Lynch syndrome (LS) accounts for 2% to 4% of colorectal carcinomas (CRCs) and is characterized by tumors with microsatellite instability (MSI) and loss of mismatch-repair (MMR) protein expression due to germline mutations in MLH1, PMS2, MSH2, and MSH6.1–4 Identification of LS facilitates screening and risk reduction strategies.5 Therefore most centers have a screening strategy for patients presenting with CRC. Screening usually begins with either immunohistochemistry (IHC) for the 4 MMR enzymes MLH1, PMS2, MSH2, and MSH6 or polymerase chain reaction (PCR) for MSI, both of which are essentially equally effective.2,3,6
It has been debated whether MMR/MSI testing should be performed on all patients with CRCs or only on those with the “red flags” of synchronous or metachronous tumors, family history, distinctive histology, or young age at onset.6,7 There is now a trend toward widespread screening strategies including those that recommend reflex IHC testing for all patients with CRC regardless of age or other risk factors.7–9 Universal screening is more expensive but identifies significantly more cases of LS than targeted approaches, perhaps up to 28% of all cases of LS.3
Loss of expression of MLH1 and PMS2 is not limited to LS but also occurs due to somatic hypermethylation of the MLH1 gene promoter causing transcriptional silencing of MLH1 in 10% to 15% of all CRCs.3 Whereas PMS2-negative/MLH1-positive tumors or tumors with negative staining for MSH2 or MSH6 and intact PMS2/MLH1 are highly correlated with LS at any age,9 the proportion of CRCs with somatic hypermethylation of MLH1 (resulting in an MLH1-negative/PMS2-negative phenotype) increases with age as the absolute risk of LS decreases. It is the further investigation by molecular testing of this large number of CRC patients who have MLH1 silencing due to somatic hypermethylation that adds significant extra downstream cost to universal LS screening by reflex MMR IHC.
Activating mutations in BRAF are found in 5% to 25% of CRCs, with the vast majority being the BRAFV600E mutation.10–14 BRAFV600E mutation occurs in two thirds of CRCs with MLH1 silencing due to somatic hypermethylation but virtually never in CRCs with MSI due to LS.4 Therefore, BRAFV600E mutation is used as a proxy marker for hypermethylation in MLH1 IHC–negative tumors. Further testing for LS is commonly offered on MLH1-negative cases only if they are BRAF wild type.9
BRAFV600E mutation also occurs in microsatellite-stable (MSS) tumors. This group has emerged as a distinct molecular and clinical phenotype with a poor prognosis.12,15–22 BRAFV600E mutation is mutually exclusive with KRAS mutation, but its presence may predict a worse or absent response to EGFR inhibition.23 Therefore, the American National Comprehensive Cancer Network guidelines now recommend consideration of BRAF testing to guide therapy in the setting of KRAS wild-type metastatic CRC.24 Assessment of BRAF status in all CRC patients may provide useful therapeutic and prognostic information, but its routine assessment is justified only if it can be delivered cheaply and efficiently.
Recently, we developed a novel mouse monoclonal mutation-specific anti-BRAFV600E antibody (clone VE1) that can be used for IHC on routinely processed formalin-fixed paraffin-embedded (FFPE) tissue.25 This antibody reacts with the protein produced by the BRAFV600E mutation but not with wild-type BRAF or other BRAF mutants and has now been shown to be robust and reliable by several groups in several different tumors, including papillary thyroid cancer, melanoma, cerebral neoplasia, ovarian tumors, and hairy cell leukemia.25–32 Importantly, BRAF IHC can potentially be performed in any diagnostic pathology laboratory that currently offers MMR IHC. Given the workflow patterns in diagnostic pathology laboratories, BRAF IHC can be performed concurrently with MMR IHC at little extra cost. It is simply a matter of performing IHC for 5 markers rather than 4.
In this study, we validate an IHC test for the BRAFV600E mutation in CRC, compare it with current PCR-based approaches for the detection of MLH1 mutation carriers, and propose a new approach to screen for LS using reflex MMR and BRAFV600E mutation–specific IHC.
