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 χ² (LR χ²) was higher for DIA that also added significantly more prognostic information to the manual scores (LR−Δχ²). 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.

Metastatic breast cancers are still incurable. Characterizing the evolutionary landscape of these cancers, including the role of metastatic axillary lymph nodes (ALNs) in seeding distant organ metastasis, can provide a rational basis for effective treatments. Here, we have described the genomic analyses of the primary tumors and metastatic lesions from 99 samples obtained from 20 patients with breast cancer. Our evolutionary analyses revealed diverse spreading and seeding patterns that govern tumor progression. Although linear evolution to successive metastatic sites was common, parallel evolution from the primary tumor to multiple distant sites was also evident. Metastatic spreading was frequently coupled with polyclonal seeding, in which multiple metastatic subclones originated from the primary tumor and/or other distant metastases. Synchronous ALN metastasis, a well-established prognosticator of breast cancer, was not involved in seeding the distant metastasis, suggesting a hematogenous route for cancer dissemination. Clonal evolution coincided frequently with emerging driver alterations and evolving mutational processes, notably an increase in apolipoprotein B mRNA–editing enzyme, catalytic polypeptide-like–associated (APOBEC-associated) mutagenesis. Our data provide genomic evidence for a role of ALN metastasis in seeding distant organ metastasis and elucidate the evolving mutational landscape during cancer progression.

Uveal melanoma is the most common primary intraocular malignancy. A vast majority of metastasizing tumors have mutations in the BAP1 gene. Here, we investigate the spatiotemporal timing of these mutations. The size of 177 uveal melanomas and 8.3 million individual tumor cells was measured. BAP1 sequencing results and BAP1 IHC were available and for 76 (43%) and 101 (57%) of these, respectively. Tumors with a BAP1 mutation had significantly larger volume (2109 vs. 1552 mm³, p = 0.025). Similarly, tumor cells with loss of BAP1 protein expression had significantly larger volume (2657 vs. 1593 μm³, p = 0.027). Using observations of the time elapsed between mitoses, the BAP1 mutation was calculated to occur when the primary tumor had a size of a few malignant cells to 6 mm³, 0.5 to 4.6 years after tumor initiation and at least 9 years before diagnosis. We conclude that BAP1 mutations occur early in the growth of uveal melanoma, well before the average tumor is diagnosed. Its timing coincides with the seeding of micrometastases.

Background: Survival in metastasized cutaneous melanoma (CM) has been improved with the advent of inhibitors of immune checkpoints CTLA4 and PD‐1. In contrast, the response rate for inhibition of these checkpoints in uveal melanoma (UM) is very low. Other checkpoints including IDO and TIGIT may be targetable.Methods: Sections from 6 patients with UM, who had undergone primary enucleation 1978—1995 and 6 paired liver metastases were stained immunohistochemically (SOX10, Melan‐A, IDO, TIGIT, and CD8). Four tumors from patients who did not develop metastasis during a mean follow‐up of 19 years, and 5 samples each of normal choroidal and liver tissue were included for comparison. The number of cells/mm² expressing IDO, TIGIT and CD8 was counted with manual and digital image analysis methods. Retrospective data on patient and tumor characteristics was reviewed.Results: The number of TIGIT positive cells was significantly higher in primary tumors from patients who eventually developed metastases (mean 4695 cells/mm²) than from patients who didn't (mean 1342 cells/mm², P < 0.01) and paired metastases (463 cells/mm², P < 0.01). The number of IDO positive cells was not significantly higher in metastatic tumors (P = 0.079), but the number of IDO and TIGIT positive cells/mm² correlated in both hot spots (R² = 0.24, P < 0.01) and full tumor sections (R² = 0.35, P < 0.01).Conclusion: The expression of immune checkpoint receptor TIGIT is increased in primary uveal melanomas that seed metastases, and correlates with the expression of checkpoint receptor IDO. Both may be future targets for therapy.

Background: Episcleral brachytherapy is the most common eye-preserving treatment for medium-sized choroidal melanomas. γ-emitting iodine-125 (¹²⁵I) and β-emitting ruthenium-106 (¹⁰⁶Ru) are widely used. The latter is however generally reserved for thinner tumours (<6 mm). In this study, we compare ocular and patient survival in thicker tumours treated with the respective radioisotope.Methods: All patients with ≥5.5 mm thick choroidal melanomas who were treated with plaque brachytherapy at a single institution between 1 November 1979 and 31 December 2015 were included (n=571). Size-controlled Cox regression HRs for postbrachytherapy enucleation, repeated brachytherapy and melanoma-related mortality were calculated, as well as Kaplan-Meier disease-specific survival and relative 10-year survival in matched subgroups.Results: 317 patients were treated with ¹⁰⁶Ru and 254 with ¹²⁵I. The rate of repeated brachytherapy was significantly higher among patients treated with ¹⁰⁶Ru (8%) than with ¹²⁵I (1%, p<0.001). Size-controlled Cox regression HRs for postbrachytherapy enucleation (¹²⁵I vs ¹⁰⁶Ru 0.7, p=0.083) and melanoma-related mortality were not significant (¹²⁵I vs ¹⁰⁶Ru 1.1, p=0.63). Similarly, Kaplan-Meier disease-specific and relative 10-year survival was comparable in matched groups of 5.5–7.4 mm (relative survival ¹⁰⁶Ru 59%, ¹²⁵I 56%) and ≥7.5 mm thick tumours (relative survival ¹⁰⁶Ru 46%, ¹²⁵I 44%).Conclusions: Rates of repeated brachytherapy were significantly higher among patients treated with ¹⁰⁶Ru versus ¹²⁵I for thick choroidal melanomas. There were, however, no significant differences in rates of enucleation or patient survival.