thyroid cancer risks have been reported to be associated with c.1100delc mutation carriers (Leedom, et al., 2016). p.i157t Truncating Mutation The I157T missense mutation is located in the FHA domain of CHEK2 gene. The protein product of this mutation was observed to be defective in its ability to bind and to phosphorylate Cdc25Q and to bind p53 and BRCA1 (Cybulski, et al., 2007). Additionally, studies have suggested that the I157T protein may have a dominant negative effect by forming heterodimers with wild-type CHEK2. A dominant negative-effect was shown to influence the clinical presentation (Cybulski, et al., 2007). When compared to the general population, the I157T missense mutation presents about 1.5 fold increased risk of breast cancer, which is lower penetrance than the c.1100delc mutation (Han, et al., 2013). Studies have shown that p.i157t carriers also developed breast cancer at an earlier age (Cybulski, et al., 2006), and I157T carriers with family history of breast cancer were at a slightly higher 2 fold increased risk of breast cancer (Cybulski, et al., 2011). p.i157t mutation carriers appear to have a higher risk for ER positive breast tumors and may have an increased risk for lobular breast cancer (Domagala, et al., 2012). They appear to have a better prognosis than the c.1100delc carriers. Also, medullary and tubular histological types have been 6

18 seen more frequently among p.i157t carrier tumors (Muranen, et al., 2016). The p.i157t mutation has also been associated with increased risk of colon, kidney, prostate, and thyroid cancers (Cybulski, et al., 2004). In particular, p.i157t mutation carriers have a 1.5 fold increased risk of colon cancer risk (Cybulski, et al., 2007). Purpose of Study Despite there being a lack of a comprehensive understanding of CHEK2 mutations, an increasing number of CHEK2 mutation carriers are being reported through HBOC multi-gene panel testing. CHEK2 mutations have accounted for 15 33% of non-brca1/2 mutations identified in multiple HBOC multi-gene panel testing cohort studies (Minion, et al., 2015), (Tung, et al., 2015). Currently, for breast cancer risk and management the NCCN Genetic/Familial High-Risk Assessment Guideline recommends CHEK2 truncating mutation carriers pursue annual mammogram with consideration of tomosynthesis and consider breast MRI with contrast starting at age 40, or 5-10 years earlier than the youngest breast cancer diagnosis in the family. The guidelines recognize I157T as having lower risk for breast cancer but no specific recommendation for management is made. The primary objective of this study is to understand if CHEK2 missense mutations carriers should have different set of surveillance guidelines from the CHEK2 truncating mutation carriers. Both c.1100delc truncating and p.i157t missense mutations are thought to compromise the chk2 protein function. However, differences in penetrance, clinical presentation, and characteristics of the disease can be observed between c.1100delc truncating and p.i157t missense mutations. This 7

19 observation raises the question of whether the difference in phenotype seen between c.1100delc and p.i157t can be applied generally to all truncating and missense mutations and whether CHEK2 missense mutations are less penetrant in general than CHEK2 truncating mutations. The aim of this study is to gain a better understanding of how truncating and missense CHEK2 mutations impart cancer risk in individuals and their families by examining both founder and non-founder CHEK2 mutations found in an ethnically diverse population. This study will also investigate if there are differences seen in CHEK2 truncating and missense mutation carrier s uptake of management options and utilization of surveillance or risk reducing surgery. To our knowledge, no study has previously explored clinical management strategies and decisions in CHEK2 mutation carriers. We hope to further the understanding of the cancer risk associated with truncating and missense CHEK2 mutations, in order to help guide clinical guidelines for surveillance and preventative surgery. 8

