Development and testing of the KnowGene scale to assess general cancer genetic knowledge related to multigene panel testing

PurposeTo develop and evaluate a measure of cancer genetics knowledge relevant to multigene panel testing.MethodsThe instrument was developed using systematic input from a national panel of genetics experts, acceptability evaluation by patient advocates, and cognitive testing. Twenty-four candidate items were completed by 591 breast or gynecological patients who had undergone genetic counseling and multigene panel testing in the past 18 months. A unidimensional item response theory model was fit with a mix of 2-parameter logistic nested response (2 plnrm) and 2-parameter logistic (2 pl) items.ResultsKey domains addressing cancer genetics knowledge were found to be overlapping. Of the 24 candidate items, 8 items were removed due to poor discrimination or local dependence. The remaining 16 items had good fit (RMSEA = 0.045, CFI = 0.946) and discrimination parameters ranging from 0.49 to 1.60. The items specified as 2 plnrm distinguish between those answering incorrect versus don’t know, with discrimination ranging from 0.51 to 1.02. Information curves were highest among those with lower knowledge.ConclusionKnowGene is a rigorously developed and effective measure of knowledge after cancer genetic counseling and multigene panel testing.Practice ImplicationsMeasuring knowledge in a systematic way will inform practice and research initiatives in cancer genetics.     

Variants of uncertain significance (VUS) in cancer predisposing genes: What are we learning from multigene panels?

One of the main factors influencing the clinical utility of genetic tests for cancer predisposition is the ability to provide actionable classifications (ie pathogenic or benign). However, a large fraction of the variants identified in cancer predisposing genes (CPGs) are of uncertain significance (VUS), and cannot be used for clinical purposes either to identify individuals at risk or to drive treatment.Here we analyze the current status of VUS identification in a subset of 24 CPGs included by the American College of Medical Genetics/Association for Molecular Pathology in the list of genes that should be considered for the return of incidental findings. To this purpose we retrieved published literature using different search strings according to the frequency of the condition and we extracted corresponding data from ClinVar.The total number of VUS has not decreased with time, due to widespread multigene panel testing, and the relative yield of VUS compared to pathogenic variants is higher in more recent studies, which tend to involve series not selected for the presence of specific high risk criteria. In addition, only few studies adopt gene specific interpretation criteria when these are available.Despite the large yield of VUS associated with multigene testing, the data obtained from such studies can be very useful for variant classification, especially for those variants that are more likely to be benign, since these are expected to be detected more frequently in a population that does not show gene specific manifestations. In addition, wider use of gene specific interpretation criteria should be promoted in order to optimize the interpretation process.     

Classification of variants of uncertain significance in BRCA1 and BRCA2 using personal and family history of cancer from individuals in a large hereditary cancer multigene panel testing cohort

PurposeGenetic testing of individuals often results in identification of genomic variants of unknown significance (VUS). Multiple lines of evidence are used to help determine the clinical significance of these variants.MethodsWe analyzed ~138,000 individuals tested by multigene panel testing (MGPT). We used logistic regression to predict carrier status based on personal and family history of cancer. This was applied to 4644 tested individuals carrying 2383 BRCA1/2 variants to calculate likelihood ratios informing pathogenicity for each. Heterogeneity tests were performed for specific classes of variants defined by in silico predictions.ResultsTwenty-two variants labeled as VUS had odds of >10:1 in favor of pathogenicity. The heterogeneity analysis found that among variants in functional domains that were predicted to be benign by in silico tools, a significantly higher proportion of variants were estimated to be pathogenic than previously indicated; that missense variants outside of functional domains should be considered benign; and that variants predicted to create de novo donor sites were also largely benign.ConclusionThe evidence presented here supports the use of personal and family history from MGPT in the classification of VUS and will be integrated into ongoing efforts to provide large-scale multifactorial classification.     

