Using a family history questionnaire to identify adult patients with increased genetic risk for sarcoma

Background Sarcomas in adults can be associated with hereditary cancer syndromes characterized by early-onset predisposition to numerous types of cancer. Because of variability in familial presentation and the largely unexplained genetic basis of sarcomas, ascertainment of patients for whom a genetics evaluation is most indicated poses challenges. We assessed the utility of a Sarcoma Clinic Genetic Screening (scgs) questionnaire in facilitating that task. Methods Between 2008 and 2012, 169 patients (median age: 53 years; range: 17–88 years) completed a selfadministered scgs questionnaire. A retrospective chart review was completed for all respondents, and descriptive statistics were reported. Probands were divided into two groups depending on whether they did or did not report a family history of Li–Fraumeni syndrome–type cancers. Results A family history of cancer (as far as 3rd-degree relatives) was reported in 113 of 163 sarcoma patients (69%). Eeles Li–Fraumeni–like (lfl) criteria were fulfilled in 46 probands (28%), Chompret lfl in 21 (13%), Birch lfl in 8 (5%), and classic Li–Fraumeni in none. In the 10 probands tested for TP53 mutations, 1 pathogenic mutation was found. Further investigation of selected families led to the discovery of germline mutations in MLH1, MSH2, and APC genes in 3 individuals. Conclusions The scgs questionnaire was useful for ascertaining probands with sarcoma who could benefit from a genetic assessment. The tool allowed us to identify high-risk families fitting the criteria for lfl and, surprisingly, other hereditary cancer syndromes. Similar questionnaires could be used in other cancer-specific clinics to increase awareness of the genetic component of these cancers.


INTRODUCTION
Sarcomas constitute a rare and heterogeneous group of tumours that make up approximately 1% of adult malignancies and 12% of childhood cancers 1,2 .In 2007, the Hereditary Cancer Clinic in the Department of Medical Genetics, McGill University Health Centre (muhc), developed a 1-page Sarcoma Clinic Genetic Screening (scgs) questionnaire to ascertain a subset of adult sarcoma patients for whom a referral to genetics might be indicated.Low referral rates for sarcoma patients have been reported in the literature 3 and, before this initiative, were also observed at our institution (WDF.Personal observation).The low rate might be attributable to a variety of factors, including the rarity of sarcoma and the lack of standardized referral criteria.In addition, it could be that health care professionals are unable to recognize probands for whom a referral is indicated.
More than 7% of children with soft-tissue sarcoma or osteosarcoma can have Li-Fraumeni syndrome (lfs) 4 .Furthermore, up to 9% of probands with sarcoma have been shown to harbour mutations in lfs genes [5][6][7] .Li-Fraumeni syndrome is a rare, autosomal-dominant hereditary cancer syndrome associated with early-onset predisposition to the following classical tumour types: soft-tissue sarcoma, osteosarcoma, premenopausal breast cancer, brain tumours, adrenocortical carcinoma, and leukemia, as well as a variety of other less-common neoplasms 8,9 .
Owing in part to the broad distribution of cancer types seen in lfs families, ascertainment of probands who might be eligible for TP53 testing poses many challenges.Although several criteria have been proposed for both classical lfs and related phenotypes ["Li-Fraumeni-like" (lfl)] [10][11][12][13] , no standard clinical definition exists.Furthermore, studies have shown that, although 60%-80% of families meeting the strictest classical lfs criteria 14,15 harbour germline mutations in TP53, other studies have shown that as few as 20% of individuals meeting the broader Chompret criteria have a detectable TP53 mutations 9,11 .Similarly, although the less-specific and more-sensitive Birch and Eeles definitions of lfl syndromes 12,13 have identified TP53 mutations in families that would have been missed by stricter criteria, a great preponderance of the families fulfilling those criteria have not been shown to harbour identifiable germline mutations 14,16 .Mutations in other genes can also sometimes predispose to osteosarcoma and other types of sarcoma 17,18 .
The goal of the present study was to assess whether implementation of a self-administered questionnaire in a busy sarcoma clinic would facilitate the process of ascertaining probands with sarcoma who could be at risk for a hereditary cancer predisposition syndrome.As a result, we also describe the sarcoma population seen at the muhc between 2008 and 2012.

