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Clinical Classification of BRCA1 and BRCA2 DNA Sequence Variants: The Value of Cytokeratin Profiles and Evolutionary Analysis—A Report From the kConFab Investigators

Amanda B. Spurdle, Sunil R. Lakhani, Sue Healey, Suzanne Parry, Leonard M. Da Silva, Ross Brinkworth, John L. Hopper, Melissa A. Brown, Davit Babikyan, Georgia Chenevix-Trench, Sean V. Tavtigian, David E. Goldgar

From the Queensland Institute of Medical Research; School of Medicine, and School of Molecular and Microbial Sciences, University of Queensland, Brisbane; Centre for Genetic Epidemiology, University of Melbourne and Peter MacCallum Cancer Centre, Melbourne, Australia; International Agency for Research on Cancer, Lyon, France; and the Department of Dermatology, University of Utah, Salt Lake City, UT

Corresponding author: Amanda B. Spurdle, PhD, Queensland Institute of Medical Research, c/o Royal Brisbane Hospital Post Office, Herston, Queensland 4029, Australia; e-mail: Amanda.Spurdle{at}qimr.edu.au

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS REFERENCES

 
Purpose Rare missense substitutions and in-frame deletions of BRCA1 and BRCA2 genes present a challenge for genetic counseling of individuals carrying such unclassified variants. We assessed the value of tumor immunohistochemical markers in conjunction with genetic and evolutionary approaches for investigating the clinical significance of unclassified variants.

Patients and Methods We studied 10 BRCA1 and 12 BRCA2 variants identified in Australian families with breast cancer. Analyses assumed a prior probability based on revised cross-species sequence alignment methods assessing amino acid evolutionary conservation and position, combined with likelihoods from data on co-occurrence with pathogenic mutations in the same gene, segregation analysis, and immunohistochemistry. We specifically explored the value of estrogen receptor, cytokeratin 5/6, and cytokeratin 14 as tumor markers of BRCA1 mutation status.

Results Posterior probabilities classified 72% of variants. BRCA1 variants IVS18+1 G>T (del exon 18) and 5632 T >A (V1838E) were classified as pathogenic, with >99% posterior probability of being deleterious, and tumor histopathology was particularly important for their classification. BRCA2 variant classification was improved over previous studies, largely by incorporating the prior probability of pathogenicity based on amino acid cross-species sequence alignments.

Conclusion Variant classification was considerably improved by analysis of estrogen receptor, cytokeratin 5/6, and cytokeratin 14 tumor expression, and use of updated methods estimating the clinical relevance of amino acid evolutionary conservation and position. These methodologies may assist genetic counseling of individuals with unclassified sequence variants.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS REFERENCES

 
Screening of the breast cancer susceptibility genes BRCA1 and BRCA2 identifies numerous nucleotide sequence changes of varying clinical significance. The effect of rare changes predicted to cause missense substitutions or in-frame exon deletions is often not clear, and presents a challenge in the clinical setting. The scale of the problem is considerable, with approximately 30% of BRCA1 and 60% of BRCA2 entries in the Breast Cancer Information Core database for BRCA1 and BRCA2 variation¹ described as unclassified variants (UVs).

An integrated approach to classification of UVs in BRCA1 and BRCA2 into high-risk mutations and neutral variants was developed to define a reliable protocol for prediction of the clinical significance of UVs.² This multifactorial likelihood model used data on co-occurrence of the UV with pathogenic mutations in the same gene, segregation in families, and amino acid physicochemical properties and evolutionary conservation. The model was used to estimate the odds of causality, a ratio of the likelihood of the observed data under the hypothesis of causality to that under the hypothesis of neutrality. Because the model cannot distinguish between variants that are truly benign and those that might be associated with modest risk, neutral variants are sometimes alternatively termed to be of low/little clinical significance (neutral/LCS). We recently revised the model to take into account relevant features of BRCA1- and BRCA2-associated tumors, including the characteristic histopathology associated with pathogenic mutations in BRCA1. Use of the revised model classified 56% of 25 unselected UVs (seven of 10 BRCA1 and seven of 15 BRCA2) from Australian families with breast cancer.³ Fewer BRCA2 UVs were classified, largely because BRCA2 evolutionary predictions were less robust (fewer cross-species sequences were available at that time, relative to BRCA1), and because BRCA2 mutations are not associated with very distinctive pathologic features. Overall, these findings suggested that further extension of the model may improve classification rates, and that classification of BRCA2 UVs particularly would be improved with more robust evolutionary predictions.

