Targeted RNA expression profiling identifies high-grade endometrial stromal sarcoma as a clinically relevant molecular subtype of uterine sarcoma.

High-grade endometrial stromal sarcoma (HGESS) may harbor YWHAE-NUTM2A/B fusion, ZC3H7B-BCOR fusion, and BCOR internal tandem duplication (ITD). NTRK3 upregulation and pan-Trk expression were reported in soft tissue lesions that share similar morphology and genetic abnormalities. To confirm these findings in HGESS, differential expression analysis was performed at gene level comparing 11 HGESS with 48 other uterine sarcomas, including 9 low-grade endometrial stromal sarcomas, 23 undifferentiated uterine sarcomas, and 16 leiomyosarcomas, using targeted RNA sequencing data. Pan-Trk immunohistochemistry was performed on 35 HGESS, including 10 tumors with RNA expression data, with genotypes previously confirmed by targeted RNA sequencing, fluorescence in situ hybridization, and/or genomic PCR. Unsupervised hierarchical clustering of the top 25% of differentially expressed probes identified three molecular groups: (1) high NTRK3, FGFR3, RET, BCOR, GLI1, and PTCH1 and low ESR1 expression; (2) low NTRK3, FGFR3, RET, BCOR, GLI1, and PTCH1 and high ESR1 expression; and (3) low NTRK3, FGFR3, RET, BCOR, GLI1, PTCH1, and ESR1 expression. Among HGESS, 64% of tumors clustered in group 1, while 27% clustered in group 2. Cytoplasmic and/or nuclear pan-Trk staining of variable extent and intensity was seen in 91% of HGESS regardless of cyclin D1 and/or BCOR positivity. ER and PR expression was seen in 44% of HGESS despite ESR1 downregulation. Two patients with ER and PR positive but ESR1 downregulated stage I HGESS were treated with endocrine therapy, and both recurred at 12 and 36 months after primary resection. By RNA expression, HGESS appear homogenous and distinct from other uterine sarcomas by activation of kinases, including NTRK3, and sonic hedgehog pathway genes along with downregulation of ESR1. Most HGESS demonstrate pan-Trk staining which may serve as a diagnostic biomarker. ESR1 downregulation is seen in some HGESS that express ER and PR which raises implications in the utility of endocrine therapy in these patients.

Evaluating the impact of universal Lynch syndrome screening in a publicly funded healthcare system.

PURPOSE: Referrals for Lynch syndrome (LS) assessment have traditionally been based on personal and family medical history. The introduction of universal screening practices has allowed for referrals based on immunohistochemistry tests for mismatch repair (MMR) protein expression. This study aims to characterize the effect of universal screening in a publicly funded healthcare system with comparison to patients referred by traditional criteria, from January 2012 to March 2017. METHODS: Patient files from the time of initiation of universal screening from 2012 to 2017 were reviewed. Patients were sorted into two groups: (a) universally screened and (b) referred by traditional methods. Mutation detection rates, analysis of traditional testing criteria met, and cascade carrier testing were evaluated. RESULTS: The mutation detection rate of the universal screening group was higher than the traditionally referred group (45/228 (19.7%) vs 50/390 (12.5%), P = .05), though each were able to identify unique patients. An analysis of testing criteria met by each patient showed that half of referred patients from the universal screening group could not meet any traditional testing criteria. CONCLUSION: The implementation of universal screening in a publicly funded system will increase efficiency in detecting patients with LS. The resources available for genetic testing and counseling may be more limited in public systems, thus inclusion of secondary screening with BRAF and MLH1 promoter hypermethylation testing is key to further optimizing efficiency.

Population-based Screening for BRAF V600E in Metastatic Colorectal Cancer Reveals Increased Prevalence and Poor Prognosis.

