Improved Staging of Ciliary Body and Choroidal Melanomas Based on Estimation of Tumor Volume and Competing Risk Analyses.

PURPOSE: The current, 8th edition of the American Joint Committee on Cancer (AJCC) anatomic classification and staging model for uveal melanoma does not fully separate survival estimates for patients with advanced stages of the disease (e.g., IIIB and IIIC). Furthermore, some tumors in higher size categories have a smaller volume than tumors in lower categories. Therefore, we developed a novel model for prognostication of metastatic mortality based on estimations of tumor volume. DESIGN: Retrospective, multicenter case series of patients with uveal melanoma involving the choroid, ciliary body, or both. PARTICIPANTS: Six thousand five hundred twenty-eight consecutively registered patients treated at 3 tertiary ocular oncology centers on 2 continents between 1981 and 2022. METHODS: Data on survival, tumor size, and extent were collected for all 6528 patients. Tumor volume was estimated using a simple equation based on largest basal diameter and thickness. Volume-based size categories and stages were developed and validated in independent patient cohorts using competing risk analyses, and correlations with cytogenetic and cytomorphologic features were examined. MAIN OUTCOME MEASURE: Cumulative incidence of metastatic death. RESULTS: The 6528 patients were distributed over 7 stages based on estimated tumor volume and anatomic extent (V stages IA, IB, IIA, IIB, IIIA, IIIB, and IIIC), with a 15-year incidence of metastatic death ranging from 7% to 77%. A new category, V1min, and corresponding stage IA, were introduced, indicating an excellent prognosis. Metastatic mortality in V stage IIIC was significantly higher than that in V stage IIIB (P = 0.03), whereas incidence curves crossed for patients in AJCC stages IIIC vs. IIIB (P = 0.53). Univariable and multivariable competing risk regressions demonstrated higher Wald statistics for V stages compared with AJCC stages (1152 vs. 1038 and 71 vs. 17, respectively). The frequency of monosomy 3, gain of chromosome 8q, and epithelioid cytomorphologic features increased with tumor volume (R2 = 0.70, R2 = 0.50, and R2 = 0.71, respectively; P < 0.001) and showed similar correlations with both AJCC and V stages. CONCLUSIONS: Anatomic classification and staging of ciliary body and choroidal melanomas based on estimation of tumor volume improves prognostication of metastatic mortality. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Melatonin Receptor Expression in Primary Uveal Melanoma.

Melatonin, noted for its anti-cancer properties in various malignancies, including cutaneous melanoma, shows promise in Uveal melanoma (UM) treatment. This study aimed to evaluate melatonin receptor expression in primary UM and its association with UM-related mortality and prognostic factors. Immunohistochemical analysis of 47 primary UM tissues showed low expression of melatonin receptor 1A (MTNR1A) and melatonin receptor 1B (MTNR1B), with MTNR1A significantly higher in patients who succumbed to UM. Analysis of TCGA data from 80 UM patients revealed RNA expression for MTNR1A, retinoic acid-related orphan receptor alpha (RORα), and N-ribosyldihydronicotinamide:quinone oxidoreductase (NQO2), but not MTNR1B or G protein-coupled receptor 50 (GPR50). Higher MTNR1A RNA levels were observed in patients with a BRCA1 Associated Protein 1 (BAP1) mutation, and higher NQO2 RNA levels were noted in patients with the epithelioid tumor cell type. However, Kaplan-Meier analysis did not show distinct survival probabilities based on receptor expression. This study concludes that UM clinical samples express melatonin receptors, suggesting a potential mechanism for melatonin's anti-cancer effects. Despite finding higher MTNR1A expression in patients who died of UM, no survival differences were observed.

Delays between Uveal Melanoma Diagnosis and Treatment Increase the Risk of Metastatic Death.