'Indirect' BRCA1/2 testing: a useful approach in hereditary breast and ovarian cancer families without a living affected relative

We report an approach for BRCA1/2 testing whereby genetic testing can be offered to families at high risk of hereditary breast and ovarian cancer but where no DNA from affected relatives is available. By testing two or more unaffected relatives at 50% risk of being heterozygous for a potential BRCA1/2 mutation, there is a chance of up to 99% of finding a mutation that would have been detectable in an affected individual from the same family. The overall likelihood of identifying a mutation is dependent on the family history, and therefore 'indirect' testing would be most applicable for families with a very high risk of carrying a BRCA1/2 mutation. Using this approach also requires balancing issues of testing resource limitations, family dynamics and adequate preparation of unaffected persons for a positive test, with the advantages of targeting screening and prophylactic surgery.     

Prevalence of pathogenic and likely pathogenic variants in the RASopathy genes in patients who have had panel testing for cardiomyopathy

RASopathies are a group of developmental disorders caused by pathogenic variants in the RAS‐MAPK pathway. Cardiomyopathy is a major feature of this group of disorders, specifically hypertrophic cardiomyopathy (HCM). HCM can be the first presenting feature in individuals with RASopathies. We conducted a retrospective study of all individuals who have had a cardiomyopathy gene panel ordered through our institution to determine the prevalence of pathogenic or likely pathogenic variants in RAS pathway genes in individuals with cardiomyopathy. We evaluated variants in the following genes: BRAF, CBL, HRAS, KRAS, MAP2K1, MAP2K2, NF1, NRAS, PTPN11, RAF1, SHOC2, and SOS1. We reviewed 74 cases with cardiomyopathy, including 32 with HCM, 24 with dilated cardiomyopathy (DCM), nine with both left ventricular noncompaction (LVNC) and DCM, four with LVNC only, two with arrhythmogenic right ventricular cardiomyopathy (ARVC) and three with unspecified cardiomyopathy. We identified four patients (5.41%) with pathogenic or likely pathogenic variants in HRAS, PTPN11 and RAF1 (two individuals). Indication for testing for all four individuals was HCM. The prevalence of pathogenic or likely pathogenic variants in RASopathy genes in our HCM patient cohort is 12.5% (4/32). We conclude that the RASopathy genes should be included on multi‐gene panels for cardiomyopathy to increase diagnostic yield for individuals with HCM.     

Experiences of predictive testing in young people at risk of Huntington's disease,familial cardiomyopathy or hereditary breast and ovarian cancer

While debate has focused on whether testing of minors for late onset genetic disorders should be carried out if there is no medical benefit, less is known about the impact on young people (<25 years) who have had predictive testing often many years before the likely onset of symptoms. We looked at the experiences of young people who had had predictive testing for a range of conditions with variable ages at onset and options for screening and treatment. A consecutive series of 61 young people who had a predictive test aged 15–25 years at the Clinical Genetic Service, Manchester, for HD, HBOC (BrCa 1 or 2) or FCM (Hypertrophic Cardiomyopathy or Dilated Cardiomyopathy), were invited to participate. Thirty-six (36/61; 59%) agreed to participate (10 HD, 16 HBOC and 10 FCM) and telephone interviews were audiotaped, transcribed and analysed using Interpretative Phenomenological Analysis. None of the participants expressed regret at having the test at a young age. Participants saw the value of pretest counselling not in facilitating a decision, but rather as a source of information and support. Differences emerged among the three groups in parent/family involvement in the decision to be tested. Parents in FCM families were a strong influence in favour of testing, in HBOC the decision was autonomous but usually congruent with the views of parents, whereas in HD the decision was autonomous and sometimes went against the opinions of parents/grandparents. Participants from all three groups proposed more tailoring of predictive test counselling to the needs of young people.     

