Willemijne A. M. E. Schrijver

Influence of decalcification procedures on immunohistochemistry and molecular pathology in breast cancer.

Distant breast cancer metastases are nowadays routinely biopsied to reassess receptor status and to isolate DNA for sequencing of druggable targets. Bone metastases are the most frequent subgroup. Decalcification procedures may negatively affect antigenicity and DNA quality. We therefore evaluated the effect of several decalcification procedures on receptor status and DNA/RNA quality. In 23 prospectively collected breast tumors, we compared ERα, PR and HER2 status by immunohistochemistry in (non-decalcified) tissue routinely processed for diagnostic purposes and in parallel tissue decalcified in Christensen’s buffer with and without microwave, EDTA and Formical-4. Furthermore, HER2 fluorescence in situ hybridization and DNA/RNA quantity and quality were assessed. We found that the percentage of ERα-positive cells were on average lower in EDTA (P=0.049) and Formical-4 (P=0.047) treated cases, compared with controls, and PR expression showed decreased antigenicity after Christensen’s buffer treatment (P=0.041). Overall, a good concordance (weighted kappa) was seen for ERα, PR and HER2 immunohistochemistry when comparing the non-decalcified control tissues with the decalcified tissues. For two patients (9%), there was a potential influence on therapeutic decision making with regard to hormonal therapy or HER2-targeted therapy. HER2 fluorescence in situ hybridization interpretation was seriously hampered by Christensen’s buffer and Formical-4, and DNA/RNA quantity and quality were decreased after all four decalcification procedures. Validation on paired primary breast tumor specimens and EDTA-treated bone metastases showed that immunohistochemistry and fluorescence in situ hybridization were well assessable and DNA and RNA yield and quality were sufficient. With this, we conclude that common decalcification procedures have only a modest negative influence on hormone and HER2 receptor immunohistochemistry in breast cancer. However, they may seriously affect DNA/RNA-based diagnostic procedures. Overall, EDTA-based decalcification is therefore to be preferred as it best allows fluorescence in situ hybridization and DNA/RNA isolation.

Receptor Conversion in Distant Breast Cancer Metastases: A Systematic Review and Meta-analysis

Background In metastatic breast cancer, hormone and/or human epidermal growth factor receptor 2 (HER2)-targeted therapy decision-making is still largely based on tissue characteristics of the primary tumor. However, a change of estrogen receptor alpha (ERα), progesterone receptor (PR), and HER2 status in distant metastases has frequently been reported. The actual incidence of this phenomenon has been debated. Methods We performed a meta-analysis including 39 studies assessing receptor conversion from primary breast tumors to paired distant breast cancer metastases. We noted the direction of change (positive to negative or vice versa) and performed subgroup analyses for different thresholds for positivity, the type of test used to assess HER2 receptor status, and metastasis location-specific differences (two-sided tests). Results Overall, the incidence of receptor conversion varied largely between studies. For ERα, PR, and HER2, we found that random effects pooled positive to negative conversion percentages of 22.5% (95% confidence interval [CI] = 16.4% to 30.0%), 49.4% (95% CI = 40.5% to 58.2%), and 21.3% (95% CI = 14.3% to 30.5%), respectively. Negative to positive conversion percentages were 21.5% (95% CI = 18.1% to 25.5%), 15.9% (95% CI = 11.3% to 22.0%), and 9.5% (95% CI = 7.4% to 12.1%). Furthermore, ERα discordance was statistically significantly higher in the central nervous system and bone compared with liver metastases (20.8%, 95% CI = 15.0% to 28.0%, and 29.3%, 95% CI = 13.0% to 53.5%, vs 14.3%, 95% CI = 11.3% to 18.1, P = .008 and P < .001, respectively), and PR discordance was higher in bone (42.7%, 95% CI = 35.1% to 50.6%, P < .001) and liver metastases (47.0%, 95% CI = 41.0% to 53.0%, P < .001) compared with central nervous system metastases (23.3%, 95% CI = 16.0% to 32.6%). Conclusions Receptor conversion for ERα, PR, and HER2 occurs frequently in the course of disease progression in breast cancer. Large prospective studies assessing the impact of receptor conversion on treatment efficacy and survival are needed. Meanwhile, reassessing receptor status in metastases is strongly encouraged.