PURPOSE: To investigate if the interval between diagnosis and treatment of posterior uveal melanoma (UM) is associated with metastatic death. DESIGN: Retrospective, single-center cohort study. PARTICIPANTS: A total of 1145 patients consecutively diagnosed with posterior UM at St. Erik Eye Hospital, Stockholm, Sweden, from 2012 to 2022, with recorded dates of diagnosis and primary treatment. This cohort represents 81% of all diagnosed patients in Sweden during this period. METHODS: Data on the interval between diagnosis and treatment were collected for all patients. Its prognostic importance was examined with univariate and multivariate competing risks regressions, and cumulative incidence analyses. MAIN OUTCOME MEASURES: Incidence of metastatic death (UM mortality) for patients with prompt (< 1 month from diagnosis) versus delayed treatment (≥ 1 month) and subdistribution hazard ratios (exp(ßj)) for every additional 10-day delay in treatment. RESULTS: The mean interval between diagnosis and treatment was 34 days (SD, 56, range, 0-932). Patients treated promptly had larger tumors at diagnosis, but there were no differences in patient age, tumor distance to the optic disc, rates of ciliary body involvement (CBI) or extraocular extension (EXE), or symptom duration before diagnosis. Those who were treated more than 1 month after diagnosis had greater UM mortality in American Joint Committee on Cancer (AJCC) stage II and III. In stage I, UM mortality for delayed treatment was lower for the first 10 years, followed by a marked spike in the 11th year. In multivariate competing risks regressions of all 1145 patients with tumor diameter, thickness, CBI, and EXE as covariates, the risk for UM mortality increased with 1% for every additional 10-day delay in treatment (exp(ßj) 1.01). Among 355 patients treated with enucleation, this delay was associated with UM mortality, independent of AJCC stage, cytomorphology, and level of immunohistochemical BAP-1 expression. CONCLUSIONS: Increasing time between diagnosis and treatment of UM is associated with a higher risk of metastatic death. These results challenge a central concept in the understanding of metastatic progression and may indicate the existence of late metastatic seeding. They also underscore the importance of prompt treatment. Validation in independent cohorts is recommended. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

A serum protein signature at the time of Uveal Melanoma diagnosis predicts long-term patient survival.

PURPOSE: To develop a prognostic test based on a single blood sample obtained at the time of uveal melanoma diagnosis. METHODS: 83 patients diagnosed with posterior uveal melanoma between 1996 and 2000 were included. Peripheral serum samples were obtained at diagnosis and kept at -80 °C until this analysis. Protein profiling of 84 cancer-related proteins was used to screen for potential biomarkers and a prognostic test that stratifies patients into metastatic risk categories was developed (serUM-Px) in a training cohort and then tested in a validation cohort. RESULTS: Low serum leptin levels and high osteopontin levels were found to identify patients with poor prognosis and were therefore selected for inclusion in the final test. In the validation cohort, patient sex and American Joint Committee on Cancer stages were similarly distributed between the low, intermediate, and high metastatic risk categories. With increasing metastatic risk category, patients had shorter metastasis-free- and overall survival, as well as greater cumulative incidence of uveal melanoma-related mortality in competing risk analysis (P = 0.007, 0.018 and 0.029, respectively). In multivariate Cox regression, serUM-Px was an independent predictor of metastasis with tumor size and patient sex as covariates (hazard ratio 3.2, 95% CI 1.5-6.9). CONCLUSIONS: A prognostic test based on a single peripheral venous blood sample at the time of uveal melanoma diagnosis stratifies patients into low, intermediate, and high metastatic risk categories. Prospective validation will facilitate its clinical utility.

Increasing demand for ophthalmic pathology: time trends in a laboratory with nationwide coverage.