20 CHAPTER II METHODS Study Population Kaiser Permanente Northern California (KPNC) is a large integrated health care system which cares for approximately 4 million members. Those members who are 18 or older, and who meet the KPNC internal guidelines that were developed by the regional cancer genetics task force based on the National Comprehensive Cancer Network (NCCN) guidelines for hereditary breast and ovarian cancer (HBOC) are offered multi-gene panel testing using the OvaNext panel of 24 genes (Ambry, Aliso Viejo CA). This test includes CHEK2 and other high and moderate penetrance cancer predisposition genes, as well as genes that are newly described. Between December 1, 2014 and March 31, 2016, 3926 members underwent multi-gene panel testing for HBOC. Of these, 84 individuals were found to have pathogenic CHEK2 mutations. Of this group, 12 had additional pathogenic mutations in a different gene and were therefore excluded from the study, as cancer risk would be difficult to assess. The objective of this study was to describe and compare the personal and family history of these 72 remaining CHEK2 carriers. Demographic information, genetic testing results, clinical histories, family histories and management decisions were reviewed retrospectively using the electronic health record (EHR). This study was approved by the IRB of Kaiser Permanente Northern California, (PI CB Powell, CN H approved ) and by the ethics board of California State University Stanislaus (Protocol # approved ). 9

21 Demographic information was collected from electronic record review and included the following variables: age, gender, ethnicity, marital status; parity, indication for genetic testing, whether they met NCCN and/or internal Kaiser Permanente criteria for HBOC genetic testing, personal history of cancer, age of onset, type of cancer, stage, histology, ER/PR/Her2neu status, presence of multiple primary cancer, presence of bilateral cancer; genetic testing results, variant classification and family cancer pedigree. In addition, the recommendations made by the Genetics Department for clinical care, including surveillance options and surgical options were recorded. Additionally, the actual surveillance, chemoprevention or surgery undertaken were all noted. Concordance of surveillance and surgery with guidelines was recorded, as well as new cancer diagnoses. Genetic Testing In the KPNC integrated health care system, care is guideline-based and standardized. If a woman meets KPNC guidelines to be offered HBOC testing, she will usually be offered OvaNext panel testing (Ambry, Aliso Viejo CA). Of the 72 women in this cohort, all underwent OvaNext panel testing. The 24 genes included in the panel were ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, DICER1, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, SMARCA4, STK11, and TP53. These genes were evaluated by next generation sequencing (NGS) or Sanger sequencing of all coding regions, and five bases into the 5 and 3 ends of all the introns and untranslated regions. Gross deletion/duplication analysis was performed for the coding regions and untranslated 10

22 regions using read-depth from NGS data with confirmatory multiplex ligationdependent probe amplification (MLPA) and/or targeted chromosomal microarray. Variants were thoroughly assessed and classified by a five-tier variant classification protocol (pathogenic mutation; variant likely pathogenic; variant of unknown significance; variant likely benign, and benign), which is based on published recommendations and guidelines by the American College of Medical Genetics and Genomics and the International Agency for Research on Cancer. Pedigree Data A cancer-focused pedigree was obtained as part of the evaluation process by genetic counselors at the five genetics departments in Kaiser Permanente Northern California; Oakland, San Francisco, Sacramento, San Jose, and Fresno. The information on the pedigree was reported by patients, and not all cancer types and ages of onset were confirmed. Statistical Data Analysis CHEK2 pathogenic and likely pathogenic mutation carriers were further classified as truncating or missense mutation carriers. The overall frequencies of truncating, missense, and all CHEK2 mutations were calculated. Phenotypes of truncating and missense mutation carriers were compared based on personal history of cancer, breast cancer characteristics, family history of breast cancer and other cancers, management of breast cancer, colon cancer, and other cancers. Descriptive statistics such as mean and percentile were used. Chi-square test or the Fisher s exact test (for values less than 5) were used when appropriate for categorical variables to 11

23 determine the significance of differences between the two groups. Analysis of difference in age of onset between the two groups was performed using the Independent t-test. For all statistical analysis, a P value of 0.05 or <0.05 was considered significant. 12

