Fit-for-Purpose Ki-67 Immunohistochemistry Assays for Breast Cancer.

New therapies are being developed for breast cancer, and in this process, some "old" biomarkers are reutilized and given a new purpose. It is not always recognized that by changing a biomarker's intended use, a new biomarker assay is created. The Ki-67 biomarker is typically assessed by immunohistochemistry (IHC) to provide a proliferative index in breast cancer. Canadian laboratories assessed the analytical performance and diagnostic accuracy of their Ki-67 IHC laboratory-developed tests (LDTs) of relevance for the LDTs' clinical utility. Canadian clinical IHC laboratories enrolled in the Canadian Biomarker Quality Assurance Pilot Run for Ki-67 in breast cancer by invitation. The Dako Ki-67 IHC pharmDx assay was employed as a study reference assay. The Dako central laboratory was the reference laboratory. Participants received unstained slides of breast cancer tissue microarrays with 32 cases and performed their in-house Ki-67 assays. The results were assessed using QuPath, an open-source software application for bioimage analysis. Positive percent agreement (PPA, sensitivity) and negative percent agreement (NPA, specificity) were calculated against the Dako Ki-67 IHC pharmDx assay for 5%, 10%, 20%, and 30% cutoffs. Overall, PPA and NPA varied depending on the selected cutoff; participants were more successful with 5% and 10%, than with 20% and 30% cutoffs. Only 4 of 16 laboratories had robust IHC protocols with acceptable PPA for all cutoffs. The lowest PPA for the 5% cutoff was 85%, for 10% was 63%, for 20% was 14%, and for 30% was 13%. The lowest NPA for the 5% cutoff was 50%, for 10% was 33%, for 20% was 50%, and for 30% was 57%. Despite many years of international efforts to standardize IHC testing for Ki-67 in breast cancer, our results indicate that Canadian clinical LDTs have a wide analytical sensitivity range and poor agreement for 20% and 30% cutoffs. The poor agreement was not due to the readout but rather due to IHC protocol conditions. International Ki-67 in Breast Cancer Working Group (IKWG) recommendations related to Ki-67 IHC standardization cannot take full effect without reliable fit-for-purpose reference materials that are required for the initial assay calibration, assay performance monitoring, and proficiency testing.

Comprehensive Clinical-Pathologic Assessment of Malignant Phyllodes Tumors: Proposing Refined Diagnostic Criteria.

The latest World Health Organization classification of breast tumors recommends diagnosing malignant phyllodes tumors (MPTs) when all 5 morphologic features are present: permeative borders, marked stromal cellularity, marked stromal cytologic atypia, ≥10 mitoses per 10 high-power fields (HPF), and stromal overgrowth. We assessed the performance of this recommendation to capture MPTs and features predictive of distant metastasis in a multi-institutional retrospective study. Of 65 MPTs, most cases had at least focally permeative borders (58, 89%), with marked stromal cellularity in 40 (61.5%), marked atypia in 38 (58.5%), ≥10 mitoses per 10 HPF in 50 (77%), and stromal overgrowth in 56 (86%). Distant metastases were observed in 20 (31%) patients (median follow-up 24.5 mo, 1 to 204). Only 13 of 65 (20%) cases had all 5 morphologic features, while only 7 of 20 (35%) cases with distant metastases had all 5 features. In univariate analysis, only marked stromal atypia ( P =0.004) and cellularity ( P =0.017) were associated with decreased distant metastasis-free survival. In multivariate Cox regression, the combination of stromal overgrowth, marked stromal cellularity, and atypia (C-index 0.721, 95% CI: 0.578, 0.863) was associated with decreased distant metastasis-free survival. The current World Health Organization recommendation will miss a significant number of MPTs with distant metastases. We propose refined diagnostic criteria for MPTs: (1) stromal overgrowth combined with ≥1 feature(s) (marked cellularity, marked atypia, or ≥10 mitoses per 10 HPF), or (2) in the absence of stromal overgrowth, marked cellularity combined with ≥1 feature(s) (permeative borders, marked atypia, or ≥10 mitoses per 10 HPF).