PURPOSE: To report the time trends in basic patient characteristics and the number of specimens received at a national referral center for ophthalmic pathology. METHODS: Data on patient sex, age at surgical resection and geographical location of the referring unit were obtained for all specimens received at the St. Erik Ophthalmic Pathology laboratory, Stockholm, Sweden, between January 1st, 1959, and December 31st, 2021. RESULTS: A total of 33 057 specimens had been received, of which 14 560 (44%) came from men and 18 477 (56%) from women (for 20 patients, the sex was not specified). The average annual percent change (AAPC) in the number specimens received was + 10.5%, whereas the Swedish population increased with 0.5% per year. Patients became older throughout the period, with an average yearly increase of patient age at surgery of 0.3 years (AAPC 0.2%). Overall, women were three years older than men at surgery (59.4 versus 56.4 years, P < 0.0001) The number of specimens increased with patient age from the first to the 8th decade, after which it decreased to zero in the 11th decade. The largest portion of patients had undergone their surgery in one of the hospitals or clinics in the capital region, with four of the five largest sources corresponding to the most populous counties in the country. CONCLUSIONS: During six decades, the growth in number of specimens sent to our national referral center for ophthalmic pathology has greatly outpaced the growth of the population, indicating an increasing demand for subspecialized services. Throughout the period, patients have become older, and a higher number of specimens have been submitted from female patients.

Risk for Acute Myocardial Infarction After Ophthalmologic Procedures.

BACKGROUND: Preoperative cardiovascular evaluations are frequently done before ambulatory ophthalmologic procedures. However, whether these procedures can trigger an acute myocardial infarction (AMI) is unknown. OBJECTIVE: To assess the short-term risk for AMI associated with ophthalmologic procedures. DESIGN: Case-crossover design. SETTING: Population-based nationwide study from Norway and Sweden. PARTICIPANTS: First-time patients with AMI, aged 40 years and older, identified via inpatient registries and linked to outpatient surgical procedures in Norway (2008 to 2014) and Sweden (2001 to 2014), respectively. MEASUREMENTS: Using self-matching, for each participant, exposure to ophthalmologic procedures in the 0 to 7 days before AMI diagnosis (hazard period) was compared with an 8-day period 30 days earlier, that is, days 29 to 36 before AMI (control period) to estimate the relative risk for an AMI the week after an ophthalmologic procedure. The odds ratios (ORs) with 95% CIs were calculated, using conditional logistic regression. Only patients who had a procedure of interest during either the hazard or control period were included. RESULTS: For the 806 patients with AMI included in this study, there was a lower likelihood of AMI in the week after an ophthalmologic procedure than during the control week (OR, 0.83; 95% CI, 0.75 to 0.91). Furthermore, there was no evidence of increased risk for AMI when analyses were stratified by surgery subtype, anesthesia (local or general), duration, invasiveness (low, intermediate, or high), patient's age (<65 years or ≥65 years), or comorbidity (none vs. any). LIMITATION: Potential bias from time-varying confounders between the hazard and the control periods. CONCLUSION: Ophthalmologic procedures done in an outpatient setting did not seem to be associated with an increased risk for AMI. PRIMARY FUNDING SOURCE: Central Norway Regional Health Authority and the Swedish Research Council.

The rationale for treating uveal melanoma with adjuvant melatonin: a review of the literature.

BACKGROUND: Uveal melanoma is a rare form of cancer with high mortality. The incidence of metastases is attributed to early seeding of micrometastases from the eye to distant organs, primarily the liver. Once these seeded clusters of dormant tumor cells grow into larger radiologically detectable macrometastases, median patient survival is about 1 year. Melatonin is an important hormone for synchronizing circadian rhythms. It is also involved in other aspects of human physiology and may offer therapeutic benefits for a variety of diseases including cancer. METHODS: Articles involving the physiological effects of melatonin, pharmacokinetics, and previous use in cancer studies were acquired using a comprehensive literature search in the Medline (PubMed) and Web of Science databases. In total, 147 publications were selected and included in the review. RESULTS: Melatonin has been observed to suppress the growth of cancer cells, inhibit metastatic spread, enhance immune system functions, and act as an anti-inflammatory in both in vitro and in vivo models. Melatonin may also enhance the efficacy of cancer treatments such as immuno- and chemotherapy. Numerous studies have shown promising results for oral melatonin supplementation in patients with other forms of cancer including cutaneous malignant melanoma. Cell line and animal studies support a hypothesis in which similar benefits may exist for uveal melanoma. CONCLUSIONS: Given its low cost, good safety profile, and limited side effects, there may be potential for the use of melatonin as an adjuvant oncostatic treatment. Future avenues of research could include clinical trials to evaluate the effect of melatonin in prevention of macrometastases of uveal melanoma.