We searched the pathology database of Royal North Shore Hospital, Sydney, for all cases of CRC treated by surgical resection during the period 2006 to 2011. Exclusion criteria included extracolonic and appendiceal location, tumors treated endoluminally, and histologic type other than adenocarcinoma as defined by the World Health Organization 2010 system.33 Tumors were independently reviewed by 2 pathologists (C.W.T. and A.J.G.) to confirm the diagnosis and to reclassify the pathologic stage according to the seventh edition 2009 AJCC/TNM system.34 For resections involving synchronous tumors, the tumor with the highest pathologic stage was selected and annotated. Tissue microarrays (TMAs) comprising duplicate 1-mm diameter cores were created from FFPE tissue blocks.
IHC for BRAFV600E was performed on FFPE whole sections from all available CRC cases from the year 2011 using a commercially available mouse monoclonal anti-BRAFV600E antibody (clone VE1; provided by A.v.D. and D.C.; available at SpringBioscience, Pleasonton, CA).25 Cases with positive IHC staining (defined as diffuse strong positive staining of >75% of malignant cells) were scored as positive. IHC scoring was performed independently by 3 pathologists (A.J.G., C.W.T., and A.C.) blinded to all clinical, molecular, and pathologic data. Discordant scores were resolved by consensus review.
Molecular testing for BRAFV600E mutation was performed on macrodissected tumor tissue from the same block used for IHC using a multiplex PCR and matrix-assisted laser desorption/ionization-time of flight mass spectrometry detection assay (Sequenom MassArray).35 This platform has been specifically validated for BRAFV600E, V600R, V600K, and V600M, and the laboratory holds full National Association of Testing Authorities Australia accreditation for this assay. The paraffin blocks of discordant cases were macrodissected a second time and reanalyzed using a real-time PCR (rt-PCR)-based assay (Roche COBAS 4800 BRAF V600 Mutation Test kit). All molecular tests were performed blinded to clinical, pathologic and IHC data.
IHC for BRAFV600E and the 4 MMR proteins (MLH1, PMS2, MSH2, and MSH6) was performed on the TMA slides for the entire cohort of 2006 to 2011. The accuracy of BRAFV600E IHC on the TMA was validated by comparison with whole-section IHC and molecular testing performed on the cases from the year 2011. TMA IHC scoring was performed independently by 2 pathologists (A.J.G. and A.C.). Discordant cases were resolved by a third pathologist (C.W.T.). All assessors were blinded to clinical, molecular, and pathologic data.
To determine whether all known LS cases demonstrated a BRAF−/MSI phenotype, the files of the hospital familial cancer clinic were searched for all patients with genetically proven LS who underwent surgery for CRC during this period.
To further investigate the role of BRAF IHC in triaging formal genetic testing in patients with suspected LS based on negative staining for MLH1 and PMS2 in an external cohort processed in different laboratories, IHC was performed on archived FFPE tissue blocks of cases from the Jeremy Jass Memorial Tissue Bank of the Australasian Colorectal Cancer Family Registry (ACCFR). This cohort includes CRCs processed in multiple different laboratories with different fixation and processing approaches collected over a period of years. The ACCFR has been previously described, comprising a richly annotated cohort, which includes MMR IHC information correlated with molecular data such as MSI status, MMR gene mutation status, MLH1 and CpG island methylator phenotype (CIMP) methylation analysis, and BRAFV600E status determined by allele-specific PCR (AS-PCR).36–38 For this arm of the study CRC samples demonstrating loss of expression for MSH2 and/or MSH6 or solitary loss of PMS2 protein expression by IHC were excluded because of their very strong association with LS. IHC was interpreted blinded to all other data independently by A.J.G. and C.W.T.
Statistical analyses were performed using IBM SPSS Statistics v20 (detailed methods presented in supplementary methods). The following measures of test performance were determined: positive percentage agreement (PPA), negative percentage agreement (NPA), and overall percentage agreement (OPA).
This study protocol was approved by the QIMR HREC under protocol P628 and the Royal North Shore Hospital Ethics Committee under protocol 1201-035M. Patients who underwent genetic testing provided informed consent.