Mesenchymal Tumors of the Breast: Fibroblastic/Myofibroblastic Lesions and Other Lesions.

Mesenchymal breast tumors are a rare and diverse group of tumors that present some of the most challenging cases for multidisciplinary breast cancer teams. As a result of overlapping morphologies and a lack of large-scale studies on these tumors, practices are often heterogeneous and slow to evolve. Herein, we present a non-systematic review that focuses on progress, or lack thereof, in the field of mesenchymal breast tumors. We focus on tumors originating from fibroblastic/myofibroblastic cells and tumors originating from less common cellular origins (smooth muscle, neural tissue, adipose tissue, vascular tissue, etc.).

EPM2AIP1 Immunohistochemistry Can Be Used as Surrogate Testing for MLH1 Promoter Methylation in Endometrial Cancer.

Immunohistochemical (IHC) evaluation of DNA mismatch repair proteins (MMR) has become routine practice for Lynch syndrome screening and/or part of diagnostic evaluation in endometrial cancer. Approximately 20% to 30% of endometrial carcinomas demonstrate microsatellite instability due to defective DNA MMR. Vast majority of MLH1/PMS2-deficient tumors are sporadic and show MLH1 promoter methylation. MLH1 methylation testing by quantitative polymerase chain reaction-based technique is time, labor, and tissue intensive with an average institutional turnaround time of 2 weeks. MLH1 and EPM2AIP1 genes share a common promoter whose methylation has been shown to affect both genes. We assessed whether IHC for EPM2AIP1 in combination with MMR proteins can serve as surrogate marker for MLH1 promoter methylation status. We performed a retrospective review of all MLH1/PMS2-deficient endometrial carcinomas that underwent MLH1 promoter methylation testing from January 1 to September 31, 2020, at our institution. Microscopic slides were reviewed and EMP2AIP1 IHC was performed. The results were correlated with MLH1 promoter methylation status (percent methylated rate). A total of 119 cases were identified and successfully tested. Nuclear EPM2AIP1 protein expression was observed in benign endometrial cells and myometrial smooth muscle cells. Loss of nuclear EPM2AIP1 staining was identified in 90/110 (81.8%) methylated tumors with additional 14/110 (12.7%) cases showing aberrant staining patterns. Only 6/110 (5.5%) tumors demonstrated intact EPM2AIP1 nuclear expression in presence of MLH1 promoter methylation. EMP2AIP1 IHC is concordant with MLH1 promoter methylation results in 95% of endometrial carcinomas (94.5% sensitivity, 98.1% positive predictive value) and shows promise as a surrogate marker for methylation testing.

The characteristics associated with upgrade on surgical pathology of conventional imaging occult DCIS diagnosed by MRI.

PURPOSE: To characterize the clinical, pathological, and imaging features of DCIS occult on conventional imaging diagnosed on MRI-guided biopsy associated with increased risk of invasive disease on surgical excision. MATERIALS AND METHODS: All consecutive patients with MRI-detected DCIS occult on conventional imaging between January 2009 and December 2018 were included. Women were divided into two groups based on final pathology: Pure DCIS or DCIS with invasive component. Clinical, imaging, and pathological risk factors for upgrade to invasion were evaluated. RESULTS: Of 50 patients who met the inclusion criteria, 12 (24%) were upgraded to invasive malignancy in the final pathology. The only parameters that showed statistically significant association with upgrade were related to kinetic characteristics: 53% of patients with the combination of fast early upstroke and either plateau or washout curve were upgraded, compared to 12% of women without this combination (p = 0.006). The sensitivity of combined kinetic features for predicting upgrade was 67% (95% CI 35-90%), specificity was 84% (CI 95% 68-94%), positive predictive value was 57% (CI 95% 37-75%), negative predictive value was 89% (CI 95% 77-95%), and OR was 78% (64-88%). CONCLUSION: Kinetic characteristics show the strongest association with upgrade to invasion in DCIS occult on mammogram and US. Larger studies should be encouraged to consolidate our findings, which may have implication for treatment planning.