No differences in the long-term prognosis of iris and choroidal melanomas when adjusting for tumor thickness and diameter.

OBJECTIVE: To assess the long-term prognosis for patients with iris melanomas and compare it with the prognosis for small choroidal melanomas. DESIGN: Retrospective observational case series. METHODS: All patients treated for iris melanomas at a single referral institution between January 1st 1986 and January 1st 2016 were included. Patients treated for small choroidal melanomas during the same period were included for comparison. The cumulative incidence of melanoma-related mortality was calculated. Patient and tumor characteristics and size-adjusted hazard ratio (HR) for melanoma-related mortality were compared between iris and small choroidal melanomas. RESULTS: Forty-five iris melanomas and 268 small choroidal melanomas were included. Twenty-four iris melanomas (53%) had been treated with local resection, 12 (27%) with Ruthenium-106 brachytherapy, 7 (16%) with enucleation and 2 (4%) with proton beam irradiation. Twenty-one (68%), 7 (16%) and 2 (4%) of the iris melanomas were of the spindle, mixed and epithelioid cell types, respectively. Twenty-three patients had deceased before the end of follow-up. Median follow-up for the 22 survivors was 13.3 years (SD 9.4). Patients with iris melanomas were more often asymptomatic at presentation and had a trend towards significantly lower age (59 versus 63 years, Student's T-tests p = 0.057). Further, iris melanomas had significantly smaller basal diameter (5.8 versus 8.0 mm, p < 0.0001) and tumor volume (79 mm3 versus 93 mm mm3, p < 0.0001) but greater thickness (3.0 versus 2.5 mm, p < 0.0001). The cumulative incidence of iris melanoma-related mortality was 5% at 5 years after diagnosis, and 8% at 10, 15 and 20 years. The incidence was not significantly different to small choroidal melanomas (Wilcoxon p = 0.46). In multivariate Cox regression with tumor diameter and thickness as covariates, patients with choroidal melanomas did not have increased HR for melanoma-related mortality (HR 2.2, 95% CI 0.5-9.6, p = 0.29). Similarly, there were no significant survival differences in matched subgroups (Wilcoxon p = 0.82). CONCLUSIONS: There are no survival differences between iris and choroidal melanomas when adjusting for tumor size. The reason for the relatively favorable prognosis of iris melanomas compared to melanomas of the choroid and ciliary body is likely that they are diagnosed at a smaller size.

Digital morphometry of tumor nuclei correlates to BAP-1 status, monosomy 3, gene expression class and survival in uveal melanoma.

Cytologic features such as the shape and size of tumor cells can predict metastatic death in uveal melanoma and other cancers but suffer from poor reproducibility. In this study, we investigate the interobserver concordance of digital morphometry, and correlate the results with BRCA associated protein-1 (BAP-1) expression and BAP-1 gene mutation status, monosomy 3, gene expression classifications and patient survival in uveal melanoma. The average number of cells analyzed in each of 107 tumors, was 1957 (SD 349). Mean time consumption was less than 2.5 min per tumor. Identical morphometric classification was obtained for ≥85% of tumors in all twelve evaluated morphometric variables (κ 0.70-0.93). The mean nucleus area, nucleus perimeter, nucleus max caliper and nucleus to cell area ratio were significantly greater in tumors with low BAP-1 expression and gene expression class 2. Patients had significantly shorter survival if their tumors had low BAP-1 (Log-Rank p = 0.002), gene expression class 2 (p = 0.004), long nucleus perimeters (p = 0.031), long nucleus max calipers (p = 0.029) and high mean nucleus to cell area ratios (p = 0.041) as defined in a training cohort and then tested in a validation cohort. Long nucleus perimeters and long nucleus max calipers correlated with monosomy 3 (Pearson Chi-Square p = 0.006 and p = 0.009, respectively). Long nucleus perimeters also correlated with BAP-1 mutation (p = 0.017). We conclude that digital morphometry can be fast and highly reproducible, that for the first time, morphometry parameters can be objectively quantitated in thousands of cells at a time in sub-µm resolutions, and that variables describing the shape and size tumor nuclei correlate to BAP-1 status, monosomy 3, gene expression class as well as patient survival.

