Validation of biomarkers and digital image analysis in breast pathology

For women worldwide, the risk of developing breast cancer is second only to that of nonmelanoma skin cancer. Significant improvements have been made in survival over the past decades and today about 80 % of the patients survive 10 years or more after their breast cancer diagnosis. Still, far from all patients enjoy the relatively good survival indicated by statistics on breast cancer patients as one homogenous group. Improving prognostication of aggressive vs. less aggressive disease, and to separate tumors based on genetic differences for optimal treatment strategies, is therefore the focus of intensive research, including this thesis. In paper I, we compared if tumor characteristics differ depending on what method of sampling the tumor that have been used for analysis. We compared routine immunohistochemistry on surgically resected breast specimens, including stains of the Estrogen receptor alpha (ER), the Progesterone receptor (PR), Human Epidermal growth factor receptor 2 (HER2) and the proliferation-associated protein Ki67, with analysis of the same stains done on material obtained from fine needle aspiration (immunocytochemistry). We found that there were substantial differences in the expression of these biomarkers between the two methods. Thus, the same rules for interpretation of biomarkers cannot be used for immunohistochemistry and immunocytochemistry, and consequently, validation of each method should be performed individually. In paper II, we explored the scope of digital image analysis in biomarker evaluations. We scored ER, PR, HER2 and Ki67 status in several different regions of breast tumors by both manual methods and digital image analysis. The outcomes of the scoring of these biomarkers were then combined into IHC surrogate subtypes and compared to PAM50 gene expression-based subtypes as well as patient survival. All tested methods of automated digital image analysis of Ki67 outperformed manual scores in terms of sensitivity and specificity for the Luminal B subtype. Comparing digital versus manual testing concordance to all breast cancer subtypes as determined by PAM50 assays, the digital approach was superior to the manual method. The manual and digital image analysis methods matched each other in hazard ratio for all-cause mortality of patients with tumors with a “high” vs “low” Ki67 index. Manual assessments of the biomarkers ER, PR, HER2 and Ki67 were in most aspects less precise than digital image analysis. In paper III, we evolved the concept of paper I with an evaluation of the concordance of consecutive Ki67 assessments performed on fine needle aspiration cytology versus resected tumor specimens. We investigated how a status of Ki67 “low” and “high” as determined by immunohistochemistry and immunocytochemistry corresponded to overall survival, respectively. Again, Ki67-index varied when the two methods were used on the same tumors, and was prone to switch the classification between low and high proliferation. ER evaluations were discordant in 5.3 % of the tumors, which in the clinical setting would mean that 1 in 20 patients would risk being left out of beneficial endocrine treatment or being given it without benefit. Ki67 “high”, as determined by immunohistochemistry, defined as a proportion of Ki67-positive cells above the 67th percentile of the material, was significantly associated with poor overall survival and a significantly higher probability of axillary lymph node metastasis. This could not be reproduced for immunocytochemistry. In summary, this study adds to the results of paper I, in which we showed discordance between the methods. By including survival data, we now conclude that not merely are the methods discordant, but immunocytochemistry fails to provide prognostic information. Consequently, immunohistochemistry should be regarded as the superior method. In paper IV, we focused on proliferation comparing the results in the tumors’ hot spot, in the tumor periphery, and as the average proportion of Ki67-positive cells across the whole tumor section. Both manual and digital scores of Ki67 and the recently described marker for mitotic activity, PHH3, were evaluated along with mitotic counts. Their sensitivity and specificity for the gene expression based Luminal B versus A breast cancer subtypes, for the high versus low transcriptomic grade, for axillary lymph node status as well as for their prognostic value for breast cancer specific and overall survival were analyzed. Digital image analysis of Ki67 in hot spots outperformed the other markers in sensitivity and specificity both for gene expression subtypes and transcriptomic grade. In contrast to mitotic counts, tumors with high expression of Ki67, as defined by digital image analysis and high numbers of PHH3-positive cells, had significantly increased HR for all-cause mortality at 10 years from diagnosis. When we replaced the manual mitotic counts with digital image analysis of Ki67 in hot spots as the marker for proliferation when determining histological grade, the differences in estimated mean overall survival between the highest and lowest grades increased. It also added significantly more prognostic information to the classic Nottingham combined histological grade. We conclude that digital image analysis of Ki67 in hot spots might be suggested as the marker of choice for proliferative activity 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)

Abstract 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

Forty-year prognosis after plaque brachytherapy of uveal melanoma

Abstract In this study, the long-term patient survival after plaque brachytherapy of uveal melanoma is examined. All patients treated between 1980 and 1999 at a single institution were included (n = 677). 533 (79%) had deceased before the end of follow-up. The median follow-up for the 144 survivors was 25.4 years (SD 5.2). Uveal melanoma-related mortality was 18% by 5 years, 28% by 10 years, 32% by 15 years, 35% by 20 years, and 36% by 25 to 40 years. 172 of 209 (82%) uveal melanoma-related deaths occurred within the first decade after brachytherapy. Relative survival rates were 74% at 5 years, 64% at 10 years, 62% at 20 years, 83% at 30 years and ≥100% at 32 to 40 years. Tumor diameter and local recurrence were independent predictors of uveal melanoma-related mortality in multivariate Cox proportional hazards analysis. In conclusion, uveal melanoma has a high mortality rate and most uveal melanoma-related deaths occur in the first decade after treatment. Long-term survivors may have a survival advantage to individuals of the same sex and age from the general population