A search of all resected CRCs from 2006 to 2011 yielded 1456 cases. After excluding cases that did not have at least 1 tumor core on TMA, 1403 cases were available for BRAF and MMR IHC. All CRCs resected in the calendar year 2011 (n=216) were used to validate BRAF IHC against the multiplex PCR and MassArray Spectrometry–based assay. The comparison of molecular analysis and BRAF IHC on the cases from 2011 is summarized in Figure 1 and Supplementary Table 1 (http://links.lww.com/PAS/A174). Briefly, 15 cases failed to amplify or provide a result by MassArray leaving 201 cases with both IHC and molecular results. No mutations other than V600E were detected by PCR. There was complete agreement between IHC and MassArray in all but 7 cases (6 negative by PCR but positive by IHC and 1 positive by PCR but negative by IHC). When these 7 discordant cases were reevaluated using rt-PCR, all cases were positive. That is, rt-PCR favored the IHC result in 6 of 7 discordant cases. IHC was repeated on the apparently false-negative case a number of times and yielded the same result. It was noted that this case was predominantly composed of signet ring cells with little nonmucinous cytoplasm.
BRAF IHC on whole sections was readily interpretable by experienced observers. Although we chose the arbitrary cutoff of requiring 75% of malignant cells to show staining to be considered positive, the great majority of positive cases actually demonstrated diffuse strong homogenous cytoplasmic staining in essentially all malignant cells, whereas the great majority of negative cases showed completely absent staining in all malignant cells (Fig. 2). Patchy nonspecific staining was not uncommonly seen in some smooth muscle cells, mucin, and non-neoplastic colonic mucosa (sometimes with a peculiar nuclear pattern of staining). Occasional positive cases demonstrated only weak but still quite diffuse cytoplasmic staining. In these positive cases the pattern of staining in the neoplastic cells was still quite distinct and different from any nonspecific staining seen in the non-neoplastic cells.
When cases from 2011 stained on the TMA were compared with whole sections, there were 5 discordant cases all of which were false negatives of the TMA attributed on review to heterogenous staining. Compared with whole-section IHC, BRAF IHC on TMA demonstrated lower PPA, although NPA and OPA were comparable (Supplementary Table 2, http://links.lww.com/PAS/A174).
The scoring of BRAF IHC on whole sections for the cases from 2011 was completely concordant between 2 observers (κ score 1). The third observer only disagreed on 1 case (κ score 0.985). BRAF IHC on the TMA of 1403 cases showed excellent concordance between 2 observers (κ score 0.964). Scores for BRAF IHC on the ACCFR cohort were fully concordant between 2 observers (κ score 1).
The clinical and pathologic details of the full cohort of 1403 CRC cases assessed on TMA is summarized in Table 1, Supplementary Table 3 (http://links.lww.com/PAS/A174) and Supplementary Figure 1 and (http://links.lww.com/PAS/A174). Briefly, the prevalence of both BRAFV600E mutation and MSI was 20% with the following phenotypes recorded: BRAF−/MSS (1029 cases, 73%), BRAF+/MSS (98 cases, 7%), BRAF+/MSI (183 cases, 13%), and BRAF−/MSI (93 cases, 7%). In MSS tumors, BRAFV600E mutation was significantly associated with higher histologic grade, higher overall stage, and a predilection for the right colon (Supplementary Table 4, http://links.lww.com/PAS/A174). These differences were absent when BRAF status was compared in MSI tumors (Supplementary Table 5, http://links.lww.com/PAS/A174).
The results of MMR IHC subcategorized for BRAF staining are presented in Supplementary Table 6 (http://links.lww.com/PAS/A174). Briefly, 69% of MLH1−/PMS2− cases showed positive staining for BRAFV600E, as did 57% of triple-negative cases (MLH1−, PMS2−, MSH6−). Other than for the triple-negative cases, no MSH6-negative case showed positive staining for BRAFV600E. It is noteworthy that there were no MLH1-negative cases that were not PMS2 negative as well and no MSH2-negative cases that were not MSH6 negative as well. That is, IHC for only PMS2 and MSH6 would have detected all MMR-negative CRCs.