Several immunohistochemical (IHC) markers, including tumor expression of estrogen receptor (ER), cytokeratin 5/6 (CK5/6), cytokeratin 14 (CK14), and cytokeratin 17 (CK17) have been shown to be indicators of BRCA1 mutation status.^4-7 We undertook a study to investigate the clinical significance of a new panel of BRCA1 and BRCA2 UVs identified in Australian families with breast cancer, specifically exploring the value of a subset of these additional IHC markers as histopathologic classifiers of BRCA1 tumors, and considering the prior probability of pathogenicity based on improved evolutionary conservation analysis methods.

	PATIENTS AND METHODS

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Subjects and Laboratory Methodology
Pedigrees with UVs in BRCA1 and BRCA2 were ascertained by the Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab).⁸ Participants provided written informed consent, and relevant ethics committees approved the study. A total of 276 individuals (probands and family members) were available for genetic screening. Cancer-free controls (n = 180) from the Australian Breast Cancer Family Study have been described previously.³ Denaturing high performance liquid chromatography and sequencing were as described previously,³ using primers detailed in Table 1. IHC was performed using standard methods.^6,9 The study included 23 families with 22 different UVs (Tables 2 and 3).

Prior Probability of Pathogenicity From Amino Acid Conservation and Location of the Mutation in Specific Known Functional Domains
Missense substitutions and in-frame deletions were classified according to their location within one of two recognized functional domains of the proteins, the C-terminus region containing the BRCA1 BRCT repeats, defined loosely as amino acids 1396 to 1862, and the BRCA2 DNA-binding domain (DBD; amino acids 2500 to 3098). Variants were further categorized according to whether the wild-type residue involved in the substitution/deletion was evolutionarily conserved through to the pufferfish Tetraodon, using multiple sequence alignments. Heterogeneity analysis of 1,433 variants in the Myriad Genetics Laboratory database was used to estimate the proportion of deleterious variants in three classifications¹³: invariant position in BRCT/DBD domain, proportion = 0.73; variable position in BRCT/DBD domain, proportion = 0.08; and position outside of BRCT/DBD domain whether invariant or variable, proportion = 0.02. The values were then used as prior probabilities of being deleterious for classification of the studied variants.

Co-Occurrence With Pathogenic Mutations
We queried the Myriad Genetic Laboratories database of approximately 90,000 full-sequence tests to determine the number of times a UV was observed, and the number of different deleterious mutations observed to co-occur with each variant, as a measure of the number of times the UV is seen in trans with a deleterious mutation. Phase of the variant and mutation was established for a subset of individuals. Observations for variants were excluded if in cis with a mutation, included if in trans with a mutation, and assumed to be in trans with at least n-1 observations for n observations with different deleterious mutations of unknown phase.¹⁴

Histopathology and Pedigree Causality Analysis
Available invasive tumor sections were analyzed by two pathologists (S.L., L.Da.S.) blind to UV status, for parameters recognized to be associated with BRCA1 or BRCA2 mutation status.^6,15,16 IHC scoring was performed by a single pathologist (S.L.) as described previously.⁶