MLH1 epimutation is a rare mechanism for Lynch syndrome: A case report and review of the literature.

Endometrial carcinoma is one of the prototypical malignancies associated with Lynch syndrome, an inherited cancer syndrome most commonly caused by germline mutations in DNA mismatch repair (MMR) genes, although rare alternative mechanisms also exist. In this report, we describe a patient first diagnosed with colorectal cancer at age 33, then vulvar squamous cell carcinoma, facial sebaceous adenoma/sebaceoma, and finally endometrial carcinoma at age 52. All tumors were MLH1/PMS2-deficient by immunohistochemistry, and MLH1 promoter methylation was identified in the endometrial cancer. Germline MLH1 testing was negative for pathogenic variants, but she was subsequently diagnosed with Lynch syndrome secondary to a germline monoallelic constitutional epimutation of the MLH1 promoter. Identification of patients displaying a Lynch syndrome phenotype but lacking germline MMR mutations is important to avoid delays in the diagnosis of Lynch syndrome as well as the initiation of appropriate cancer screening and genetic counseling.

Review of the current state of digital image analysis in breast pathology.

Advances in digital image analysis have the potential to transform the practice of breast pathology. In the near future, a move to a digital workflow offers improvements in efficiency. Coupled with artificial intelligence (AI), digital pathology can assist pathologist interpretation, automate time-consuming tasks, and discover novel morphologic patterns. Opportunities for digital enhancements abound in breast pathology, from increasing reproducibility in grading and biomarker interpretation, to discovering features that correlate with patient outcome and treatment. Our objective is to review the most recent developments in digital pathology with clear impact to breast pathology practice. Although breast pathologists currently undertake limited adoption of digital methods, the field is rapidly evolving. Care is needed to validate emerging technologies for effective patient care.

Breast specimen handling and reporting in the post-neoadjuvant setting: challenges and advances.

Neoadjuvant systemic therapy is becoming more commonly used in patients with earlier stages of breast cancer. To assess tumour response to neoadjuvant chemotherapy, pathological evaluation is the gold standard. Depending on the treatment response, the pathological examination of these specimens can be quite challenging. However, a uniform approach to evaluate post-neoadjuvant-treated breast specimens has been lacking. Furthermore, there is no single universally accepted or endorsed classification system for assessing treatment response in this setting. Recent initiatives have attempted to create a standardised protocol for evaluation of post-neoadjuvant breast specimens. This review outlines the necessary information that should be collected prior to macroscopic examination of these specimens, the recommended and most pragmatic approach to tissue sampling for microscopic examination, describes the macroscopic and microscopic features of post-therapy breast specimens, summarises two commonly used systems for classifying treatment response and outlines the critical variables that should be included in the final pathology report.

The dynamic DNA methylation landscape of the mutL homolog 1 shore is altered by MLH1-93G>A polymorphism in normal tissues and colorectal cancer.