Of the 1403 cases in the TMA cohort, 11 cancers from 10 patients were confirmed to be associated with LS by molecular testing performed during routine care. All of these cancers demonstrated a BRAF−/MSI phenotype (Supplementary Table 7, http://links.lww.com/PAS/A174).
There were 51 CRC cases from 49 individuals obtained from both population-based and clinic-based recruitment from the ACCFR. Of the 51 CRCs, 39 (76%) demonstrated loss of staining for MLH1 and PMS2 by IHC, and 23 (45%) were positive for BRAFV600E mutation by AS-PCR (Table 2). Of the 23 CRCs demonstrating BRAFV600E mutation, 1 was associated with MLH1 mutation (c.790+2dupT r.[678_790del, 678_884del] p.?), 11 with MLH1 methylation, and the remaining 11 cases were MMR proficient. The CRC from the individual with both MLH1 germline mutation and BRAFV600E mutation was methylated at the RUNX3, CACNA1G, SOCS1, NEUROG1, and IGF2 loci, thereby demonstrating high levels of CIMP (CIMP-H). BRAF IHC was concordant with BRAFV600E determined by AS-PCR in all but 1 case, including the LS case with BRAFV600E mutation. The discordant case was positive by BRAF IHC but negative by AS-PCR and occurred in a patient with LS (MLH1 mutation c.678-1G>C r.spl? p.?). It did not demonstrate hypermethylation of the MLH1 gene promoter or a CIMP-H phenotype. Repeat testing of both the AS-PCR and BRAF IHC from freshly cut sections and extracted DNA from the same block did not change the findings of either test.
Personalized medicine strategies and increased recognition of familial cancer syndromes have created an urgent need for rapid and accurate molecular characterization of cancers in the clinical diagnostic setting. Many techniques have been developed to identify BRAF mutations. These methods are all PCR based, relatively time consuming, require significant infrastructure, are technically demanding, and are subject to tissue heterogeneity, sampling error, and suboptimal preservation of DNA in FFPE tissue. For these reasons, BRAFV600E testing has been difficult to deploy clinically particularly in resource poor settings.
In this study we have demonstrated that simple IHC performed on routinely processed FFPE tissue in a standard surgical pathology laboratory compares favorably to 2 different but commonly used molecular platforms. There was high agreement between IHC and a multiplex PCR-based assay in the determination of BRAF status on FFPE tissue [PPA=97.4%, 95% confidence interval (CI)=86.5%-99.5%; NPA=96.3%, 95% CI=92.2%-98.3%; OPA=96.5%, 95% CI=93.0%-98.3%). These figures are comparable to published data comparing an AS-PCR-based assay to sequencing (PPA=93.3%, 95% CI=66.0%-99.7%)30 and rt-PCR-based assay (Roche COBAS 4800 BRAF Mutation Test kit) to sequencing (PPA=96.4%, 95% CI=93.1%-98.2%; NPA=80.0, 95% CI=74.1%-84.8%; OPA=88.5, 95% CI=85.1%-91.1%).39
In fact our results suggest that BRAF IHC may outperform PCR MassArray in the routine clinical setting. For example, IHC provided a result in all cases (versus a failure rate of 7% for MassArray), and, using rt-PCR as the gold standard in discordant cases, IHC provided the “correct” result in 200 of 201 cases (99.5%), whereas MassArray was “correct” in only 195 of 201 cases (97%).
The BRAF antibody we used only reacts with the protein product of the V600E mutant and not with proteins associated with other mutations of BRAF.25 This makes it ideal for use in CRC because BRAF mutations other than V600E are rare and not associated with somatic hypermethylation.10–14
Royal North Shore Hospital in Sydney, Australia, performs centralized pathology testing for 2 quaternary referral hospitals with dedicated colorectal surgery units as well as 4 community hospitals. Therefore the cases processed at this center represent a true snapshot of CRC in the community rather than being biased toward the patient populations commonly seen in study cohorts. In conjunction with the increased sensitivity of BRAFV600E IHC, this older unselected population with its tendency to somatic hypermethylation may account for our relatively high BRAF mutation rate (20%) and low rate of confirmed LS (0.8%).10–14 It is noteworthy that even in this cohort in which there were 276 MSI cases (20%), LS may be considered virtually excluded in the 183 cases that showed positive staining for BRAFV600E, leaving only 93 CRCs (7% of the entire CRC population) requiring formal genetic counseling and further testing.