Study Population
A retrospective chart review was conducted for a clinicbased sample of patients with adult-onset sarcoma who completed the self-administered scgs questionnaire between March 2008 and May 2012.Within the time frame of the study, 374 sarcoma patients (100 from the bone sarcoma clinic and 274 from the soft-tissue sarcoma clinic) were seen at the Montreal General Hospital (one of the muhc hospitals) as part of routine clinical care.Questionnaires were available in French or English and were distributed by the pivot nurse (nurse navigator) at the clinic.Of the 374 patients seen, 169 probands (45.2%) completed the 1-page questionnaire (Table i) and consented to the use of their demographic and medical information for research purposes.Questionnaires were then forwarded to the Hereditary Cancer Clinic by the pivot nurse and triaged by the clinical team (LK, LP, WDF).
A decision to offer an appointment for genetic counselling was based on fulfilment of at least one of the lfs or lfl clinical diagnostic criteria, or the presence of an unusual constellation of cancers in the proband's personal or family history, or both (Table ii).Individuals who received an appointment were part of the clinical sample.Patients seen in the sarcoma clinic during the study period who did not complete the questionnaire were not included in the analysis.It is important to note that during the study period, only 1 sarcoma patient outside of the study sample was seen (as an inpatient) for genetic counselling.That patient was being treated for a sarcoma at the same institution and also had a personal history of breast cancer.A germline TP53 mutation was identified; however, given that the patient had not been ascertained using the scgs questionnaire, the patient's data were not included in the final analysis.All other referrals received from the sarcoma clinic during the study period came via completion of the scgs questionnaire.
Ethics approval to complete the chart review was obtained from the Research Ethics Board of the muhc.

Data Collection
The scgs questionnaire collected data about cancer history for both the patient and the patient's family.Patients were asked to report whether they or other members of their family had been diagnosed with breast cancer, sarcoma, bone cancer, lung cancer, brain cancer, adrenal cancer, prostate cancer, or another cancer, and at what age.All data were summarized and, when necessary, verified using the muhc electronic health record system.Data for all patients were analyzed.
Descriptive statistics were used to summarize the sample.Probands were classified into two main groups: those with only a personal history of sarcoma, and those with a personal history of sarcoma and a family history of one or more lfs-type cancers.Subsequently, criteria fulfilment assessed disease status and whether the patient fulfilled one or more of the recognized lfs or lfl diagnostic criteria.All cancer diagnoses in the probands were confirmed by review of pathology reports.Although cancer diagnoses in the relatives of patients not eventually seen in the Hereditary Cancer Clinic were based on verbal description and not verified by pathology report, cancer diagnoses of relatives of probands seen for genetic counselling were confirmed by pathology report when possible.
The scgs questionnaire was also used as a tool for the ascertainment of individuals who could benefit from an assessment by the medical genetics service.From a review of the forms, a decision was made to offer genetic evaluations to 14 probands, 2 of whom declined.Of 12 patients seen, 10 were offered commercial TP53 genetic testing, using sequencing and mlpa (multiplex ligationdependent probe amplification).A pathogenic mutation Because of the high suspicion of lfs, sequencing and mlpa of the TP53 gene was performed, revealing a deleterious missense mutation, c.730G>A (p.Gly244Ser).Both daughters were subsequently tested and were also found to carry the familial TP53 mutation.To rule out other genetic causes of cancer, the proband was also offered testing of the BRCA1/2 genes; no mutation was identified.
The origin of the TP53 mutation in this family is unconfirmed, because both of the proband's parents are deceased.However, it is hypothesized that the TP53 mutation either derives from the maternal lineage or could be de novo.