Unravelling site-specific breast cancer metastasis: a microRNA expression profiling study

Distant metastasis is still the main cause of death from breast cancer. MicroRNAs (miRs) are important regulators of many physiological and pathological processes, including metastasis. Molecular breast cancer subtypes are known to show a site-specific pattern of metastases formation. In this study, we set out to determine the underlying molecular mechanisms of site-specific breast cancer metastasis by microRNA expression profiling. To identify a miR signature for metastatic breast carcinoma that could predict metastatic localization, we compared global miR expression in 23 primary breast cancer specimens with their corresponding multiple distant metastases to ovary (n=9), skin (n=12), lung (n=10), brain (n=4) and gastrointestinal tract (n=10) by miRCURY microRNA expression arrays. For validation, we performed quantitative real-time (qRT) PCR on the discovery cohort and on an independent validation cohort of 29 primary breast cancer specimens and their matched metastases. miR expression was highly patient specific and miR signatures in the primary tumor were largely retained in the metastases, with the exception of several differentially expressed, location specific miRs. Validation with qPCR demonstrated that hsa-miR-106b-5p was predictive for the development of lung metastases. In time, the second metastasis often showed a miR upregulation compared to the first metastasis. This study discovered a metastatic site-specific miR and found miR expression to be highly patient specific. This may lead to novel biomarkers predicting site of distant metastases, and to adjuvant, personalized targeted therapy strategies that could prevent such metastases from becoming clinically manifest.

Progressive APOBEC3B mRNA expression in distant breast cancer metastases

Background APOBEC3B was recently identified as a gain-of-function enzymatic source of mutagenesis, which may offer novel therapeutic options with molecules that specifically target this enzyme. In primary breast cancer, APOBEC3B mRNA is deregulated in a substantial proportion of cases and its expression is associated with poor prognosis. However, its expression in breast cancer metastases, which are the main causes of breast cancer-related death, remained to be elucidated. Patients and methods RNA was isolated from 55 primary breast cancers and paired metastases, including regional lymph node (N = 20) and distant metastases (N = 35). APOBEC3B mRNA levels were measured by RT-qPCR. Expression levels of the primary tumors and corresponding metastases were compared, including subgroup analysis by estrogen receptor (ER/ESR1) status. Results Overall, APOBEC3B mRNA levels of distant metastases were significantly higher as compared to the corresponding primary breast tumor (P = 0.0015), an effect that was not seen for loco-regional lymph node metastases (P = 0.23). Subgroup analysis by ER-status showed that increased APOBEC3B levels in distant metastases were restricted to metastases arising from ER-positive primary breast cancers (P = 0.002). However, regarding ER-negative primary tumors, only loco-regional lymph node metastases showed increased APOBEC3B expression when compared to the corresponding primary tumor (P = 0.028). Conclusion APOBEC3B mRNA levels are significantly higher in breast cancer metastases as compared to the corresponding ER-positive primary tumors. This suggests a potential role for APOBEC3B in luminal breast cancer progression, and consequently, a promising role for anti-APOBEC3B therapies in advanced stages of this frequent form of breast cancer.

Loss of steroid hormone receptors is common in malignant pleural and peritoneal effusions of breast cancer patients treated with endocrine therapy