BACKGROUND: Colorectal cancers (CRCs) undergo distinct genetic and epigenetic alterations. Expression of mutL homolog 1 (MLH1), a mismatch repair gene that corrects DNA replication errors, is lost in up to 15% of sporadic tumours due to mutation or, more commonly, due to DNA methylation of its promoter CpG island. A single nucleotide polymorphism (SNP) in the CpG island of MLH1 (MLH1-93G>A or rs1800734) is associated with CpG island hypermethylation and decreased MLH1 expression in CRC tumours. Further, in peripheral blood mononuclear cell (PBMC) DNA of both CRC cases and non-cancer controls, the variant allele of rs1800734 is associated with hypomethylation at the MLH1 shore, a region upstream of its CpG island that is less dense in CpG sites. RESULTS: To determine whether this genotype-epigenotype association is present in other tissue types, including colorectal tumours, we assessed DNA methylation in matched normal colorectal tissue, tumour, and PBMC DNA from 349 population-based CRC cases recruited from the Ontario Familial Colorectal Cancer Registry. Using the semi-quantitative real-time PCR-based MethyLight assay, MLH1 shore methylation was significantly higher in tumour tissue than normal colon or PBMCs (P < 0.01). When shore methylation levels were stratified by SNP genotype, normal colorectal DNA and PBMC DNA were significantly hypomethylated in association with variant SNP genotype (P < 0.05). However, this association was lost in tumour DNA. Among distinct stages of CRC, metastatic stage IV CRC tumours incurred significant hypomethylation compared to stage I-III cases, irrespective of genotype status. Shore methylation of MLH1 was not associated with MSI status or promoter CpG island hypermethylation, regardless of genotype. To confirm these results, bisulfite sequencing was performed in matched tumour and normal colorectal specimens from six CRC cases, including two cases per genotype (wildtype, heterozygous, and homozygous variant). Bisulfite sequencing results corroborated the methylation patterns found by MethyLight, with significant hypomethylation in normal colorectal tissue of variant SNP allele carriers. CONCLUSIONS: These results indicate that the normal tissue types tested (colorectum and PBMC) experience dynamic genotype-associated epigenetic alterations at the MLH1 shore, whereas tumour DNA incurs aberrant hypermethylation compared to normal DNA.

MLH1 region polymorphisms show a significant association with CpG island shore methylation in a large cohort of healthy individuals.

Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation. We previously demonstrated that SNPs (rs1800734, rs749072, and rs13098279) in the MLH1 gene region are associated with MLH1 promoter island methylation, loss of MLH1 protein expression, and microsatellite instability (MSI) in colorectal cancer (CRC) patients. Recent studies have identified less CpG-dense "shore" regions flanking many CpG islands. These shores often exhibit distinct methylation profiles between different tissues and matched normal versus tumor cells of patients. To date, most epigenetic studies have focused on somatic methylation events occurring within solid tumors; less is known of the contributions of peripheral blood cell (PBC) methylation to processes such as aging and tumorigenesis. To address whether MLH1 methylation in PBCs is correlated with tumorigenesis we utilized the Illumina 450 K microarrays to measure methylation in PBC DNA of 846 healthy controls and 252 CRC patients from Ontario, Canada. Analysis of a region of chromosome 3p21 spanning the MLH1 locus in healthy controls revealed that a CpG island shore 1 kb upstream of the MLH1 gene exhibits different methylation profiles when stratified by SNP genotypes (rs1800734, rs749072, and rs13098279). Individuals with wild-type genotypes incur significantly higher PBC shore methylation than heterozygous or homozygous variant carriers (p<1.1×10(-6); ANOVA). This trend is also seen in CRC cases (p<0.096; ANOVA). Shore methylation also decreases significantly with increasing age in cases and controls. This is the first study of its kind to integrate PBC methylation at a CpG island shore with SNP genotype status in CRC cases and controls. These results indicate that CpG island shore methylation in PBCs may be influenced by genotype as well as the normal aging process.

Promoter methylation of Wnt antagonists DKK1 and SFRP1 is associated with opposing tumor subtypes in two large populations of colorectal cancer patients.