24 CHAPTER III RESULTS The overall frequency of CHEK2 positive in the multi-gene panel (OvaNext) cohort was 84/3926 (2.1%), including 12 individuals carrying additional mutations in non-chek2 genes. The final cohort included the 72/3926 (1.8%) individuals who had CHEK2 mutations and no pathogenic or likely pathogenic mutations in other genes. All but one subject was female. The majority were Caucasian (n=55, 76.4%). Other ethnicities included; Ashkenazi Jewish (n=10, 13.9%), Asian (n=2), Hispanic (n=1), Middle Eastern (n=1), and Mixed ethnicity (n=3) The majority of CHEK2 positive had met the NCCN guidelines for testing (n=71, 98.6%). Indication for testing were; personal history of cancer only (n=15, 20.8%), family history or cancer only (n=15, 20.8%), or both (n=42, 58.3%). Of the 72 CHEK2 mutation carriers, 43 (59.7%) were truncating mutation carriers, and 29 (40.3%) were missense mutation carriers. The most commonly detected truncating mutations were the two founder mutations c.1100delc, and EX8_9del which represented 67.4% of the total number of truncating mutations observed. The most commonly detected missense mutations were also the two founder mutations p.i157t and p.s428f, which represented 75.9% of the total number of truncating mutations observed. Personal History of Cancer The frequency of cancer diagnosis in individuals with CHEK2 mutations, and the types of cancer diagnosed are listed in Table 1. Among the 72 individuals with 13

25 CHEK2 mutations in this study, 58 (80.6%) individuals had a personal history of cancer, and 17 (23.6%) individuals had multiple primary cancers. The most common type of cancer was breast cancer (ductal carcinoma in situ included), which represented 82.9% (n=68, mean age of diagnosis: 50 years) of the total number of cancer diagnoses. Ovarian cancer represented 6.1% (n=5, mean age of diagnosis: 58.2 years), followed by thyroid cancer (n=2), endometrial cancer (n=1), melanoma (n=1), and others (n=5). Among truncating mutation carriers, 34 (79.1%) of 43 had a personal history of cancer, and 10 (23.3%) had multiple primary cancer diagnosis. Breast cancer represented 85.1% (n=40, mean age of diagnosis: 51.1 years), ovarian cancer represented 6.4% (n=3) of cancer diagnosis among truncating mutation carriers. 24 (82.8%) of 29 missense mutation carriers had personal history of cancer, and 7 (24.1%) had multiple primary cancer diagnosis. Breast cancer represented 80% (n=28, mean age of diagnosis: 47.9 years), ovarian cancer represented 5.7% (n=2) of cancer diagnosis among missense mutation carriers. The difference between truncating mutation carriers and missense mutation carriers in cancer diagnosis, presence of multiple primary cancer, breast cancer diagnosis, mean age of diagnosis of breast cancer and all cancers were not statically significant. 14

26 Table 1. Personal history of cancer comparison Personal History Any CHEK2 Mutation Positive Truncating Missense No./Total Mean age of No./Total Mean age No./Total No. % onset* No. % of onset No. % Mean age of onset Truncating vs. Missense p-value Any Cancer 58/ / / Multiple primary 17/ / / No. of cancer diagnosed Breast (+DCIS) 68/ / / Ovarian 5/ / / Thyroid 2/ / / Endometrial 1/ /47 0 1/ N/A Melanoma 1/ /47 0 1/ N/A Other 5/ / / * age at earliest diagnosis 15