Case 10
A genetic consultation was offered to a 48-year-old woman who presented with a leiomyosarcoma of the right pectoralis major at age 37 and who later developed a colonic tubulovillous adenoma with high-grade dysplasia at age 40.Immunohistochemistry for the mismatch repair proteins of both the leiomyosarcoma [Figure 2(A)] and the colonic tubulovillous adenoma (not shown) revealed deficient staining of MSH2 with a corresponding lack of MSH6 expression.Genetic testing by mlpa and high-resolution melting showed a germline MSH2 mutation in exon 4: c.649_650dupA [Figure 2(B)].
Because of the risk for gynecologic cancer associated with Lynch syndrome, the patient underwent prophylactic total hysterectomy and bilateral oophorectomy at age 49.Pathology subsequently revealed endometriosis, focal adenomyosis, leiomyomata, and an incidental endometrial carcinoma (endometrioid type) without myometrial invasion (Federation Internationale de Gynecologie et d'Obstetrique grade 1), which required no further therapy.Her mother had synchronous cancers of the right ovary (reported as a low-grade serous papillary cystadenocarcinoma) and endometrium (adenocarcinoma) at age 36 and ultimately died of metastatic colorectal cancer at age 49.The patient's maternal grandfather reportedly died of stomach cancer in his 50s, and a maternal aunt was thought to have had a gastrointestinal malignancy at age 67 [Figure 2(C)].

Case 11
A 48-year-old man reported a leiomyosarcoma at age 39 and osteosarcoma at age 42.In his questionnaire, he also indicated a personal and family history of colorectal cancer (Figure 3, MLH1 pedigree).The patient declined a consultation because he had previously been seen at the genetics centre of Université de Montréal.Genetic testing at that centre had revealed a germline MLH1 mutation in c.2195_2198dupAACA, previously reported to be associated with Lynch syndrome 19 .His daughter developed  Family history included a brother with a sarcoma and 3 sisters with breast cancer (all later-onset). c Family history included a first-degree cousin with sarcoma at 35 years of age.
a glioblastoma at age 18. Genetic testing was also performed for TP53, which was negative.

Case 12
A 22-year-old adopted woman was ascertained because of a desmoid tumour at age 22 and was seen in the sarcoma clinic because the referring diagnosis had been a sarcoma, which was later revised after a biopsy review.At age 11, the patient had been diagnosed with familial adenomatous polyposis based on the presence of florid colorectal polyps, and she underwent a total colectomy with ileoanal anastomosis.She also had a history of stomach and duodenal polyps.Upon assessment, genetic testing for familial adenomatous polyposis was performed using a combination of high-resolution melting, protein truncation test, and mlpa.The latter test identified a whole-gene deletion of APC (Figure 4).

DISCUSSION
Of a series of 169 patients with sarcoma who completed a 1-page self-administered scgs questionnaire, 164 patients were analyzed, 14 were offered a genetic consultation, and 12 pursued genetic testing.Of the 10 patients tested for TP53, 1 was found to carry a deleterious mutation.Of the pedigrees for the 10 tested patients, 6 were consistent with the Chompret criteria.None of the patients in the series, including those selected for a genetic consultation, met the classical lfs criteria.Because the questionnaire was designed to target lfs, which is associated with a broad spectrum of cancers, the questionnaire was able to identify patients at high risk for other genetic conditions (for example, Lynch syndrome and familial adenomatous polyposis).Without the use of the questionnaire, such patients could otherwise have been missed because of their atypical constellation of cancers and family history [Figures 1, 2(C), 3, and 4].As a result, the questionnaire identified one family with lfs and other families that harboured mutations in genes which are usually more relevant to susceptibility to other cancers.The distribution of sarcoma subtypes observed in our clinical sample was consistent with those reported in the literature 20 .
The scgs questionnaire was successful as a tool for referral.It was designed to include cancer-related questions targeting features of lfs and lfl for both individuals and their families.One strength of the questionnaire is that it allows for inclusion of distant relatives; however, it has limitations in that it relies on accurate reporting by the patient and on the collaboration of hospital staff.A useful modification of the questionnaire would be the inclusion of an indication of maternal or paternal lineage for the family history section.
The simplicity of the questionnaire made a referral to the genetics clinic much more straightforward and allowed patients requiring an assessment to be selected.Thus, the scgs questionnaire proved more useful than a typical referral from a treating physician.Although the sample size was quite large, a weakness of the study is that only 45.2% of the target population was sampled.During the entire duration of the study, the same pivot nurse was responsible for distributing the scgs questionnaire to sarcoma patients seen, but it is possible that some patients were overlooked or were not interested in completing the questionnaire ( "nonresponders").Similarly, some patients might have self-selected as being at "low risk" (for example, elderly patients with a negative family history) and might therefore have chosen not to complete the questionnaire.
A recent 2013 international prospective cohort study found that about 3.6% of unselected adult sarcoma probands had TP53 mutations.However, 41% of those with an identifiable TP53 mutation did not meet the classical lfs or Chompret lfl criteria 17 .The fact that our only patient with a TP53 mutation met neither of the foregoing diagnostic criteria illustrates the challenge in detecting TP53 Moreover, our study suggests that germline mutations in cancer susceptibility genes other than TP53 are likely to make a contribution to adult sarcoma distinct from any contributions seen in childhood sarcoma.
Testing everyone with a sarcoma for a TP53 mutation would not be cost-effective, and therefore tools such as the scgs questionnaire can help to devote resources to the patients with suggestive personal and family histories.Owing to the large range of detection frequencies for mutations families, there is interest in further studies to assess the robustness of the current lfs and lfl diagnostic criteria.The scgs questionnaire could not only be used as a method of referral for sarcoma probands to hereditary cancer services at the muhc and other facilities, but-if suitably modified-could be implemented as a tool in other adult and pediatric specialty cancer clinics.