PURPOSE: BRAF V600E mutations portend poor prognosis in metastatic colorectal cancer (mCRC); however, the true prevalence and prognosis are unknown, as unwell patients may not undergo BRAF sequencing. EXPERIMENTAL DESIGN: We reviewed a population-based cohort of 1,898 patients with colorectal cancer that underwent reflexive IHC mismatch repair (MMR) and BRAF V600E testing. Outcomes among IHC-detected BRAF V600E mCRC (BRAF IHC) were compared with patients with next-generation sequencing (NGS)-identified BRAF V600E-mutated mCRC from two institutions (BRAF NGS) with patients spanning from 2004 to 2018. RESULTS: All-stage population prevalence of BRAF V600E was 12.5% (238/1,898) and did not differ between early and metastatic stages (P = 0.094). Prevalence among mCRC was 10.6% (61/575), of whom 51 (83.6%) were referred to oncology and 26 (42.6%) had NGS testing. BRAF IHC had worse median overall survival (mOS) than BRAF NGS [5.5 vs. 20.4 months; HR, 2.90; 95% confidence interval (CI), 1.89-4.45; P < 0.0001], which persisted in multivariate analysis (P < 0.0001). Across a combined NGS and IHC cohort, BRAF V600E tumors with deficient MMR showed worse mOS compared with MMR proficient tumors (8.9 vs. 17.2 months; HR, 1.46; 95% CI, 0.96-2.27; P = 0.043). In this combined cohort, first-line progression-free survival was 5.9 months, with minimal differences between regimens. Within the population-based cohort, attrition between treatment lines was high with only 60.7% receiving first-line chemotherapy and 26.2% receiving second line. CONCLUSIONS: Patients with BRAF V600E-mutated mCRC have a worse prognosis than previously suggested, potentially arising from referral bias for testing. High attrition between lines of therapy suggests efficacious therapies need to be prioritized early for patients to benefit.

Mismatch repair deficiency and prognostic significance in patients with low-risk endometrioid endometrial cancers.

OBJECTIVES: Mismatch repair deficiency is observed in 25%-30% of all endometrial cancers. This can be detected by the absence of mismatch repair protein staining on immunohistochemistry, and is used as a screen for Lynch syndrome. Only 10% of women with mismatch repair deficiency have Lynch syndrome, but mismatch repair deficiency may still have prognostic significance. The objective of this study was to compare clinical outcomes between mismatch repair-deficient and mismatch repair-proficient low-risk endometrioid endometrial cancers (stage IA, grade 1 or 2). METHODS: This was a retrospective population-based cohort study of all low-risk endometrioid endometrial cancers (stage IA, grade 1 or 2) from the Vancouver Coastal Health Authority region from February 2011 to January 2016 that were assessed for mismatch repair deficiency. Any other histology, stage, or grade was excluded from the study. Primary outcome measures were progression-free survival and overall survival calculated using Kaplan-Meier method and log-rank tests. Cox proportional hazards model estimated the association between mismatch repair deficiency and recurrence and death after adjustment for covariates, expressed as hazard ratios (HRs). Secondary outcome measures were recurrence rates expressed per 100 person-years (p100py). RESULTS: There were 475 patients diagnosed with low-risk endometrioid endometrial cancer, including 131 with mismatch repair-deficient (27.6%) and 344 with mismatch repair-proficient (72.4%) tumors. Women with mismatch repair-deficient tumors had worse progression-free survival (24 months; p=0.0082) and higher recurrence rates (3.56 p100py) compared with those with mismatch repair-proficient tumors (27 months; 1.21 p100py, p=0.04). The absolute number of recurrences was overall low. There were 11 recurrences out of 131 mismatch repair-deficient cases (8.4%) and 14 out of 344 mismatch repair proficient cases (4.1%). After adjustment for age, lymphovascular space invasion status, adjuvant therapy, and post-operative grade, mismatch repair-deficient status remained associated with a higher risk of recurrence (HR 3.56, 95% CI 2.01 to 5.95). There was no significant difference in overall survival between mismatch repair groups (mismatch repair-proficient group 27.5 months vs 25.0 months in the deficient group) (HR 1.23, 95% CI 0.49 to 3.10). CONCLUSION: In low-risk stage IA grade 1 or 2 endometrioid endometrial cancers, mismatch repair deficiency is associated with a higher recurrence rate than mismatch repair proficiency after adjustment for covariates, implying that mismatch repair deficiency reflects a different biology in endometrial cancer.

Should you repeat mismatch repair testing in cases of tumour recurrence? An evaluation of repeat mismatch repair testing by the use of immunohistochemistry in recurrent tumours of the gastrointestinal and gynaecological tracts.