27 Breast Cancer Characteristics The breast cancer characteristics of the individuals with a CHEK2 truncating mutation and CHEK2 missense mutation are listed in Table 2. No significant phenotypic differences were observed between truncating and missense mutation carriers. 53 CHEK2 positive women were identified to have had personal history of breast cancer. 31 had truncating mutations, and 22 had missense mutations. The mean age of diagnosis for breast cancer in the 53 CHEK2 positive women was 46 years. Mean age of diagnosis of breast cancer was 47.3 years in truncating mutation carriers and 44.2 years in missense mutation carriers. There were seven individuals with bilateral breast cancer (13.2%); five (16.1%) had truncating mutations and two (9.1%) had missense mutations. 13 individuals had multiple primary breast cancers (24.5%); seven (22.6%) had truncating mutations and six (27.3%) had missense mutations. 67.9% (n=36) of CHEK2 positive individuals with a personal history of breast cancer had a family history of cancer. 64.5% (n=20) of truncating mutation carriers and 72.7% (n=16) of missense mutation carriers had positive family history of breast cancer. Ductal cancer was the most common type of cancer found in both truncating mutation carriers (55%) and missense mutation carriers (64.5%). Mammary breast cancer was observed in higher frequency among missense mutation carriers (14.3%) than truncating mutation carriers (2.5%) but the difference is not statistically significant (P=0.15). 16

28 Table 2. Breast cancer clinical characteristic comparison Any CHEK2+ with BC (n=53) Truncating+ with BC (n=31) Missense+ with BC (n=22) Truncating vs. Missense Breast Cancer Characteristic No. % No. % No. % p-value Age at dx*, years Mean < ,> Bilateral BC (+DCIS) Multiple primary BC (No DCIS) Positive family history Number of BC diagnosis Histology Ductal Lobular Mammary (Ductal, Lobular) Tubulobular Inflammatory Micropapillary Mucinous DCIS ER Positive PR Positive Her2 Positive Triple negative Size, cm < , > Unknown *age of diagnosis; earliest diagnosis of BC The majority of breast cancers in both truncating mutation carriers and missense mutation carriers were ER positive (80%, 82.1%), and PR positive (62.5%, 67.9%). 12.5% of breast cancers in truncating mutation carriers, and 14.3% of breast 17

29 cancers in missense mutation carriers were Her2 positive. Only two individuals had triple negative breast cancer. Family History of Breast Cancer Family history of breast cancer in first-, second-, and third-degree relatives for truncating CHEK2 mutation carriers and missense CHEK2 mutation carriers are listed in table 3. Family history of breast cancer was present in 31(73.8%) of 42 individuals with a truncating CHEK2 mutation, and in 22 (75.9%) of 29 individuals with a missense CHEK2 mutation. Among truncating mutation carriers, 33.3% had one relative with breast cancer, and 40.5% had more than one relative with breast cancer. Of missense mutation carriers, 34.5% had one relative with breast cancer, and 41.4% had more than one relatives with breast cancer. 23 (54.8%) truncating mutation carriers had first-degree relative with breast cancer, which included 15 (35.7%) mother, 7 (16.7%) sister, one daughter affected. 12 (41.4%) missense mutation carriers had first-degree relative with breast cancer, which included 9 (31%) mother, 6 (20.7%) sister, one daughter affected. Missense mutation carriers were more likely to have more than one first-degree relative affected with breast cancer than truncating mutation carriers (10.3% v 0%, respectively; P=0.03). When looking at seconddegree relatives with breast cancer; 18 (42.9%) truncating mutation carriers had second-degree relative with breast cancer which included seven (16.7%) on father s side, and 12 (28.6%) on the mother s side of the family; 14 (48.3%) missense mutation carriers had second-degree relative with breast cancer which included two (6.9%) on father s side, and 11 (37.9%) on the mother s side of the family. 18

30 Table 3. Family history of breast cancer comparison Total (n=71) History of breast cancer in first- and/or second and/or third- degree relative Truncating+ (n=42) Missense+ (n=29) Truncating vs. Missense No. % No. % No. % p-value Negative Positive No. of relatives with breast cancer > First-degree relative with breast cancer Mother affected Sister affected Daughter affected >1 first-degree relative affected Second-degree relative with breast cancer Father's side Mother's side Half sister Unknown First-, second- and third-degree relative with breast cancer Although the difference was not statistically significant, missense mutation carriers had more second-degree relatives with breast cancer on the mother s side, and truncating mutation carriers had more second-degree relatives with breast cancer on the father s side. Total of four (5.6%) individuals were found to have first, second, and third relatives with breast cancer. Of the individuals who did not have any family history of breast cancer, 14 (77.8%) had a personal history of breast cancer. Prostate (n=7), colon (n=6), and ovary (n=5) cancers were the most common cancers found in relatives of the individuals with negative family history of breast cancer. Family History of All Cancer 19