Density of PAS positive patterns in uveal melanoma: Correlation with vasculogenic mimicry, gene expression class, BAP-1 expression, macrophage infiltration, and risk for metastasis.

Purpose: Periodic acid-Schiff (PAS) positive patterns of vasculogenic mimicry (VM) have been associated with poor prognosis in uveal melanoma (UM). We examined these patterns with digital image analysis and transmission electron microscopy, and correlated them with BAP-1 expression, gene expression class, macrophage infiltration, and metastatic disease in full tumor cross-sections and intratumor regions. Methods: Thirty-two enucleated eyes with UM were stained immunohistochemically (BAP-1, laminin, CD31, and CD68) and with PAS without hematoxylin counterstain. Retrospective data on gene expression class and patient survival were retrieved. Tumor sections were digitally scanned and analyzed with the QuPath Bioimage analysis software, and imaged with transmission electron microscopy. Results: The mean area proportion covered by CD31, laminin, and PAS positive patterns in tumor cross-sections was 0.9% (SD 0.6), 3.0% (SD 1.9), and 8.4% (SD 5.9), respectively. PAS density was statistically significantly greater in tumors with gene expression class 2 (p=0.02). The cumulative 5-year metastasis-free survival decreased for each quartile of increased PAS density (1.0, 0.75, 0.40, and 0.17, p=0.004). Forty percent of the tumors had heterogeneous BAP-1 expression. Intratumor regions with low BAP-1 expression were more likely to harbor VM (p<0.0001), and had statistically significantly greater PAS density (p<0.0001) and number of CD68 positive cells (p=0.01). Conclusions: PAS positive patterns in UM are composed of a mixture of blood vessels and extracellular matrix (ECM), including VM. Increased density of PAS positive patterns correlated with gene expression class and metastasis, and colocated to tumor regions with macrophage infiltration and low BAP-1 expression.

Digital image analysis of Ki67 in hot spots is superior to both manual Ki67 and mitotic counts in breast cancer.

AIMS: During pathological examination of breast tumours, proliferative activity is routinely evaluated by a count of mitoses. Adding immunohistochemical stains of Ki67 provides extra prognostic and predictive information. However, the currently used methods for these evaluations suffer from imperfect reproducibility. It is still unclear whether analysis of Ki67 should be performed in hot spots, in the tumour periphery, or as an average of the whole tumour section. The aim of this study was to compare the clinical relevance of mitoses, Ki67 and phosphohistone H3 in two cohorts of primary breast cancer specimens (total n = 294). METHODS AND RESULTS: Both manual and digital image analysis scores were evaluated for sensitivity and specificity for luminal B versus A subtype as defined by PAM50 gene expression assays, for high versus low transcriptomic grade, for axillary lymph node status, and for prognostic value in terms of prediction of overall and relapse-free survival. Digital image analysis of Ki67 outperformed the other markers, especially in hot spots. Tumours with high Ki67 expression and high numbers of phosphohistone H3-positive cells had significantly increased hazard ratios for all-cause mortality within 10 years from diagnosis. Replacing manual mitotic counts with digital image analysis of Ki67 in hot spots increased the differences in overall survival between the highest and lowest histological grades, and added significant prognostic information. CONCLUSIONS: Digital image analysis of Ki67 in hot spots is the marker of choice for routine analysis of proliferation in breast cancer.

Exome sequencing of primary breast cancers with paired metastatic lesions reveals metastasis-enriched mutations in the A-kinase anchoring protein family (AKAPs).