Pedigree Causality Analysis, Derivation of Probabilities, Multifactorial Likelihood Scoring
Bayes factor analysis was as described previously.^3,13,17 Probabilities were derived for each of the following components, under the assumption that each factor was independent. For the co-occurrence component, we estimated the likelihood that a UV was causal as described previously.² The Bayes factor was included directly as a likelihood ratio (LR) score for the pedigree analysis component. For BRCA1, ER, CK5/6, and CK14 expression was used for calculating histopathologic LR scores, based on the previously reported prevalence of the combined immunotypes of these independent predictors of BRCA1 mutation status in breast tumors.⁶ The likelihoods for causality were: ER positive (irrespective of CK score) = 0.14:1; negative for all three markers = 0.87:1; ER negative, CK14 negative, CK5/6 positive = 5.6:1; ER negative, CK14 positive, CK5/6 negative = 2.6:1; ER negative, CK14 positive, CK5/6 positive = 27.4:1. Categories were collapsed for scoring the single ER-negative, CK14-positive tumor lacking CK5/6 data (LR, 8.8:1). The likelihood was calculated from ER expression and grade (LR, 2.95:1), as previously described,³ for the single ER-negative grade 3 tumor lacking CK data. For BRCA2, tubule formation was used for LR estimates, based on the previously reported prevalence of this independent predictor of BRCA2 mutation status.¹⁶ Likelihoods were: tubule formation in less than 10% of tumor = 1.2:1; tubule formation 10% = 0.5:1, as used previously.³

The individual LRs were multiplied to calculate an overall multifactorial likelihood ratio assuming statistical independence of the sources of information. Bayes rule was then used to calculate a posterior probability that the variant was deleterious from the multifactorial LR, and the prior probability determined from sequence alignment. Variants with a posterior probability 0.99 were classified as pathogenic, and those with probability .001 were classified as neutral/LCS. Example calculations are provided in Tables 2 and 3.

	RESULTS

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We analyzed a total of 10 BRCA1 UVs and 12 BRCA2 UVs in this study (Tables 2 and 3). All UVs were predicted missense substitutions, except for two in-frame exon deletions, BRCA1 IVS18+1 G>T (del exon 18) and BRCA2 9345 G>A (del exon 23). All UVs fell in regions reported to interact with at least one other protein thought to be involved in DNA repair. Protein modeling predictions were possible for only two variants: BRCA1 5632 T>A (V1838E) in the BRCT domain, and BRCA2 5506 T>G (S1760A) in the BRAF35 interaction domain. The BRCA1 V1838E substitution creates a charged hydrophilic group in a hydrophobic patch, and the glutamine side chain is predicted to clash with the Leu1790 side chain, and to a lesser extent with the Phe1761 side chain. This alteration might thus be expected to have serious effects on protein structure. The BRCA2 S1760A substitution is between the BRCA2 repeat 5 (1664 to 1698) and repeat 6 (1837 to 1871), and would not cause a major effect on protein structure, but may be important in that it would remove a potential N-linked glycosylation site. Screening of controls for all UVs under study identified a single UV, BRCA1 1767 A>C (N550H), in 1/180 controls.

Amino acid position and sequence alignment analysis was used to group missense substitutions into categories, as described in the Patients and Methods section. Overall, eight variants fell within the BRCT/DBD domains, only two of which were conserved to Tetraodon (Tables 2 and 3). Prior probabilities were assigned according to category, for inclusion in the multifactorial analysis.

All the UVs seen in these families have been identified at least once by Myriad Genetic Laboratories Inc. Five BRCA1 and five BRCA2 UVs were considered to co-occur in trans with at least one known deleterious mutation in the same gene, and phase was proven to be in trans at least once for all but one (BRCA2 3743 C>T [S1172L]). LRs based on co-occurrence alone were indicative of neutrality/LCS for five of five BRCA1 UVs and three of five BRCA2 UVs with co-occurrent mutations. Furthermore, a homozygote with BRCA2 3031 G>A (D935N) and no clinical features of Fanconi anemia has been identified in the Myriad data set, further suggesting that this variant is neutral/LCS. Segregation analysis provided firm evidence regarding causality for eight UVs, including the single family carrying the BRCA1 655 A>G (Y179C), 1575 T>C (F486L), and 1767 A>C (N550H) variants in cis.