31 Frequency of cancer diagnosis and the distribution of cancers observed in first-, second-, and third degree-relatives are shown in table 5. The most common cancers diagnosed in first-degree relatives were breast (n=42, 34.4%), prostate (n=11, 9%), ovarian (n=9, 7.4%), colon (n=7, 5.7%), and melanoma (n=6, 4.9%). Prostate cancer was more common in first-degree relatives of truncating mutation carriers than in missense mutation carriers (13.3% v 2.1%, respectively; P=0.05). Of the firstdegree relatives of truncating mutation carriers, there were 25 (33.3%) breast cancers, five (6.7%) ovarian cancers, five (6.7%) colon cancers, and four (5.3%) melanoma observed. Of the first-degree relatives of missense mutation carriers, there were 17 (36.2%) breast cancers, four (8.5%) ovarian cancers, two (4.3%) colon cancers, and one (2.1%) melanoma observed. Other than prostate cancer, there was no statistically significant difference in frequency of certain cancer diagnosis between the two groups. The most common cancers diagnosed in second- and/or third-degree relatives were breast (n=60, 34.4%), colon (n=17, 8.7%), ovarian (n=15, 7.7%), prostate (n=14, 7.1%), and melanoma (n=7, 3.6%). The difference in frequency of certain cancer diagnosis between second- and/or third degree relatives of truncating mutation carriers and missense carriers are not statistically significant. However, prostate cancer was observed more frequently in second- and/or third degree relatives of truncating mutation carriers than missense mutation carriers (9.5% v 3.8%). In contrast, colon cancer (12.5% v 6%), melanoma (6.3% v 1.7%), pancreatic cancer (5% v 0.8%) were observed more frequently in second- and/or third degree relatives 20

32 of missense mutation carriers than truncating mutation carriers. The distribution of cancers in all relatives is shown in Figure 1. Table 4. Family history all cancer comparison Total Truncating Missense Mean Mean Mean age of age of age of No. % onset No. % onset No. % onset No. of first-degree relatives with cancer No. of first-degree relatives with multiple cancer No. of all cancer diagnosed in first-degree relatives Truncating vs. Missense p-value Breast Prostate N/A 0.05 Ovarian Colon N/A 0.71 Melanoma N/A 0.65 Uterine Pancreas Kidney Thyroid Other Unknown No. of second- and/or thirddegree relatives with cancer No. of second- and/or thirddegree relatives with multiple cancer No. of all cancer in secondand/or third-degree relatives Breast Prostate N/A 0.16 Ovarian Colon Melanoma N/A Uterine Pancreas N/A Kidney Thyroid Other Unknown

33 No. of cancer Distribution of cancer in relatives All relatives 1st degree Figure 1. Distribution of cancer in relatives Breast Cancer Management Comparison of breast cancer management can be seen in table 5. Communication of NCCN guidelines for genetic/familial high-risk assessment: Breast and Ovarian for CHEK2 mutation carriers was documented in the medical record of 61 (84.7%) of CHEK2 mutation carriers. A surveillance plan of yearly mammogram and breast MRI was offered to 60 (83.3%) CHEK2 mutation carriers, and surveillance of only annual mammogram was offered to 1(1.4%). 11 CHEK2 mutation carriers (15.3%) were not offered any breast surveillance. Risk-reducing mastectomy was offered to 25 (34.7%) CHEK2 mutation carriers. Of those who were offered MRI and mammogram, 32 (44.4%) completed mammograms and breast MRIs, 8 (11.1%) completed mammogram only, 4 (5.6%) completed breast MRI only, and 17 (23.6%) 22