Aberrant activation of canonical Wnt signaling is a hallmark event in colorectal carcinogenesis. The Dickkopf-1 (DKK1) and Secreted Frizzled Related Protein 1 (SFRP1) genes encode extracellular inhibitors of Wnt signaling that are frequently silenced by promoter hypermethylation in colorectal cancer (CRC). These methylation events have been identified as prognostic markers of patient outcome and tumor subtype in several cancers but similar roles in CRC have not been comprehensively examined. In CRC, the microsatellite instability (MSI) subtype associates with favorable disease outcome but the molecular events that are responsible remain poorly understood. Consequently, we quantified promoter methylation status of the Wnt antagonist genes DKK1 and SFRP1 in a large population-based cohort of CRCs from Ontario (n = 549) and Newfoundland (n = 696) stratified by MSI status. We examined the association between methylation status and clinicopathological features including tumor MSI status and patient survival. DKK1 and SFRP1 were methylated in 13 and 95% of CRCs, respectively. In Ontario, DKK1 methylation was strongly associated with MSI tumors after adjustment for age, sex and tumor location [odds ratio (OR) = 13.7, 95% confidence interval (CI) = 7.8-24.2, P < 0.001]. Conversely, SFRP1 methylation was inversely associated with MSI tumors after these adjustments (OR = 0.3, 95% CI = 0.1-0.9, P = 0.009). Similar results were obtained in Newfoundland. There were no independent associations with recurrence-free survival. This is the first large study to identify associations between Wnt antagonist promoter hypermethylation and CRC MSI subtype. These events provide insight into subtype-specific epigenetic mediation of Wnt signaling in CRC.

Functional effects of the MLH1-93G>A polymorphism on MLH1/EPM2AIP1 promoter activity.

Defective mismatch repair leads to the microsatellite instability (MSI) phenotype of colorectal cancer (CRC). We previously showed that the MLH1-93G>A promoter polymorphism is strongly associated with MSI tumours, suggesting a modifier role for this polymorphism in CRC. The MLH1 promoter is bi-directional with the EPM2AIP1 gene located on the antisense strand. In order to evaluate the functional effects of this polymorphism, we transfected a panel of CRC, endometrial cancer and non-tumourigenic cell lines with MLH1 luciferase promoter constructs. We used constructs in reverse orientation to assess the effect of this polymorphism on EPM2AIP1. The luciferase activities were compared using a two-sided Student's t-test. Electrophoretic mobility shift assays (EMSAs) were used to evaluate whether differential protein binding was responsible for the differences in promoter activity. We observed a higher level of activity with the -93G allele in all the cell lines observed; including the CRC cell line, HCT116 (P=0.002), the endometrial cancer cell line, HEC-1-A (P<0.001) and the normal colonic cell line, CCD-841-CoTr (P=0.002). This polymorphism also affected EPM2AIP1 transcription with the -93A allele demonstrating higher promoter activity in the HCT116 (P=0.007) and HEC-1-A (P=0.004) cells. The EMSA results suggest that this polymorphism alters the affinity of nuclear factors that bind to this region. Our findings indicate that the -93G>A polymorphism modifies the efficiency of MLH1/EPM2AIP1 transcription.

Specific variants in the MLH1 gene region may drive DNA methylation, loss of protein expression, and MSI-H colorectal cancer.

BACKGROUND: We previously identified an association between a mismatch repair gene, MLH1, promoter SNP (rs1800734) and microsatellite unstable (MSI-H) colorectal cancers (CRCs) in two samples. The current study expanded on this finding as we explored the genetic basis of DNA methylation in this region of chromosome 3. We hypothesized that specific polymorphisms in the MLH1 gene region predispose it to DNA methylation, resulting in the loss of MLH1 gene expression, mismatch-repair function, and consequently to genome-wide microsatellite instability. METHODOLOGY/PRINCIPAL FINDINGS: We first tested our hypothesis in one sample from Ontario (901 cases, 1,097 controls) and replicated major findings in two additional samples from Newfoundland and Labrador (479 cases, 336 controls) and from Seattle (591 cases, 629 controls). Logistic regression was used to test for association between SNPs in the region of MLH1 and CRC, MSI-H CRC, MLH1 gene expression in CRC, and DNA methylation in CRC. The association between rs1800734 and MSI-H CRCs, previously reported in Ontario and Newfoundland, was replicated in the Seattle sample. Two additional SNPs, in strong linkage disequilibrium with rs1800734, showed strong associations with MLH1 promoter methylation, loss of MLH1 protein, and MSI-H CRC in all three samples. The logistic regression model of MSI-H CRC that included MLH1-promoter-methylation status and MLH1 immunohistochemistry status fit most parsimoniously in all three samples combined. When rs1800734 was added to this model, its effect was not statistically significant (P-value  = 0.72 vs. 2.3×10(-4) when the SNP was examined alone). CONCLUSIONS/SIGNIFICANCE: The observed association of rs1800734 with MSI-H CRC occurs through its effect on the MLH1 promoter methylation, MLH1 IHC deficiency, or both.