AIMS: The role of mismatch repair (MMR) testing has evolved from identifying Lynch syndrome patients to predicting response to immune checkpoint inhibitors. This has led to requests from clinicians to retest recurrences of MMR-proficient primary tumours in the hope that the recurrence may show a different MMR status and qualify the patient for treatment. We aimed to determine whether repeat testing is warranted. METHODS AND RESULTS: We evaluated recurrent tumours (local recurrences or metastases) from 137 patients with MMR-proficient primary tumours of the gastrointestinal and gynaecological tracts. The local recurrences and metastases all occurred at least 30 days after resection of the primary tumour. We used a combination of a tissue microarray and whole slide staining to perform immunohistochemistry (IHC) for PMS2, MLH1, MSH2, and MSH6, and compared the results with the MMR status of the primary tumour. Three of 137 (2%) initially showed a discordant staining pattern. However, further investigation showed that these discordances were attributable to some of the known pitfalls associated with MMR IHC interpretation - post-radiotherapy loss of MSH6 expression and subclonal loss of MLH1 staining. We did not identify any cases with a genuine discordance in MMR status. CONCLUSION: We conclude that repeat MMR IHC testing of recurrences is not warranted, as MMR status does not change relative to that of the primary tumour.

Immunohistochemistry Critical Assay Performance Controls (ICAPC) Reduce Interobserver Variability in the Interpretation of BRAFV600E Immunohistochemistry.

The utility of prognostic and predictive immunohistochemistry biomarkers in the context of cancer is plagued by inconsistent interpretation of results which can lead to poor rates of adoption or inappropriate use of novel therapeutic strategies. To monitor immunohistochemistry assay performance, a new on-slide control motif, Immunohistochemistry Critical Assay Performance Controls (ICAPC) was developed. We hypothesized that the use of these controls by the diagnosing pathologist to interpret BRAFV600E would result in reduced interobserver and intraobserver interpretation errors. A cross-sectional, sequentially obtained sample of surgical pathology cases stained for BRAFV600E was assembled from a single hospital in Vancouver, British Columbia. Half of the cases had normal on-slide controls and the remainder with ICAPC. Results from 6 independent and blinded readers were compared with each other and to the gold-standard pathologic diagnosis with the goal of demonstrating superior interrater agreement with ICAPC relative to standard on-slide controls. Cohen's κ was used to compute pair-wise reader agreements, whereas Fleiss' κ was used to compare to the gold standard. The implementation of ICAPC resulted in statistically significant improvements in the interobserver agreement of BRAF mutation status ascertained by BRAFV600E immunohistochemistry. Half of the readers demonstrated significant improvements in agreement with the gold-standard diagnosis with the addition of ICAPC. Across all readers, the mean increase in κ was 0.14 with a 95% confidence interval of 0.01-0.28 (P=0.04). This study demonstrates that the addition of ICAPC serves to significantly reduce interobserver variability in the assessment of BRAFV600E immunohistochemistry. As such, we recommend that this approach should be used as part of a comprehensive quality management strategy in the setting of histopathology.

Does MMR status in endometrial cancer influence response to adjuvant therapy?

OBJECTIVE: Mismatch repair (MMR) deficiency occurs in 20-40% of endometrial cancers but its therapeutic implication remains uncertain. Our objective was to compare clinical outcomes after adjuvant therapy between MMR deficient and proficient endometrial cancers from a population-based study. METHODS: This was a retrospective population-based cohort study of all endometrial cancers from the Vancouver Coastal Health authority region from 2011 to 2016, for which adjuvant therapy (radiotherapy and/or chemotherapy) was administered. Primary outcome measure was recurrence rates, expressed per 100 person-years (p100 py). Progression free survival (PFS) and overall survival (OS) rates were compared using Kaplan-Meier method and log-rank tests, and covariates were evaluated using Cox proportional hazards regression. RESULTS: There were 535 patients who received adjuvant therapy (radiotherapy and/or chemotherapy), including 162 (30.3%) and 373 (69.7%) with MMR-deficient and proficient tumors, respectively. Demographic variables were similar except MMR-deficient patients were younger (62.0 vs. 64.8, p = 0.01). Patients with MMR-deficient tumors were more likely to have endometrioid histotype (85.8% vs. 61.4%), more likely to have Stage I disease (62.3% vs 54.7%), and LVSI (65.4% vs. 53.4%) compared to those with MMR-proficient tumors. There was a trend for MMR-proficient group to have higher recurrence rates (10.7 p100 py vs 5.9 p100 py) and MMR deficiency was associated with better OS and PFS, but on multivariable analysis, MMR status was no longer significant. CONCLUSION: Women with MMR-deficient endometrial cancers who receive adjuvant therapy have a lower rate of recurrence compared to those with MMR-proficient cancers. However, on multivariable analysis, MMR status does not remain associated with differences in PFS or OS.