There was at least one tumor sample available for pathology review and IHC screening for all but two BRCA1 and one BRCA2 UVs (Tables 2 and 3). The specific BRCA1 histopathologic features included in LR estimates provided evidence that BRCA1 5632 T>A V1838E and IVS18+1 G>T (del exon 18) were likely pathogenic. All these tumors were ER negative and high grade, all five with available CK5/6 data expressed this basal marker of BRCA1 mutation status, and CK14 was expressed in four of seven tumors. No other tumors from carriers of BRCA1 UVs displayed cytokeratin expression consistent with a BRCA1 mutation. Of the 14 BRCA2 tumors screened, one of two tumors for K513R had histopathologic features of BRCA1 tumors (high grade, ER negativity, focal positivity for CK14). This result was consistent with the fact that this individual (but not the other relative screened) was found to carry a BRCA1 pathogenic mutation during the course of this study.

The combined odds of causality for each variant were derived as described in the Patients and Methods section, including LRs for pathology, co-occurrence, and segregation data, to generate an intermediate classification. Of the 10 BRCA1 UVs studied, five were classified as neutral/LCS and two as pathogenic. The overall classification rate was less for the 12 BRCA2 UVs, with six classified as neutral/LCS. After combination of the multifactorial odds with prior probabilities based on sequence analysis, two BRCA1 variants (2878 T>C [V920A]; 3827 T>G [N1236K]), and four BRCA2 variants (2048 A>C [K607T]; 5506 T>G [S1760A]; 8801 A>G [Q2858R]; 9345 G>A [del exon 23]) remained unclassified.

	DISCUSSION

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Our study shows that multifactorial likelihood analysis, in combination with prior probabilities based on evolutionary and physicochemical conservation, is a valuable tool to evaluate clinically problematic BRCA1/2 sequence variants. This approach classified 72% of the 22 UVs studied. Only two BRCA1 UVs (5632 T>A [V1838E] and IVS18+1 G>T [del exon 18]) were classified as pathogenic, and there was strong evidence for pathogenicity (86% posterior probability of being deleterious) for one BRCA2 variant, 9345 G>A (del exon 23). This highlights the general understanding that the majority of UVs are individually of little clinical importance, based on the fact that the number of UVs far exceeds the proportion of families linked to BRCA1 or BRCA2 but with no identified pathogenic mutation. Both BRCA1 variants fall within or span the BRCT motifs, domains well recognized to be critical for BRCA1 function, while BRCA2 exon 23 spans the BUBR1A/FilaminA/BCCI p-alpha interaction sites and contains residues conserved to Arabidopsis.

The interpretation from protein modeling data was also consistent with final classification. BRCA1 V1838E was predicted to have a serious effect on protein structure, and this variant was classified as pathogenic. Likewise, evidence from modeling of BRCA2 5506 T>G (S1760A) was more equivocal, and this variant remained unclassified. Results from control screening supported our previous suggestion that this approach might be a simple adjunct to UV evaluation for nonfounder populations,³ with a neutral/LCS classification for the single UV observed in controls. While underpowered to evaluate pathogenicity of rare variants, it is a useful prescreen to exclude common variation in little studied population groups.

As shown previously, the most informative components of the multifactorial likelihood predictions were co-occurrence and pedigree analyses. Co-occurrence scores alone were sufficient to classify six UVs (27%), and odds from segregation analysis classified eight UVs (36%), including three variants in cis. A total of 11 UVs (50%) could be classified considering only co-occurrence and segregation data.