34 had not done any surveillance. Following genetic testing, 16 CHEK2 mutation carriers (22.2%) pursued risk-reducing mastectomy (13 unilateral, 3 bilateral) Table 5. Breast cancer management comparison CHEK2 + (n=72) Truncating (n=43) Missense (n=29) Truncating vs. Missense Breast cancer management No. % No. % No. % p-value NCCN guideline communicated Offered to patients Mammogram and MRI Mammogram only Risk Reducing Mastectomy Breast surveillance not offered Surveillance after genetic testing Mammogram and MRI Mammogram only MRI only No surveillance after genetic testing Both breasts removed Following management guidelines* Mammogram and MRI Breast cancer identified Risk Reducing Mastectomy after genetic testing Unilateral Bilateral * NCCN guideline communicated and surveillance completed within 18 months of genetic testing Of the 61 CHEK2 carriers to whom NCCN guideline was communicated, 32 individuals were observed to complete the recommended surveillance plan within 18 months of receiving the recommendation following genetic testing results. Breast cancer was identified in two individuals (2.8%) through breast cancer surveillance. No significant breast cancer management differences were observed between truncating mutation carriers and missense mutation carriers. 23

35 Risk Reducing Mastectomy Mammogram and MRI Mammogram only 1 8 MRI only 0 4 None Offered Executed No. of CHEK2+ Figure 2. Management of breast cancer in CHEK2 mutation positive patients Colon Cancer Management Since the establishment of the NCCN guideline for hereditary colon cancer surveillance in CHEK2 mutation carriers in 2015, this guideline was communicated to 35 (48.6%) CHEK2 mutation carriers (Table 6). Colonoscopy was offered to 54 (75%) individuals. 41 (56.9%) of all CHEK2 mutation carriers completed a colonoscopy after receiving the genetic testing results and 27 (37.5%) did not complete a colonoscopy. During surveillance, colorectal cancer was identified in one CHEK2 mutation carrier, and polyps were identified in 12 (16.7%) CHEK2 mutation carriers. The average age at the time of polyp discovery was 55 years, with ages 24

36 ranging from 47 years to 69 years. Of the 26 polyps found, 12 were tubular adenomas, 4 sessile serrated, and 1 tubulovillous. Of the 35 cases where documentation of CHEK2 NCCN guidelines colorectal guidelines communication, 28 individuals followed the management guidelines. Of CHEK2 truncating mutation carriers, 24 (55.8%) underwent colonoscopy while 17 (58.6%) of missense mutation carriers had a colonoscopy within the study period. Table 6. Colon cancer management comparison Total Truncating Missense Truncating vs. Missense Colon cancer management No. % No. % No. % p-value NCCN guideline communicated Colonoscopy offered Surveillance after genetic testing Colonoscopy No surveillance after genetic testing Current age < Following management guidelines* Colorectal cancer identified Individuals with polyps Polyps identified Tubular Adenoma Sessile Serrated Tubulovillous *NCCN guideline communicated, and surveillance completed Other Cancer Management As seen in table 7, more than half (59.7%) of individuals with CHEK2 mutation were offered more extensive surveillance than what was recommended based on the NCCN guideline at the time. Risk reducing bilateral salpingo- 25

37 oophorectomy was offered to 22 (30.6%), thyroid ultrasound to 15 (20.8%), kidney ultrasound to 7 (9.7%), transvaginal ultrasound to 1 (1.4%), chest wall screening to 3 (4.2%), and skin cancer screening was offered to 10 (13.9%). Three CHEK2 mutation carriers underwent risk-reducing salpingo-oophorectomy with no other obvious indication other than being CHEK2 mutation carriers. 5 (6.9%) had transvaginal ultrasound, 4 (5.6%) completed kidney ultrasounds, and 8 (11.1%) completed thyroid ultrasounds. Information on chest wall and skin surveillance was not available. One thyroid cancer was identified during thyroid ultrasound. Overall, the utilization of all cancer surveillance and surgery was similar in between truncating and missense mutation carriers. 26