BACKGROUND: Tumor heterogeneity in breast cancer tumors is today widely recognized. Most of the available knowledge in genetic variation however, relates to the primary tumor while metastatic lesions are much less studied. Many studies have revealed marked alterations of standard prognostic and predictive factors during tumor progression. Characterization of paired primary- and metastatic tissues should therefore be fundamental in order to understand mechanisms of tumor progression, clonal relationship to tumor evolution as well as the therapeutic aspects of systemic disease. METHODS: We performed full exome sequencing of primary breast cancers and their metastases in a cohort of ten patients and further confirmed our findings in an additional cohort of 20 patients with paired primary and metastatic tumors. Furthermore, we used gene expression from the metastatic lesions and a primary breast cancer data set to study the gene expression of the AKAP gene family. RESULTS: We report that somatic mutations in A-kinase anchoring proteins are enriched in metastatic lesions. The frequency of mutation in the AKAP gene family was 10% in the primary tumors and 40% in metastatic lesions. Several copy number variations, including deletions in regions containing AKAP genes were detected and showed consistent patterns in both investigated cohorts. In a second cohort containing 20 patients with paired primary and metastatic lesions, AKAP mutations showed an increasing variant allele frequency after multiple relapses. Furthermore, gene expression profiles from the metastatic lesions (n = 120) revealed differential expression patterns of AKAPs relative to the tumor PAM50 intrinsic subtype, which were most apparent in the basal-like subtype. This pattern was confirmed in primary tumors from TCGA (n = 522) and in a third independent cohort (n = 182). CONCLUSION: Several studies from primary cancers have reported individual AKAP genes to be associated with cancer risk and metastatic relapses as well as direct involvement in cellular invasion and migration processes. Our findings reveal an enrichment of mutations in AKAP genes in metastatic breast cancers and suggest the involvement of AKAPs in the metastatic process. In addition, we report an AKAP gene expression pattern that consistently follows the tumor intrinsic subtype, further suggesting AKAP family members as relevant players in breast cancer biology.

Intra-tumor heterogeneity in breast cancer has limited impact on transcriptomic-based molecular profiling.

BACKGROUND: Transcriptomic profiling of breast tumors provides opportunity for subtyping and molecular-based patient stratification. In diagnostic applications the specimen profiled should be representative of the expression profile of the whole tumor and ideally capture properties of the most aggressive part of the tumor. However, breast cancers commonly exhibit intra-tumor heterogeneity at molecular, genomic and in phenotypic level, which can arise during tumor evolution. Currently it is not established to what extent a random sampling approach may influence molecular breast cancer diagnostics. METHODS: In this study we applied RNA-sequencing to quantify gene expression in 43 pieces (2-5 pieces per tumor) from 12 breast tumors (Cohort 1). We determined molecular subtype and transcriptomic grade for all tumor pieces and analysed to what extent pieces originating from the same tumors are concordant or discordant with each other. Additionally, we validated our finding in an independent cohort consisting of 19 pieces (2-6 pieces per tumor) from 6 breast tumors (Cohort 2) profiled using microarray technique. Exome sequencing was also performed on this cohort, to investigate the extent of intra-tumor genomic heterogeneity versus the intra-tumor molecular subtype classifications. RESULTS: Molecular subtyping was consistent in 11 out of 12 tumors and transcriptomic grade assignments were consistent in 11 out of 12 tumors as well. Molecular subtype predictions revealed consistent subtypes in four out of six patients in this cohort 2. Interestingly, we observed extensive intra-tumor genomic heterogeneity in these tumor pieces but not in their molecular subtype classifications. CONCLUSIONS: Our results suggest that macroscopic intra-tumoral transcriptomic heterogeneity is limited and unlikely to have an impact on molecular diagnostics for most patients.

Digital image analysis outperforms manual biomarker assessment in breast cancer.