Information from tumors was available for most UVs, but only added weight to the final classification for the subset of pathogenic BRCA1 UVs. This indicates that ER and cytokeratin IHC would be very helpful in the classification of likely pathogenic BRCA1 UVs. Although cytokeratin IHC is not yet routinely carried out in all diagnostic laboratories, the scores for agreement on staining positivity of CK5/6 and CK14 reported for a large data set were 0.74 to 0.82 (SE, 0.06 to 0.07), corresponding to concordance rates of 88% to 92%.⁶ This suggests that cytokeratin staining could be introduced into routine histopathologic practice, especially in larger centers, to provide information for evaluation of BRCA1 UVs. In our experience, CK5/6 and CK14 IHC can be implemented relatively easily if appropriate quality controls are in place—as most pathology laboratories practice routinely. We recognize that the IHC approach relies on the underlying assumption that missense and in-frame deletions will exhibit the same tumor histopathologic characteristics as truncating mutations, but have confidence that it is valid for classification of high-risk BRCA1 mutations from our published^17a and unpublished data showing that functionally deficient BRCA1 RING and BRCT domain missense substitutions exhibit immunohistopathologic profiles consistent with a BRCA1 mutation.

Pathology scores for BRCA2 UVs were not very informative individually, but in two instances contributed to the final classification. In this study, we did not include analysis of tumor loss of heterozygosity for BRCA1 and BRCA2, as done previously,³ because analysis of this and our previous data set revealed statistically significant increased loss of the variant compared with what is expected for the underlying hypothesis used previously to calculate likelihood estimates.

Sequence alignment analysis was useful for refining the classification, especially for BRCA2 variants. The increased value of this approach over our previous study³ was largely due to increasing the informativeness with additional sequences in the BRCA2 alignment, and the incorporation of estimates of prior probabilities based on both alignment and location within functionally important domains. BRCA1 and BRCA2 protein multiple sequence alignments used for this analysis (or updated versions thereof) are publically available through the web-based program and alignments at http://agvgd.iarc.fr (International Agency for Research on Cancer; Align GVHD). Thus, investigators could use this resource to estimate prior probabilities for any BRCA1 or BRCA2 missense substitutions of interest. However, the clinical application of the methods presented in this study should be undertaken only with consideration of the caveats associated with the approaches, principally that prior probabilities and likelihood ratio estimates were derived assuming that variants were either neutral or of similar high risk as the average BRCA1/2 mutation,¹⁸ and from analysis of single data sets for the prior probability, co-occurrence and histopathology components, and that likelihoods are valid for patients with familial breast cancer. Moreover, it is likely that further development of sequence information-based estimates of prior probability to incorporate the physicochemical characteristics of unclassified variants as assessed by Align GVGD analysis¹⁴ will refine different classes of substitutions, and thus improve the precision of the prior probabilities associated with individual substitutions.

Few of the UVs studied here have been analyzed previously, and none using the range of approaches we examined. Comparisons to larger studies with several points of evidence are not inconsistent with our findings. Co-occurrence and evolutionary conservation analysis of BRCA1 missense variants^14,19 classified BRCA1 Y179C, F486L, N550H, and P1099L as neutral/LCS, and another study found F486L to be absent in 1054 ethnicity-matched controls, but failed to classify it using a multifactorial approach based on odds derived from tumor information, bilaterality, and family history of ovarian cancer.²⁰ The BRCA1 T826K and BRCA2 S1172L, T2515I, and A2717S UVs were all reported to be present in the proband from at least one German family with breast/breast-ovarian cancer but absent from 200 ethnically matched controls.²¹ The BRCA2 K2950N variant was observed in a patient with familial prostate cancer, but also in two of 340 normal chromosomes, supporting the neutral/LCS classification from this study.²²

Few variants have been studied functionally. The splice site variant BRCA2 9345 G>than A has been reported to cause an in-frame deletion of exon 23 from reverse transcription polymerase chain reaction studies of peripheral blood lymphocyte RNA.²³ The T2515I amino acid substitution was shown to be associated with normal expression levels, complete ablation of cell survival activity, partial inactivation of homologous recombination and centrosome regulatory functions, and a cytoplasmic-nuclear localization profile intermediate between the aberrant predominantly nuclear localization of known mutations and the cytoplasmic localization for wild-type BRCA2.²⁴ These data suggest this alteration has a subtle effect on BRCA2 function, and is thus unlikely to be associated with a high risk of cancer. Indeed, the same study reported low odds in favor of causality from segregation analysis,²⁴ and our multifactorial analysis classified T2515I as neutral/LCS.