BRCA1/2 testing in hereditary breast and ovarian cancer families: effectiveness of problem-solving training as a counseling intervention

It remains uncertain whether members of hereditary breast and ovarian cancer (HBOC) families experience psychological distress with genetic testing and whether pre-test counseling can have a moderating effect on client well-being. One purpose of this study was to assess change in psychological well-being from baseline to 6-9 months follow-up and the effect of a problem-solving training (PST) intervention on psychological well-being. Two hundred and twelve members of 13 HBOC families were offered BRCA1/2 testing for a previously identified family mutation. Participants received education and were randomized to one of two counseling interventions; PST or client-centered counseling. Psychological well-being was assessed at baseline and again at 6-9 months following the receipt of test results, or at the equivalent time for those participants who chose not to undergo testing. Well-being was assessed using measures of depressive symptoms (CESD), intrusive thoughts (IES), cancer worries, and self-esteem. Comparisons were made between those who chose testing and those who did not as well as between those who received positive and negative test results. One hundred eighty one participants elected to undergo genetic testing (85%) and 47 of these (26%) were identified as BRCA1/2 mutation carriers. Breast and ovarian cancer worries decreased significantly (p = 0.007 and 0.008, respectively) in those who tested negative while there was no appreciable change in psychological well-being from baseline to follow-up in either those who tested positive or in non-testers. Among all participants, particularly testers, those randomized to PST had a greater reduction in depressive symptoms than those randomized to client-centered counseling (p < 0.05 and p = 0.02, respectively). Regardless of the decision to test, individuals with a personal history of cancer (n = 22) were more likely to have an increase in breast cancer worries compared to those who had never been diagnosed with cancer (p < 0.001). Results suggest that a problem-solving counseling intervention may help to enhance psychological well-being following testing and that a personal history of cancer may increase psychological distress associated with genetic testing.     

Pre‐test genetic counseling services for hereditary breast and ovarian cancer delivered by non‐genetics professionals in the state of Florida

Genetic counseling and testing for hereditary breast and ovarian cancer now includes practitioners from multiple healthcare professions, specialties, and settings. This study examined whether non‐genetics professionals (NGPs) perform guideline‐based patient intake and informed consent before genetic testing. NGPs offering BRCA testing services in Florida (n = 386) were surveyed about clinical practices. Among 81 respondents (response rate = 22%), approximately half reported: sometimes scheduling a separate session for pre‐test counseling lasting 11–30 min prior to testing, discussing familial implications of testing, benefits and limitations of risk management options, and discussing the potential psychological impact and insurance‐related issues. Few constructed a three‐generation pedigree, discussed alternative hereditary cancer syndromes, or the meaning of a variant result. This lack of adherence to guideline‐based practice may result in direct harm to patients and their family members. NGPs who are unable to deliver guideline adherent cancer genetics services should focus on identification and referral of at‐risk patients to in person or telephone services provided by genetics professionals.     

BRCA1/2 testing in newly diagnosed breast and ovarian cancer patients without prior genetic counselling: the DNA-BONus study

Germline BRCA1/2 testing of breast and ovarian cancer patients is growing rapidly as the result affects both treatment and cancer prevention in patients and relatives. Through the DNA-BONus study we offered BRCA1/2 testing and familial risk assessment to all new patients with breast (N=893) or ovarian (N=122) cancer diagnosed between September 2012 and April 2015, irrespective of family history or age, and without prior face-to-face genetic counselling. BRCA1/2 testing was accepted by 405 (45.4%) and 83 (68.0%) of the patients with breast or ovarian cancer, respectively. A pathogenic BRCA1/2 variant was found in 7 (1.7%) of the breast cancer patients and 19 (22.3%) of the ovarian cancer patients. In retrospect, all BRCA1/2 mutation carriers appeared to fulfill current criteria for BRCA1/2 testing. Hospital Anxiety and Depression Scale (HADS) scores showed that the mean levels of anxiety and depression were comparable to those reported for breast and gynecological cancer patients in general, with a significant drop in anxiety symptoms during a 6-month follow-up period, during which the test result was forwarded to the patients. These results show that BRCA1/2 testing is well accepted in newly diagnosed breast and ovarian cancer patients. Current test criteria based on age and family history are sufficient to identify most BRCA1/2 mutation carriers among breast cancer patients. We recommend germline BRCA1/2 testing in all patients with epithelial ovarian cancer because of the high prevalence of pathogenic BRCA1/2 variants.     