SUMMARY
The results of our study suggest the importance of self-administered clinic-based surveys in screening for hereditary cancer syndromes.The scgs questionnaire analyzed here successfully identified 1 individual with a germline TP53 mutation and 2 others with mutations in cancer susceptibility genes (MSH2 and APC) that might otherwise have been missed.

FIGURE 1
FIGURE 1 Pedigree of a man with a germline TP53 mutation.Arrowhead = proband; square = man; circle = woman; diamond = sex unknown; filled symbol = cancer-affected individual (age at diagnosis shown beside cancer type); diagonal line = deceased individual (age at death or at time of study shown immediately below); double diagonal line = divorced or separated parents; + = mutation carrier (heterozygous for the TP53 mutation); -= mutation non-carrier (wild-type TP53); ca = cancer; dx = diagnosed.

FIGURE 2
FIGURE 2 Phenotype, genotype, and pedigree of a 48-year-old woman with a germline MSH2 mutation.(A) Immunohistochemistry staining for MLH1, MSH2, MSH6, and PMS2 in leiomyosarcoma tumour tissue.Blue nuclei indicate a lack of target protein expression; this tissue shows deficient MSH2 and MSH6 expression.(B) Chromatograms from sequencing the proband and wild-type MSH2 illustrates the c.649_650dupA mutation (asterisk) and frameshift in the proband.(C) The proband's family cancer history fulfils both the Eeles and Lynch criteria.Arrowhead = proband; square = man; circle = woman; diamond = sex unknown; filled symbol = cancer-affected individual (age at diagnosis shown beside cancer type); half-filled symbol = individual with cancer-associated trait; diagonal line = deceased individual (age at death or at time of study shown immediately below); double diagonal line = divorced or separated parents; + = mutation carrier (heterozygous for the mutation); -= mutation non-carrier (wildtype gene); PSU = primary site unknown; HGD = high-grade dysplasia.

TABLE I
Sarcoma Clinic Genetic Screening questionnaire the TP53 gene in exon 7 [c.730G>A(p.Gly244Ser)] was identified in the peripheral blood dna of only 1 individual (case 1, Tableiii).A 57-year-old man with a history of melanoma of the left arm at 38, leiomyosarcoma of the right thigh at 47, prostate cancer at 54, and a recurrence of the sarcoma in the right forearm at age 54 (Figure1) presented for genetic consultation after completing the scgs questionnaire.He also had an extensive family history of cancer including in his two daughters: one diagnosed with a brain tumour (oligoastrocytoma) at age 30, and one diagnosed with breast cancer at age 29 (invasive ductal carcinoma with ductal carcinoma in situ).Other family members with cancer included the proband's mother (confirmed ovarian cancer at age 48), a maternal aunt (lung cancer), a paternal uncle (prostate cancer), and a paternal aunt (intestinal cancer).
k. other types of cancer not listed above (if YES, describe below) Please list any OTHER type of cancer here If you have answered Yes to any of these, please list family member(s) and their relationship to you (example: maternal cousin, paternal grandmother, sister, daughter, etc.) as well as age at diagnosis and type of cancer.Please use your best guess: Current Oncology, Vol.22,No. 5, October 2015 © 2015 Multimed Inc.in

TABLE II
Characteristics of the sarcoma probands investigated