Endometrial Cancer Presentation and Outcomes Based on Mismatch Repair Protein Expression From a Population-Based Study.

OBJECTIVE: There is uncertainty about the prognostic significance of mismatch repair (MMR) deficiency in endometrial cancer. The objective was to evaluate clinical characteristics and outcomes of endometrial cancers based on MMR status within a population-based study. METHODS: This was a retrospective population-based cohort study of all endometrial cancer cases from the Vancouver Coastal Health Authority region, evaluated for 4 MMR proteins using immunohistochemistry from 2012 to 2015. Patients were classified as MMR deficient (dMMR, any MMR protein absent) or MMR proficient (pMMR), Demographics, tumor characteristics, recurrences, and survival rates were compared according to MMR status. RESULTS: There were 892 patients, with 650 pMMR (72.5%) and 242 dMMR tumors. The dMMR group had more endometrioid tumors (87.6% vs 74.0%, P < 0.001), lymphovascular space invasion (43.8% vs 30.8%, P = 0.001), and dedifferentiation (5.9% vs 1.5%, P < 0.001), but fewer grade 1 tumors compared with the pMMR group (31.8% vs 40.8%, P < 0.001). Median progression-free survival and overall survival have not been reached. After a median follow-up of 31 months (1-99 months), there was no difference in progression or recurrence rates between pMMR and dMMR tumors (19.5% vs 16.5%; P = 0.31). However, among those with nonendometrioid tumors, recurrence and mortality rates were significantly higher for pMMR than dMMR tumors (42.0% vs 10.0%, P = 0.001, and 36.1% vs 13.1%, P = 0.01, respectively), despite similar stage and lymphovascular space invasion distributions. DISCUSSION: In this population-based study, there were no significant differences in recurrence or survival outcomes according to MMR status in endometrial cancer. However, among those with nonendometrioid tumors, there were lower recurrence and mortality rates associated with MMR-deficient compared with MMR-proficient tumors.

Basal biomarkers nestin and INPP4b identify intrinsic subtypes accurately in breast cancers that are weakly positive for oestrogen receptor.

AIMS: Recent evidence indicates that weakly positive immunohistochemical staining of oestrogen receptor (ER) is not associated reliably with a luminal subtype, with the majority reclassified as basal-like by gene expression profile. In this study we assessed the capacity of recently identified immunohistochemical markers of basal-like subtype not dependent upon ER status - positive expression of nestin or loss of inositol polyphosphate-4-phosphatase (INPP4b) - to discriminate intrinsic subtypes, focusing on clinically problematic cases with weak ER positivity. METHODS AND RESULTS: Formalin-fixed paraffin-embedded blocks, enriched for large proportions of ER-negative and ER weakly positive breast cancers, were selected from two previous studies conducted in the period 2008-13 and used for (i) RNA extraction for 50-gene subtype predictor (PAM50) intrinsic subtyping and (ii) tissue microarray construction for immunohistochemical assessment of nestin and INPP4b. Fifty-eight cases were weakly positive for ER (Allred 3-5), among which 28 (48%) were assigned as basal-like by PAM50 gene expression. In these 58 cases, the nestin/INPP4b panel identified 23 basal-like cases with a positive predictive value of 87% [95% confidence interval (CI) 78-95%] and excluded luminal subtype with a negative predictive value of 95% (95% CI 88-100%). Weakly positive ER patients assigned as basal-like by nestin/INPP4b definition demonstrated a median survival time of 45.8 months, significantly lower than 65 months among other ER weakly positive cases (P = 0.012). CONCLUSIONS: Immunohistochemical assessment of nestin and INPP4b provides an accurate, accessible and inexpensive tool to identify basal-like breast cancer subtype in the clinically problematic setting of weak ER positivity. This panel identifies poor prognosis patients who might need strong considerations for non-endocrine-based therapies.

Molecular subtype profiling of invasive breast cancers weakly positive for estrogen receptor.