Characterization of BRCA1 and BRCA2 deleterious mutations and variants of unknown clinical significance in unilateral and bilateral breast cancer: the WECARE study

BRCA1 and BRCA2 screening in women at high‐risk of breast cancer results in the identification of both unambiguously defined deleterious mutations and sequence variants of unknown clinical significance (VUS). We examined a population‐based sample of young women with contralateral breast cancer (CBC, n=705) or unilateral breast cancer (UBC, n=1398). We identified 470 unique sequence variants, of which 113 were deleterious mutations. The remaining 357 VUS comprised 185 unique missense changes, 60% were observed only once, while 3% occurred with a frequency of >10%. Deleterious mutations occurred three times more often in women with CBC (15.3%) than in women with UBC (5.2%), whereas combined, VUS were observed in similar frequencies in women with CBC and UBC. A protein alignment algorithm defined 16 rare VUS, occurring at highly conserved residues and/or conferring a considerable biochemical difference, the majority located in the BRCA2 DNA‐binding domain. We confirm a multiplicity of BRCA1 and BRCA2 VUS that occur at a wide range of allele frequencies. Although some VUS inflict chemical differences at conserved residues, suggesting a deleterious effect, the majority are not associated with an increased risk of CBC. © 2010 Wiley‐Liss, Inc.     

Mutational analysis of RAD51C and RAD51D genes in hereditary breast and ovarian cancer families from Murcia (southeastern Spain)

RAD51C and RAD51D have been defined as susceptibility genes for hereditary breast and ovarian cancer syndrome in several studies. In the present study, a mutation analysis of these genes was performed on non BRCA1/2 families. RAD51C and RAD51D genes were analyzed in 141 and 77 families, respectively. The analysis included direct sequencing and multiple ligation probe analysis. The RAD51C pathogenic variant c.404G?>?A was identified in a breast and ovarian cancer family (0.7%), while the RAD51D pathogenic variant c.694C?>?T was described in an ovarian cancer family (1.3%). Moreover, three unknown clinical significance variants were detected: c.307T?>?G in RAD51C, and c.413A?>?G and c.715C?>?T in RAD51D. No large genomic rearrangements (LGRs) were found. RAD51D carriers suffered from premenopausal ovarian tumors. These results increase our knowledge about the RAD51C and RAD51D mutation spectrum and support the notion that these genes should be included in the gene panel testing performed on patients with hereditary breast and ovarian cancer syndrome.     

Hereditary truncating mutations of DNA repair and other genes in BRCA1/BRCA2/PALB2‐negatively tested breast cancer patients

Hereditary breast cancer comprises a minor but clinically meaningful breast cancer (BC) subgroup. Mutations in the major BC‐susceptibility genes are important prognostic and predictive markers; however, their carriers represent only 25% of high‐risk BC patients. To further characterize variants influencing BC risk, we performed SOLiD sequencing of 581 genes in 325 BC patients (negatively tested in previous BRCA1/BRCA2/PALB2 analyses). In 105 (32%) patients, we identified and confirmed 127 truncating variants (89 unique; nonsense, frameshift indels, and splice site), 19 patients harbored more than one truncation. Forty‐six (36 unique) truncating variants in 25 DNA repair genes were found in 41 (12%) patients, including 16 variants in the Fanconi anemia (FA) genes. The most frequent variant in FA genes was c.1096_1099dupATTA in FANCL that also show a borderline association with increased BC risk in subsequent analysis of enlarged groups of BC patients and controls. Another 81 (53 unique) truncating variants were identified in 48 non‐DNA repair genes in 74 patients (23%) including 16 patients carrying variants in genes coding proteins of estrogen metabolism/signaling. Our results highlight the importance of mutations in the FA genes' family, and indicate that estrogen metabolism genes may reveal a novel candidate genetic component for BC susceptibility.     

The updated ASCO/CAP guideline recommendations for HER2 testing in the management of invasive breast cancer: a critical review of their implications for routine practice

The American Society of Clinical Oncology and the College of American Pathologists have issued joint updated comprehensive guideline recommendations for human epidermal growth factor receptor 2 (HER2) testing in breast cancer. The update not only provides guidelines for the test performance parameters, with the aim of improving test accuracy, reproducibility, and precision, but also provides comprehensive recommendations on the post‐analytical interpretation of the results, and requires improved communication among healthcare providers. The updated guidelines are targeted at testing laboratories, pathologists, oncologists, surgeons, and, indirectly, other healthcare providers. Although the guidelines contribute to the improved analytical validity and clinical utility of laboratory assays required for successful molecularly targeted therapy in the era of personalized medicine, the implications of such recommendations have to be acknowledged. Certain recommendations, particularly those related to repeating the test and pathological concordance, have lower levels of supportive evidence than existing key recommendations, and the associated workload implications will be challenging to support in most healthcare systems. In this commentary, we critically address the key updated recommendations and their impact on service provision and patient care.     