In the spectrum of breast cancers, categorization according to the four gene expression-based subtypes 'Luminal A,' 'Luminal B,' 'HER2-enriched,' and 'Basal-like' is the method of choice for prognostic and predictive value. As gene expression assays are not yet universally available, routine immunohistochemical stains act as surrogate markers for these subtypes. Thus, congruence of surrogate markers and gene expression tests is of utmost importance. In this study, 3 cohorts of primary breast cancer specimens (total n=436) with up to 28 years of survival data were scored for Ki67, ER, PR, and HER2 status manually and by digital image analysis (DIA). The results were then compared for sensitivity and specificity for the Luminal B subtype, concordance to PAM50 assays in subtype classification and prognostic power. The DIA system used was the Visiopharm Integrator System. DIA outperformed manual scoring in terms of sensitivity and specificity for the Luminal B subtype, widely considered the most challenging distinction in surrogate subclassification, and produced slightly better concordance and Cohen's κ agreement with PAM50 gene expression assays. Manual biomarker scores and DIA essentially matched each other for Cox regression hazard ratios for all-cause mortality. When the Nottingham combined histologic grade (Elston-Ellis) was used as a prognostic surrogate, stronger Spearman's rank-order correlations were produced by DIA. Prognostic value of Ki67 scores in terms of likelihood ratio χ(2) (LR χ(2)) was higher for DIA that also added significantly more prognostic information to the manual scores (LR-Δχ(2)). In conclusion, the system for DIA evaluated here was in most aspects a superior alternative to manual biomarker scoring. It also has the potential to reduce time consumption for pathologists, as many of the steps in the workflow are either automatic or feasible to manage without pathological expertise.

Gene protein detection platform--a comparison of a new human epidermal growth factor receptor 2 assay with conventional immunohistochemistry and fluorescence in situ hybridization platforms.

Human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are widely used semiquantitative assays for selecting breast cancer patients for HER2 antibody therapy. However, both techniques have been shown to have disadvantages. Our aim was to test a recent automated technique of combined IHC and brightfield dual in situ hybridization-gene protein detection platform (GPDP)-in breast cancer HER2 protein, gene, and chromosome 17 centromere status evaluations, comparing the results in accordance to the American Society of Clinical Oncology/College of American Pathologists recommendations for HER2 testing in breast cancer from both 2007 and 2013. The GPDP technique performance was evaluated on 52 consecutive whole slide invasive breast cancer cases with HER2 IHC 2/3+ scoring results. Applying in turns the American Society of Clinical Oncology/College of American Pathologists recommendations for HER2 testing in breast cancer from 2007 and 2013 to both FISH and GPDP DISH assays, the HER2 gene amplification results showed 100% concordance among amplified/nonamplified cases, but there was a shift in 4 cases toward positive from equivocal results and toward equivocal from negative results. This might be related to the emphasis on the average HER2 copy number in the 2013 criteria. HER2 expression by IVD market IHC kit (Pathway®) has a strong correlation with GPDP HER2 protein, including a full concordance for all cases scored as 3+ and a reduction from 2+ to 1+ in 7 cases corresponding to nonamplified cases. Gene protein detection platform HER2 protein "solo" could have spared the need for 7 FISH studies. In addition, the platform offered advantages on interpretation reassurance including selecting areas for counting gene signals paralleled with protein IHC expression, on heterogeneity detection, interpretation time, technical time, and tissue expense.

Low concordance of biomarkers in histopathological and cytological material from breast cancer.

AIMS: The aim of this study was to investigate in primary breast cancer the congruency of routine clinical predictive biomarker evaluations, including ER, PR and Ki67, obtained using immunocytochemistry (ICC) and immunohistochemistry (IHC). METHODS AND RESULTS: Clinicopathological data were collected on all women diagnosed with primary breast cancer at Karolinska University Hospital in 2011. A total of 346 patients were included in a retrospective paired comparison of predictive biomarker evaluations on direct smear ICC and IHC. This showed a low congruency between findings with the two methods, especially evident for Ki67 (κ = 0.35-0.42). By suggested adjustments to ICC cut-offs, we managed to improve the inter-rater agreement of Ki67 classification slightly to κ = 0.46. CONCLUSIONS: Our findings suggest that routine clinical ICC and IHC evaluations of predictive biomarkers produce discordant results. Consequently, basing therapeutic decisions on cytology with cut-offs defined for IHC induces a risk that patients will receive suboptimal therapy. However, our analysis shows that local adjustments to biomarker cut-off levels may improve congruency and increase the probability of correct classifications.