38 Table 7. Other cancer management comparison Total Truncating Missense Truncating vs. Missense Other cancer management No. % No. % No. % p-value Offered more than current NCCN guidelines Offered to patients Risk reducing bilateral salpingooophorectomy Transvaginal US Kidney ultrasound Thyroid ultrasound Chest wall Skin Surveillance after genetic testing* Risk reducing bilateral salpingooophorectomy Transvaginal US Kidney ultrasound Thyroid ultrasound Chest wall Unknown N/A Unknown N/A Unknown N/A N/A Skin Unknown N/A Unknown N/A Unknown N/A N/A No. of cancer identified through screening Ovarian cancer N/A Kidney cancer N/A Thyroid cancer

39 CHAPTER IV DISCUSSION Personal History of Cancer Based on prior studies, truncating mutation carriers were expected to have more instances of breast cancer with a higher likelihood of developing unilateral and bilateral breast cancer compared to missense mutation carriers (Kleibl and Kristensen, 2016). However, the differences in number of breast cancer (47 v 35) and bilateral breast cancer diagnosis (5 v 2) between the two groups were not significant. Although previous work reports a significant association between the CHEK2 p.i157t variant and increased risk of lobular type breast tumors (Liu, et al., 2012), there was only one lobular cancer among the missense mutation carriers in the current study. Consistent with breast characteristics of CHEK2 c.1100delc and pi157t mutations reported by other authors, CHEK2 truncating and missense mutation carriers were both observed at a median age of 45 years compared to the expected median age in the general population of 62 years. Also, in accordance with prior literature, ER and PR positive tumors were common with 80.9% of breast cancers being ER positive and 64.4% being PR positive, with similar results in truncating and missense mutations. Family History of Cancer When considering family history of breast cancer, missense mutation carriers were more likely to have more than one first-degree relative with breast cancer compared to truncating mutation carriers (p=0.03). This was unexpected given that 28

40 c.1100delc truncating mutation carriers are thought to be at a higher risk of breast cancer than the p.i157t missense mutation carriers. Two of the three individuals who had more than one first-degree relative with breast cancer, were carriers of nonfounder missense mutations, and the other one had the p.i157t missense mutation. In a study that looked at risk of breast cancers in CHEK2 mutation carriers with and without family history of breast cancer (Cybulski et al 2011), a higher risk of cancer was associated with the missense mutation if the cancer in a relative was present on the father s side rather than on the mother s side. It was speculated that the I157T mutation may play a role of imprinting or that there may be an X-linked modifier. However, although not significant, an opposite effect was observed in this study in which more relatives with breast cancer were seen on the mother s side compared to the father s side in the family history of missense mutation carriers. When considering family histories of all cancers, truncating mutation carriers were found more likely to have a first-degree relative with prostate cancer compared to missense mutation carriers (p=0.05). This finding is consistent with studies that report higher penetrance for familial prostate cancer in c.1100delc truncating mutations than in p.i157t missense mutations. (Cybulski et al., 2004A). However, when first-, second- and third-degree relatives were all included, the difference in frequency of prostate cancer between the two groups did not reach significance. Studies have suggested that the p.i157t mutation may be associated with increased risk of colorectal cancer, while truncating mutations may not confer an increased risk (Gronwald, et al., 2009). Our study however, showed no difference in frequency of 29