We note that only selected cases in the TMA cohort underwent genetic testing as part of their clinical care. Therefore, this cohort may include some cases of unrecognized LS—a potential limitation of this study. However, it is reassuring that all 11 CRCs known to be associated with LS were identified by our IHC approach and that the yield of targeted molecular testing in this BRAF−/MSI group is therefore at least 12% (11 of 93).
We caution that all screening strategies have limited sensitivity and specificity. For example, although MMR IHC and formal MSI analysis demonstrate similar sensitivity and specificity, neither will identify all cases of LS.2,3 Similarly, BRAFV600E-mutant CRCs can occur in LS patients, albeit in only a few reported cases (<1%).10–14 These potential pitfalls are well illustrated in our targeted examination of cases from the ACCFR. In this cohort there was 1 false-positive result of BRAFV600E IHC compared with AS-PCR in a patient with LS and 1 true-positive result of BRAFV600E IHC in a tumor from a patient with LS which also harbored this mutation. Therefore, regardless of IHC findings, we recommend that formal genetic testing be considered in individuals at very high risk for LS based on clinical findings.
BRAF IHC has been shown to be robust and reliable by several different groups studying different tumors. Although occasional false-negative staining has been reported, false positives (which would result in LS being incorrectly discounted) are rare in experienced hands.26–32 However, we caution that deployment of BRAFV600E IHC in the clinical setting should be subject to an appropriate quality assurance program with prospective validation and that great care should be taken in optimizing the antibody for use in individual laboratories. We found that the antibody performed very well but only after ideal dilutions, and antigen retrieval procedures were formulated for our particular laboratory conditions. Although there was commonly nonspecific staining of mucus, non-neoplastic epithelium, and sometimes smooth muscle cells, nonspecific staining was not found in malignant cells once conditions were optimized. During the optimization process or if conditions are not ideal, we would recommend paying careful attention to the degree of nonspecific staining in non-neoplastic cells compared with the degree of staining in neoplastic cells before cases that show weak staining are classified as positive or negative. Because BRAFV600E is a cytoplasmic stain, particular care is recommended in interpreting cases with a mucinous histology with minimal cytoplasm.
Identification of the BRAFV600E mutation in MSS tumors may emerge as a beneficial by-product of this approach to universal LS screening in light of studies that demonstrate that these tumors are associated with a significantly worse outcome.12,15–19,21,22
We estimate the primary antibody costs of performing BRAFV600E IHC as being <US$10 per case with minimal additional labor costs as MMR IHC is concurrently performed. It is noteworthy that our study also confirmed the findings of others suggesting that, when interpreted with care by an experienced pathologist, IHC for PMS2 and MSH6 alone identifies all cases of MMR.40–42 Therefore the cost of BRAFV600E IHC could potentially be completely offset by omitting IHC for MLH1 and MSH2 from LS screening programs.
In conclusion, IHC for BRAFV600E mutation is highly concordant with current PCR-based approaches and effective in the diagnosis of LS. We propose that BRAF and MMR IHC can be performed on all CRC patients at point-of-care diagnostic testing on surgical excision specimens. One potential algorithm is presented in Figure 3. There are clear advantages in terms of laboratory workflow and triaging referral for formal genetic analysis in performing both MMR and BRAF IHC together rather than sequentially, and this approach has the added benefit of identifying the emerging poor prognostic group of BRAFV600E-mutated, MSS tumors.
The authors thank all study participants of the ACCFR and study coordinator Judi Maskiell, data managers Kelly Aujard, Maggie Angelakos, and David Packenas, and laboratory staff Belinda Nagler and Sally-Ann Pearson. The authors also acknowledge the contributions of the late Professor Jeremy Jass to the study including performing pathology reviews for cases.
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