In summary, we have provided evidence for the classification of 16 of 22 different BRCA1 or BRCA2 sequence variants. Our findings support the general understanding¹³ that the majority of unclassified variants of BRCA1 and BRCA2 are not associated with a high risk of disease as associated with classical truncating mutations. Classification of individual variants is necessary to identify the subset demonstrating features of classical mutations with high risk of disease, and is considerably improved by analysis of ER, CK5/6, and CK14 tumor expression, and updated methods to estimate the clinical relevance of amino acid evolutionary conservation and position. These methodologies may be easily implemented, and together with supporting information provided by additional studies such as functional assays, may assist genetic counseling of unclassified sequence variants.

	AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS REFERENCES

 
The author(s) indicated no potential conflicts of interest.

	AUTHOR CONTRIBUTIONS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS REFERENCES

 
Conception and design: Amanda B. Spurdle

Financial support: Amanda B. Spurdle, Melissa A. Brown

Administrative support: Amanda B. Spurdle, Sue Healey

Provision of study materials or patients: John L. Hopper

Collection and assembly of data: Amanda B. Spurdle, Sunil R. Lakhani, Sue Healey, Suzanne Parry, Leonard M. Da Silva, Ross Brinkworth, Davit Babikyan, Georgia Chenevix-Trench, David E. Goldgar

Data analysis and interpretation: Amanda B. Spurdle, Sunil R. Lakhani, Sue Healey, Suzanne Parry, Ross Brinkworth, Melissa A. Brown, Georgia Chenevix-Trench, Sean V. Tavtigian, David E. Goldgar

Manuscript writing: Amanda B. Spurdle, Sunil R. Lakhani, Sue Healey, Georgia Chenevix-Trench, Sean V. Tavtigian, David E. Goldgar

Final approval of manuscript: Amanda B. Spurdle, Sunil R. Lakhani, Sue Healey, Suzanne Parry, Leonard M. Da Silva, Ross Brinkworth, John L. Hopper, Melissa A. Brown, Davit Babikyan, Georgia Chenevix-Trench, Sean V. Tavtigian, David E. Goldgar

	ACKNOWLEDGMENTS

 
We thank Heather Thorne, Eveline Niedermayr, the Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab) research nurses and staff, heads and staff of the Family Cancer Clinics, and the clinical follow-up study for their contributions to kConFab, and the many families who contribute to kConFab. We thank Amie Deffenbaugh for data on variant co-occurrence with mutations from the Myriad Genetic Laboratories database.

	NOTES

 
The Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab) is supported by grants from the National Breast Cancer Foundation, the National Health and Medical Research Council (NHMRC) and by the Queensland Cancer Fund, the Cancer Councils of New South Wales, Victoria, Tasmania, and South Australia, and the Cancer Foundation of Western Australia. The kConFab clinical follow-up study was funded by NHMRC Grants No. 145684 and 288704; and by a grant from the Susan G. Komen Breast Cancer Foundation, and the NHMRC. J.L.H. and G.C-T. are NHMRC senior principal research fellows; A.B.S. is funded by an NHMRC career development award; L.Da.S. is supported by a fellowship from the Ludwig Institute for Cancer Research; D.B. is a recipient of a post-doctoral fellowship from the International Agency for Research on Cancer; and S.V.T. and D.E.G. were supported in part by the INHERIT BRCAs programme from the Canadian Institute for Health Research, and a subaward agreement from the Mayo Clinic, Rochester, MN.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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Submitted June 26, 2007; accepted December 5, 2007.

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