Discordance in estrogen receptor alpha (ERα), progesterone receptor (PR), androgen receptor (AR) and human epidermal growth factor receptor 2 (HER2) status between primary breast cancers and solid distant metastases (“conversion”) has been reported previously. Even though metastatic spread to the peritoneal and pleural cavities occurs frequently and is associated with high mortality, the rate of receptor conversion and the prognostic implications thereof remain elusive. We therefore determined receptor conversion in 91 effusion metastases (78 pleural, 13 peritoneal effusions) of 69 patients by immunohistochemistry (IHC) and in situ hybridization. Data were coupled to clinical variables and treatment history. ERα, PR and AR receptor status converted from positive in the primary tumor to negative in the effusion metastases or vice versa in 25-30%, 30-35% and 46-51% of cases for the 1% and 10% thresholds for positivity, respectively. 19-25% of patients converted clinically relevant from “ERα+ or PR+” to ERα-/PR- and 3-4% from ERα-/PR- to “ERα+ or PR+”. For HER2, conversion was observed in 6% of cases. Importantly, receptor conversion for ERα (p = 0.058) and AR (p < 0.001) was more often seen in patients adjuvantly treated with endocrine therapy. Analogous to this observation, HER2-loss was more frequent in patients adjuvantly treated with trastuzumab (p < 0.001). Alike solid distant metastases, receptor conversion for ERα, PR, AR and HER2 is a frequent phenomenon in peritoneal and pleural effusion metastases. Adjuvant endocrine and trastuzumab therapy imposes an evolutionary selection pressure on the tumor, leading to receptor loss in effusion metastases. Determination of receptor status in malignant effusion specimens will facilitate endocrine treatment decision-making at this lethal state of the disease, and is hence recommended whenever possible.

The prognostic effect of DDX3 upregulation in distant breast cancer metastases

Metastatic breast cancer remains one of the leading causes of death in women and identification of novel treatment targets is therefore warranted. Functional studies showed that the RNA helicase DDX3 promotes metastasis, but DDX3 expression was never studied in patient samples of metastatic cancer. In order to validate previous functional studies and to evaluate DDX3 as a potential therapeutic target, we investigated DDX3 expression in paired samples of primary and metastatic breast cancer. Samples from 79 breast cancer patients with distant metastases at various anatomical sites were immunohistochemically stained for DDX3. Both cytoplasmic and nuclear DDX3 expression were compared between primary and metastatic tumors. In addition, the correlation between DDX3 expression and overall survival was assessed. Upregulation of cytoplasmic (28%; OR 3.7; p = 0.002) was common in breast cancer metastases, especially in triple negative (TN) and high grade cases. High cytoplasmic DDX3 levels were most frequent in brain lesions (65%) and significantly correlated with high mitotic activity and triple negative subtype. In addition, worse overall survival was observed for patients with high DDX3 expression in the metastasis (HR 1.79, p = 0.039). Overall, we conclude that DDX3 expression is upregulated in distant breast cancer metastases, especially in the brain and in TN cases. In addition, high metastatic DDX3 expression correlates with worse survival, implying that DDX3 is a potential therapeutic target in metastatic breast cancer, in particular in the clinically important group of TN patients.

Prospects of targeting the gastrin releasing peptide receptor and somatostatin receptor 2 for nuclear imaging and therapy in metastatic breast cancer

Background The gastrin releasing peptide receptor (GRPR) and the somatostatin receptor 2 (SSTR2) are overexpressed on primary breast cancer (BC), making them ideal candidates for receptor-mediated nuclear imaging and therapy. The aim of this study was to determine whether these receptors are also suitable targets for metastatic BC. Methods mRNA expression of human BC samples were studied by in vitro autoradiography and associated with radioligand binding. Next, GRPR and SSTR2 mRNA levels of 60 paired primary BCs and metastases from different sites were measured by quantitative reverse transcriptase polymerase chain reaction. Receptor mRNA expression levels were associated with clinico-pathological factors and expression levels of primary tumors and corresponding metastases were compared. Results Binding of GRPR and SSTR radioligands to tumor tissue correlated significantly with receptor mRNA expression. High GRPR and SSTR2 mRNA levels were associated with estrogen receptor (ESR1)-positive tumors (p<0.001 for both receptors). There was no significant difference in GRPR mRNA expression of primary tumors versus paired metastases. Regarding SSTR2 mRNA expression, there was also no significant difference in the majority of cases, apart from liver and ovarian metastases which showed a significantly lower expression compared to the corresponding primary tumors (p = 0.02 and p = 0.03, respectively). Conclusion Targeting the GRPR and SSTR2 for nuclear imaging and/or treatment has the potential to improve BC care in primary as well as metastatic disease.