The estrogen receptor (ER) is a key predictive biomarker in the treatment of breast cancer. There is uncertainty regarding the use of hormonal therapy in the setting of weakly positive ER by immunohistochemistry (IHC). We report intrinsic subtype classification on a cohort of ER weakly positive early-stage breast cancers. Consecutive cases of breast cancer treated by primary surgical resection were retrospectively identified from 4 centers that engage in routine external proficiency testing for breast biomarkers. ER-negative (Allred 0 and 2) and ER weakly positive (Allred 3-5) cases were included. Gene expression profiling was performed using qRT-PCR. Intrinsic subtype prediction was made based upon the PAM50 gene expression signature. 148 cases were included in the series: 60 cases originally diagnosed as ER weakly positive and 88 ER negative. Of the cases originally assessed as ER weakly positive, only 6 (10 %) were confirmed to be of luminal subtype by gene expression profiling; the remaining 90 % of cases were classified as basal-like or HER2-enriched subtypes. This was not significantly different than the fraction of luminal cases identified in the IHC ER-negative cohort (5 (5 %) luminal, 83(95 %) non-luminal). Recurrence-free, and overall, survival rates were similar in both groups (p = 0.4 and 0.5, respectively) despite adjuvant hormonal therapy prescribed in the majority (59 %) of weakly positive ER cases. Weak ER expression by IHC is a poor correlate of luminal subtype in invasive breast cancer. In the setting of highly sensitive and robust IHC methodology, cutoffs for ER status determination and subsequent systemic therapy should be revisited.

Quantitative Detection and Resolution of BRAF V600 Status in Colorectal Cancer Using Droplet Digital PCR and a Novel Wild-Type Negative Assay.

A need exists for robust and cost-effective assays to detect a single or small set of actionable point mutations, or a complete set of clinically informative mutant alleles. Knowledge of these mutations can be used to alert the clinician to a rare mutation that might necessitate more aggressive clinical monitoring or a personalized course of treatment. An example is BRAF, a (proto)oncogene susceptible to either common or rare mutations in codon V600 and adjacent codons. We report a diagnostic technology that leverages the unique capabilities of droplet digital PCR to achieve not only accurate and sensitive detection of BRAF(V600E) but also all known somatic point mutations within the BRAF V600 codon. The simple and inexpensive two-well droplet digital PCR assay uses a chimeric locked nucleic acid/DNA probe against wild-type BRAF and a novel wild-type-negative screening paradigm. The assay shows complete diagnostic accuracy when applied to formalin-fixed, paraffin-embedded tumor specimens from metastatic colorectal cancer patients deficient for Mut L homologue-1.

Improved breast cancer biomarker detection through a simple, high frequency, low cost external proficiency testing program.

AIMS: We describe a simple, low cost, high frequency immunohistochemistry external proficiency testing program, and show how its use can lead to improved breast cancer biomarker detection. METHODS: Over a 30 month period in British Columbia, Canada, we used tissue microarray slides to follow the performance of twelve clinical laboratories in nine separate external proficiency testing runs. Sensitivity for detection of oestrogen receptor (ER), progesterone receptor (PR), and HER2 were calculated for each laboratory, biomarker, and run. RESULTS: Mean sensitivities for detection of ER, PR, and HER2 were 97.1%, 84.8%, and 90.7%, respectively. HER2 sensitivity improved over time, from 87.0% to 92.9% (p=0.04), with a trend towards improvement seen for PR (81.9-88.1%, p=0.13). ER sensitivities were high throughout the test period. Improvements occurred without mandating any specific laboratory changes. CONCLUSIONS: This simple, low cost, high frequency external proficiency testing program is highly sustainable and can be implemented in any multi-institutional group or region.

Implementation of a Canadian external quality assurance program for breast cancer biomarkers: an initiative of Canadian Quality Control in immunohistochemistry (cIQc) and Canadian Association of Pathologists (CAP) National Standards Committee/Immunohistochemistry.

Immunohistochemistry results for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 are used to guide breast carcinoma patient management and it is essential to monitor these tests in external quality assurance (EQA) programs. Canadian Immunohistochemistry Quality Control is a web-based program with novel approach to EQA. Canadian Immunohistochemistry Quality Control RUN2 included tissue microarray slides with 38 samples tested by 18 immunohistochemical laboratories. Deidentified results were posted for viewing at www.ciqc.ca including all used protocols matched with scanned slides for virtual microscopy and garrattograms. Sensitivity, specificity, Kendall W test (concordance between laboratories), and kappa statistics (agreement with designated reference values) were calculated. Kappa values were within the target range (>0.8, or "near perfect" agreement) for 85% results. Kendall coefficient was 0.942 for estrogen receptor, 0.930 for progesterone receptor, and 0.958 for human epidermal growth factor receptor 2. The anonymous participation, quick feedback, and unrestricted full access in EQA results provides rapid insight into technical or interpretive deficiencies, allowing appropriate corrective action to be taken whereas the use of tissue microarrays enables meaningful statistical analysis.