教学医院内临床解剖实验室建设及意义——以唐山市第二医院为例

人体解剖学是一门基础医学学科,是医学生的必修课程;而医学生毕业从事临床工作后,很少再有机会回到解剖室,没有条件带着临床中遇到的问题再次学习解剖。在教学医院内建设临床解剖实验室能为临床医生提供根据临床需要进行解剖研究的场所和条件,也能促进临床手术技术和诊疗水平的提高,意义重大,值得推广。     

Analysis of BRCA1 and BRCA2 genes in Spanish breast/ovarian cancer patients: a high proportion of mutations unique to Spain and evidence of founder effects

We screened index cases from 410 Spanish breast/ovarian cancer families and 214 patients (19 of them males) with breast cancer for germ-line mutations in the BRCA1 and BRCA2 genes, using SSCP, PTT, CSGE, DGGE, and direct sequencing. We identified 60 mutations in BRCA1 and 53 in BRCA2. Of the 53 distinct mutations observed, 11 are novel and 12 have been reported only in Spanish families (41.5%). The prevalence of mutations in this set of families was 26.3%, but the percentage was higher in the families with breast and ovarian cancer (52.1%). The lowest proportion of mutations was found in the site-specific female breast cancer families (15.4%). Of the families with male breast cancer cases, 59.1% presented mutations in the BRCA2 gene. We found a higher frequency of ovarian cancer associated with mutations localized in the 5' end of the BRCA1 gene, but there was no association between the prevalence of this type of cancer and mutations situated in the ovarian cancer cluster region (OCCR) region of exon 11 of the BRCA2 gene. The mutations 187_188delAG, 330A>G, 5236G>A, 5242C>A, and 589_590del (numbered after GenBank U14680) account for 46.6% of BRCA1 detected mutations whereas 3036_3039del, 6857_6858del, 9254_9258del, and 9538_9539del (numbered after GenBank U43746) account for 56.6% of the BRCA2 mutations. The BRCA1 330A>G has a Galician origin (northwest Spain), and BRCA2 6857_6858del and 9254_9258del probably originated in Catalonia (northeast Spain). Knowledge of the spectrum of mutations and their geographical distribution in Spain will allow a more effective detection strategy in countries with large Spanish populations.     

Tissue microarray technology in the routine assessment of HER-2 status in invasive breast cancer: a prospective study of the use of immunohistochemistry and fluorescence in situ hybridization

Aims:  To compare tissue microarray (TMA) and whole-section (WS) techniques in the routine assessment of HER-2 status in invasive breast cancer by immunohistochemistry and fluorescence in situ hybridization (FISH).
Methods and results:  HER-2 status was assessed prospectively in 106 consecutive cases of excised high-grade and/or node-positive breast carcinoma using both WS- and TMA-based techniques. Whole sections were assessed by immunohistochemistry with FISH being performed on equivocal cases (scoring 2+ on Hercep^®Test) and randomly selected 3+ cases included for quality assurance. Five 0.6-mm cores from each tumour allowed accurate immunohistochemical and FISH testing in >95% of cases. Ninety-seven per cent concordance was achieved between WS and TMA approaches to FISH analysis, the only discrepancies being in cases that were borderline or near borderline by both techniques. TMA and WS approaches were comparable in terms of time for preparation and scoring.
Conclusions:  TMA technology is a robust method of assessing HER-2 status in invasive breast cancer. This is directly comparable to the current standard methodology using whole sections. The use of TMA technology offers several advantages over existing full-section methods in terms of cost, quality control, facilitation of future research and the facility to provide a high-throughput testing methodology.