Gustav Stålhammar

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 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.

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

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).

Evolutionary history of metastatic breast cancer reveals minimal seeding from axillary lymph nodes

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.

Prognostic value of Ki67 analysed by cytology or histology in primary breast cancer

Aims The accuracy of biomarker assessment in breast pathology is vital for therapy decisions. The therapy predictive and prognostic biomarkers oestrogen receptor (ER), progesterone receptor, HER2 and Ki67 may act as surrogates to gene expression profiling of breast cancer. The aims of this study were to investigate the concordance of consecutive biomarker assessment by immunocytochemistry on preoperative fine-needle aspiration cytology versus immunohistochemistry (IHC) on the corresponding resected breast tumours. Further, to investigate the concordance with molecular subtype and correlation to stage and outcome. Methods Two retrospective cohorts comprising 385 breast tumours with clinicopathological data including gene expression-based subtype and up to 10-year overall survival data were evaluated. Results In both cohorts, we identified a substantial variation in Ki67 index between cytology and histology and a switch between low and high proliferation within the same tumour in 121/360 cases. ER evaluations were discordant in only 1.5% of the tumours. From cohort 2, gene expression data with PAM50 subtype were used to correlate surrogate subtypes. IHC-based surrogate classification could identify the correct molecular subtype in 60% and 64% of patients by cytology (n=63) and surgical resections (n=73), respectively. Furthermore, high Ki67 in surgical resections but not in cytology was associated with poor overall survival and higher probability for axillary lymph node metastasis. Conclusions This study shows considerable differences in the prognostic value of Ki67 but not ER in breast cancer depending on the diagnostic method. Furthermore, our findings show that both methods are insufficient in predicting true molecular subtypes.

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

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).

Sebaceous epithelial-myoepithelial carcinoma of the parotid gland: a case report of a new histologic variant.

Epithelial-myoepithelial carcinoma (EMCa) is a double-cell layered low-grade malignant tumor, representing approximately 1% of all salivary gland tumors (Barnest et al. 2005 [1], Brocheriou et al. 1991 [2], Fonte et al. 2001 [3]). Its histologic characteristic is that of an inner layer of cuboidal epithelial cells with dense granular cytoplasm and central or basal rounded nucleus, and an outer layer of clear, polygonal myoepithelial cells, together forming ductal structures in a lobulated papillary or cystic pattern. Although solid components of clear cells are not uncommon, and squamous differentiation, spindle cells, and oncocytic appearance are well-documented histologic features of EMCa, sebaceous differentiation as a precise histologic variant has, to our knowledge, only been suggested by Shinozaki et al [4] in 2008. In this report, we present a case of a carcinoma of the parotid gland in a 59-year old female patient with an immunophenotype supporting the proposed entity of sebaceous EMCa.

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

BackgroundTumor 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.MethodsWe 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.ResultsWe 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).ConclusionSeveral 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.

Tumour thickness, diameter, area or volume? The prognostic significance of conventional versus digital image analysis-based size estimation methods in uveal melanoma

The aim of this study was to compare conventional and novel size estimation methods’ ability to predict survival in uveal melanoma (UM).

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

BackgroundTranscriptomic 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.MethodsIn 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.ResultsMolecular 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.ConclusionsOur results suggest that macroscopic intra-tumoral transcriptomic heterogeneity is limited and unlikely to have an impact on molecular diagnostics for most patients.

Response to correspondence on ‘Low concordance of biomarkers in histopathological and cytological material from breast cancer’

tumours would most likely be relegated to group 3. According to the recommendation of Kawasaki et al., only one case, morphologically consistent with smallcell carcinoma, would be left in group 2. For group 3, Kawasaki et al. suggested that tumours showing characteristic NE morphologies (solid growth of tumour cells with abundant fine-granular eosinophilic cytoplasm, peripheral palisading, and fibrovascular stroma) and diffuse NE marker expression would correspond to NE tumour, well-differentiated (group 1). Three of the group 3 tumours showed morphological features similar to those described above, and low or intermediate grade. We did not observe definite peripheral palisading of tumour cells. Before publication of the revised WHO classification, these morphological features were described in the literature under the name NE carcinoma of the breast. We have applied strict criteria to group 1; therefore, these cases were initially considered to be group 3, because they did not show a carcinoid-like pattern. Finally, as suggested by Kawasaki et al., we reclassified 59 breast carcinomas with NE features in accordance with the morphological subclassification of the 2012 WHO edition: six cases of NE tumour, well differentiated (group 1); one case of NE carcinoma, poorly differentiated/small-cell carcinoma (group 2); and 52 cases of IBC with NE differentiation (group 3). All of the cases in groups 1 and 2 showed diffuse expression of at least one NE marker, and group 3 tumours showed focal (35 cases, 67.3%) or diffuse (17 cases, 32.7%) expression. We reinvestigated patient OS and DFS in the modified NE subgroups; however, we did not observe statistically significant differences in OS and DFS between group 1 (one recurrence and one death) and group 3 (18 recurrences and eight deaths). There seems to be some overlap and confusion between the NE morphologies described in the revised WHO classification (particularly group 1) and those reported in the previous literature before 2012. Nevertheless, we would like to highlight the major findings of our study: (i) the majority of breast carcinomas with NE features do not have typical NE morphology; and (ii) patients with NE differentiation have worse OS and DFS than those without NE differentiation, irrespective of NE morphology and the extent of immunoreactivity for NE markers.