MSH2 118T>C and MSH6 159C>T promoter polymorphisms and the risk of colorectal cancer.

The most important indicator of colorectal cancer (CRC) risk is the presence of family history of the disease. Inherited genetic changes, such as single nucleotide polymorphisms, in key candidate genes may contribute to CRC risk. We investigated whether promoter polymorphisms in DNA mismatch repair (MMR) genes MSH2 and MSH6 are associated with the risk of CRC. We genotyped 929 CRC patients and 1098 control subjects from Ontario, and 467 patients and 344 controls from Newfoundland and Labrador, for two promoter polymorphisms in the MMR genes MSH2 and MSH6 using the fluorogenic 5' nuclease assay. We used unconditional logistic regression to evaluate the association between each polymorphism and CRC after adjusting for age and sex. The associations between polymorphisms and tumor clinicopathological features were evaluated with a Pearson's chi-squared test or Fisher's exact test. All statistical tests were two sided. We observed strong associations between the MSH2 -118T>C polymorphism and family history of CRC based on the Amsterdam criteria I (P = 0.005) and Amsterdam criteria I and II (P = 0.036) among cases from Ontario. This association was especially evident among female CRC patients in Ontario (for Amsterdam criteria I, and I and II combined, P = 0.003 and P = 0.0001, respectively). The MSH2 -118T>C polymorphism was associated with strong family history of CRC in Ontario patients.

MLH1 -93G>A promoter polymorphism and the risk of microsatellite-unstable colorectal cancer.

BACKGROUND: Although up to 30% of patients with colorectal cancer have a positive family history of colorectal neoplasia, few colorectal cancers can be explained by mutations in high-penetrance genes. We investigated whether polymorphisms in DNA mismatch repair genes are associated with the risk of colorectal cancer. METHODS: We genotyped 929 case patients and 1098 control subjects from Ontario and 430 case patients and 275 control subjects from Newfoundland and Labrador for five polymorphisms in the mismatch repair genes MLH1 and MSH2 with the fluorogenic 5' nuclease assay. Tumor microsatellite instability (MSI) was determined with a polymerase chain reaction-based method; MSI status was assigned as high (MSI-H, > or = 30% unstable markers among all markers tested), low (MSI-L, <30% markers unstable), or stable (MSS, no unstable markers). We used unconditional logistic regression to evaluate the association between each polymorphism and colorectal cancer after adjusting for age and sex. The associations between polymorphisms and tumor clinicopathologic features were evaluated with a Pearson's chi-square or Fisher's exact test. All statistical tests were two-sided. RESULTS: We observed strong associations between the MLH1 -93G>A polymorphism and MSI-H tumors among case patients from Ontario (P = .001) and Newfoundland (P = .003). When compared with the control populations, homozygosity for the MLH1 -93G>A variant allele was associated with MSI-H tumors among case patients in Ontario (adjusted odds ratio [OR] = 3.23, 95% confidence interval [CI] = 1.65 to 6.30) and in Newfoundland (OR = 8.88, 95% CI = 2.33 to 33.9), as was heterozygosity among case patients in Ontario (OR = 1.84, 95% CI = 1.20 to 2.83) and in Newfoundland (OR = 2.56, 95% CI = 1.14 to 5.75). Genotype frequencies were similar among case patients with MSS and MSI-L tumors and control subjects, and the majority of homozygous variant carriers had MSS tumors. Among case patients from Ontario, an association between the MLH1 -93G>A polymorphism and a strong family history of colorectal cancer (for Amsterdam criteria I and II, P = .004 and P = .02, respectively) was observed. CONCLUSION: In two patient populations, the MLH1 -93G>A polymorphism was associated with an increased risk of MSI-H colorectal cancer.