41 colorectal cancers in family members of CHEK2 truncating and missense mutation carriers. Multiple studies report no increased risk of ovarian cancer in individuals with CHEK2 mutations. Yet despite ovarian cancer being rare, it was the third most common cancer identified in relatives of CHEK2 mutation carriers in this study. This may be due to an inherent bias that individuals who have relatives with ovarian cancer meet the NCCN guidelines and therefore were offers genetic testing. Observations of CHEK2 truncating and missense mutation carriers Overall, no significant differences were observed between personal cancer histories, breast cancer characteristics, and family cancer histories of CHEK2 truncating and missense mutation carriers. This suggests that the phenotypic difference reported in CHEK2 c.1100delc and pi157t mutation cannot be generalized and expected in all CHEK2 truncating and missense mutation carriers. There are three possible explanations for this outcome. First, the small sample size could mask potential differences that would be significant in a data set with a larger sample size. A second possibility is that all CHEK2 mutation carriers may have similar risk and phenotype except for the p.i157t mutation carriers. A study done by Leedom et al, 2016 looked at the difference between breast cancer risks and phenotype of CHEK2 founder and non-founder mutation carriers. No significant differences were observed between founder and non-founder CHEK2 mutations which suggests that breast cancer risks reported for founder mutations may be generalized to both founder and non-founder CHEK2 mutation carriers. p.i157t mutation however, was excluded from their analysis suggesting that all other CHEK2 30

42 mutation carriers have similar penetrance and clinical presentation except for p.i157t mutation carriers. Lastly, it is possible that each specific CHEK2 missense mutation, due to varying effects on the function of the protein, have different risk and phenotype. Studies have suggested that the Ashkenazi Jewish founder missense mutation S428F has a twofold elevated risk of breast cancer which is slightly higher than that of p.i157t (Shaag, et al, 2005). Another study estimates that approximately half of the CHEK2 mutation associated risk is attributable to protein-truncating variants and the remainder to rare missense substitutions (Taxitigian and Chenevix- Trench, 2014). Each missense mutation can impact the chk2 protein differently and show difference in penetrance and clinical presentation. Thus generalizing the characteristics of p.i157t to all missense mutation carriers may be a gross oversimplification. Management There were no significant differences observed between cancer risk management of breast cancer, colon cancer, and other cancers of CHEK2 truncating and missense mutation carriers. The current management recommendations for CHEK2 mutation carriers are based on risk of founder truncating mutation. The risks for missense mutations are unclear, however the risk for p.i157t missense mutation carriers is lower than for truncating mutation carriers. This suggests that despite the knowledge that p.i157t missense mutation carriers have a lower risk, or lack of evidence about missense mutation risks overall, clinical recommendations were not adjusted accordingly, indicating a potential for overtreatment. 31

43 In this study, individuals who went through unilateral risk-reducing mastectomy were those who removed one breast as part of cancer treatment and then chose to remove the other due to concern of contralateral breast cancer. Despite unclear evidence of risk for bilateral breast cancer for the missense mutation carriers including p.i157t mutation carriers, five individuals with a missense mutation chose to proceed with risk-reducing mastectomy on the unaffected breast. Although it is difficult to know how and why these individuals came to their decision, there is a possibility that the knowledge of elevated risk for bilateral breast cancer in CHEK2 truncating mutation could have influenced the decision. Risk of thyroid cancer and kidney cancer in CHEK2 mutation carriers is still unclear and the NCCN guidelines currently do not provide any recommendations. In the study, a high uptake of recommended thyroid and kidney cancer surveillance was observed. With CHEK2 mutation being the only indication, kidney ultrasound was offered to seven individuals and four individuals proceeded with the ultrasound. Thyroid ultrasound was offered to 15 individuals and eight individuals elected to undergo the procedure. This is evidence of the concern that unnecessary additional surveillance measures may potentially be prescribed. CHEK2 mutation carriers are thought not to be at increased risk for ovarian cancer. However, we see that 22 (30.6%) individuals were offered to undergo risk reducing bilateral salpingo-oophorectomies (RRBSO) and three individuals chose to proceed with the process with no other indication other than the presence of CHEK2 mutation. The respective ages of the three women who elected RRBSO, were 40, 52, 32