FOXA1 levels are decreased in pleural breast cancer metastases after adjuvant endocrine therapy, and this is associated with poor outcome

Estrogen receptor‐alpha (ERα)‐positive breast cancer is often treated with antihormonal regimens. However, resistance to treatment is common, leading to metastatic disease. ERα activity requires the functional involvement of pioneer factors FOXA1 and GATA3, which enable ERα–chromatin binding and are crucial for ERα‐driven cell proliferation. FOXA1 was found increased in metastatic breast cancers in relation to the primary tumor, but a comprehensive clinical assessment thereof, in relation to different metastatic sites and endocrine therapy usage, is currently lacking. Prior cell line‐based reports, however, have revealed that FOXA1 is required for tamoxifen‐resistant tumor cell proliferation. We studied expression levels of ERα, GATA3, and FOXA1 by immunohistochemistry in samples from both primary tumors and various metastatic sites. For all factors, expression levels varied between the metastatic sites. For pleural metastases, strong variation was found in FOXA1 and GATA3 levels. Although GATA3 levels remained unaltered between primary breast cancer and pleural metastases, FOXA1 levels were reduced exclusively in metastases of patients who received endocrine therapies in the adjuvant setting, even though ERα was still expressed. Importantly, decreased FOXA1 levels in pleural metastases correlated with hormone irresponsiveness in the palliative setting, while no such correlation was found for GATA3. With this, we show divergent clinical correlations of the two ERα pioneer factors FOXA1 and GATA3 in metastatic breast cancer, where endocrine therapy resistance was associated with decreased FOXA1 levels in pleural metastases.

Mutation Profiling of Key Cancer Genes in Primary Breast Cancers and Their Distant Metastases

Although the repertoire of somatic genetic alterations of primary breast cancers has been extensively catalogued, the genetic differences between primary and metastatic tumors have been less studied. In this study, we compared somatic mutations and gene copy number alterations of primary breast cancers and their matched metastases from patients with estrogen receptor (ER)-negative disease. DNA samples obtained from formalin-fixed paraffin-embedded ER-negative/HER2-positive (n = 9) and ER-, progesterone receptor (PR-), HER2-negative (n = 8) primary breast cancers and from paired brain or skin metastases and normal tissue were subjected to a hybridization capture-based massively parallel sequencing assay, targeting 341 key cancer genes. A large subset of nonsynonymous somatic mutations (45%) and gene copy number alterations (55%) was shared between the primary tumors and paired metastases. However, mutations restricted to either a given primary tumor or its metastasis, the acquisition of loss of heterozygosity of the wild-type allele, and clonal shifts of genes affected by somatic mutations, such as TP53 and RB1, were observed in the progression from primary tumors to metastases. No metastasis location-specific alterations were identified, but synchronous metastases showed higher concordance with the paired primary tumor than metachronous metastases. Novel potentially targetable alterations were found in the metastases relative to their matched primary tumors. These data indicate that repertoires of somatic genetic alterations in ER-negative metastatic breast cancers may differ from those of their primary tumors, even by the presence of driver and targetable somatic genetic alterations.Significance: Somatic genetic alterations in ER-negative breast cancer metastases may be distinct from those of their primary tumors, suggesting that for treatment-decision making, genetic analyses of DNA obtained from the metastatic lesion rather than from the primary tumor should be considered. Cancer Res; 78(12); 3112-21. ©2018 AACR.

Molecular diagnostics in breast cancer routine practice

The portfolio of adjuvant systemic treatment of breast cancer nowadays contains novel anti-hormonal and chemotherapeutic drugs, immunotherapeutic approaches and small molecules that are only effective in a limited number of patients and are often associated with high costs and significant side effects. Therefore, a personalised approach based on individual tumour biomarkers is required to arrive at the optimal balance between effectiveness on the one hand, and costs and side effects on the other. The aim of this paper is to provide an overview of the molecular biomarkers and associated molecular tests that are currently relevant in pathology of invasive breast cancer.