Intratumor Heterogeneity in Uveal Melanoma BAP-1 Expression

Malignant tumors are rarely homogenous on the morphological, genome, transcriptome or proteome level. In this study, we investigate the intratumor heterogeneity of BAP-1 expression in uveal melanoma with digital image analysis of 40 tumors. The proportion of BAP-1 positive cells was measured in full tumor sections, hot spots, cold spots and in scleral margins. The mean difference between hot spots and cold spots was 41 percentage points (pp, SD 29). Tumors with gene expression class 1 (associated with low metastatic risk) and 2 (high metastatic risk) had similar intratumor heterogeneity. Similarly, the level of intratumor heterogeneity was comparable in tumors from patients that later developed metastases as in patients that did not. BAP-1 measured in any tumor region added significant prognostic information to both American Joint Committee on Cancer (AJCC) tumor size category (p ≤ 0.001) and gene expression class (p ≤ 0.04). We conclude that there is substantial intratumor heterogeneity in uveal melanoma BAP-1 expression. However, it is of limited prognostic importance. Regardless of region, analysis of BAP-1 expression adds significant prognostic information beyond tumor size and gene expression class

Digital morphometry and cluster analysis identifies four types of melanocyte during uveal melanoma progression

Abstract Background Several types of benign and malignant uveal melanocytes have been described based on their histological appearance. However, their characteristics have not been quantified, and their distribution during progression from normal choroidal melanocytes to primary tumors and metastases has not been reported. Methods A total of 1,245,411 digitally scanned melanocytes from normal choroid, choroidal nevi, primary uveal melanomas, and liver metastases were entered into two-step cluster analyses to delineate cell types based on measured morphometric characteristics and expression of protein markers. Results Here we show that a combination of the area and circularity of cell nuclei, and BAP-1 expression in nuclei and cytoplasms yields the highest silhouette of cohesion and separation. Normal choroidal melanocytes and three types of uveal melanoma cells are outlined: Epithelioid (large, rounded nuclei; BAP-1 low; IGF-1R, IDO, and TIGIT high), spindle A (small, elongated nuclei; BAP-1 high; IGF-1R low; IDO, and TIGIT intermediate), and spindle B (large, elongated nuclei; BAP-1, IGF-1R, IDO, and TIGIT low). In normal choroidal tissue and nevi, only normal melanocytes and spindle A cells are represented. Epithelioid and spindle B cells are overrepresented in the base and apex, and spindle A cells in the center of primary tumors. Liver metastases contain no normal melanocytes or spindle A cells. Conclusions Four basic cell types can be outlined in uveal melanoma progression: normal, spindle A and B, and epithelioid. Differential expression of tumor suppressors, growth factors, and immune checkpoints could contribute to their relative over- and underrepresentation in benign, primary tumor, and metastatic samples

Gain of Chromosome 6p Correlates with Severe Anaplasia, Cellular Hyperchromasia, and Extraocular Spread of Retinoblastoma

Gain of chromosome 6p has been associated with poor ocular survival in retinoblastoma and histopathologic grading of anaplasia with increased risk of metastatic spread and death. This study examined the correlation between these factors and other chromosomal abnormalities as well as results of whole genome sequencing, digital morphometry, and progression-free survival. Retrospective cohort study from 2 United States tertiary referral centers. Forty-two children who had undergone enucleation for retinoblastoma from January 2000 through December 2017. Status of chromosomes 6p, 1q, 9q, and 16q was evaluated with fluorescence in situ hybridization, the degree of anaplasia and presence of histologic high-risk features were assessed by ocular pathologists, digital morphometry was performed on scanned tumor slides, and whole genome sequencing was performed on a subset of tumors. Progression-free survival was defined as absence of distant or local metastases or tumor growth beyond the cut end of the optic nerve. Correlation between each of chromosomal abnormalities, anaplasia, morphometry and sequencing results, and survival. Forty-one of 42 included patients underwent primary enucleation and 1 was treated first with intra-arterial chemotherapy. Seven tumors showed mild anaplasia, 19 showed moderate anaplasia, and 16 showed severe anaplasia. All tumors had gain of 1q, 18 tumors had gain of 6p, 6 tumors had gain of 9q, and 36 tumors had loss of 16q. Tumors with severe anaplasia were significantly more likely to harbor 6p gains than tumors with nonsevere anaplasia (P 0.5). Patients with gain of 6p showed significantly shorter progression-free survival (P = 0.03, Wilcoxon test). Gain of chromosome 6p emerges as a strong prognostic biomarker in retinoblastoma because it correlates with severe anaplasia, quantifiable changes in tumor cell staining characteristics, and extraocular spread