Jelle Wesseling

Subtyping of breast cancer by immunohistochemistry to investigate a relationship between subtype and short and long term survival: a collaborative analysis of data for 10,159 cases from 12 studies

Paul Pharoah and colleagues evaluate the prognostic significance of immunohistochemical subtype classification in more than 10,000 breast cancer cases with early disease, and examine the influence of a patients survival time on the prediction of future survival.

Reversible and adaptive resistance to BRAF(V600E) inhibition in melanoma

Treatment of BRAF(V600E) mutant melanoma by small molecule drugs that target the BRAF or MEK kinases can be effective, but resistance develops invariably. In contrast, colon cancers that harbour the same BRAF(V600E) mutation are intrinsically resistant to BRAF inhibitors, due to feedback activation of the epidermal growth factor receptor (EGFR). Here we show that 6 out of 16 melanoma tumours analysed acquired EGFR expression after the development of resistance to BRAF or MEK inhibitors. Using a chromatin-regulator-focused short hairpin RNA (shRNA) library, we find that suppression of sex determining region Y-box 10 (SOX10) in melanoma causes activation of TGF-β signalling, thus leading to upregulation of EGFR and platelet-derived growth factor receptor-β (PDGFRB), which confer resistance to BRAF and MEK inhibitors. Expression of EGFR in melanoma or treatment with TGF-β results in a slow-growth phenotype with cells displaying hallmarks of oncogene-induced senescence. However, EGFR expression or exposure to TGF-β becomes beneficial for proliferation in the presence of BRAF or MEK inhibitors. In a heterogeneous population of melanoma cells having varying levels of SOX10 suppression, cells with low SOX10 and consequently high EGFR expression are rapidly enriched in the presence of drug, but this is reversed when the drug treatment is discontinued. We find evidence for SOX10 loss and/or activation of TGF-β signalling in 4 of the 6 EGFR-positive drug-resistant melanoma patient samples. Our findings provide a rationale for why some BRAF or MEK inhibitor-resistant melanoma patients may regain sensitivity to these drugs after a ‘drug holiday’ and identify patients with EGFR-positive melanoma as a group that may benefit from re-treatment after a drug holiday.

Magnetic Resonance Imaging Response Monitoring of Breast Cancer During Neoadjuvant Chemotherapy: Relevance of Breast Cancer Subtype

PURPOSE To evaluate the relevance of breast cancer subtypes for magnetic resonance imaging (MRI) markers for monitoring of therapy response during neoadjuvant chemotherapy (NAC). PATIENTS AND METHODS MRI examinations were performed in 188 women before and during NAC. MRI interpretation included lesion morphology at baseline, changes in morphology, size, and contrast uptake kinetics (initial and late enhancement). By using immunohistochemistry, tumors were divided into three subtypes: triple negative, human epidermal growth factor receptor 2 (HER2) positive, and estrogen receptor (ER) positive/HER2 negative. Tumor response was assessed dichotomously (ie, presence or absence of residual tumor in the surgical specimen). Complementary, a continuous scale assessment was used (the breast response index [BRI], representing the relative change in tumor stage). Multivariate regression analysis and receiver operating characteristic analysis were employed to establish significant associations. RESULTS Residual tumor at pathology was present in 31 (66%) of 47 triple-negative tumors, 23 (61%) of 38 HER2-positive tumors, and 96 (93%) of 103 ER-positive/HER2-negative tumors. Multivariate analysis of residual disease showed significant associations between breast cancer subtype and MRI (area under the curve [AUC], 0.84; P < .001). BRI also showed significant correlation among breast cancer subtype, MRI, and age (Pearsons r = 0.465; P < .001). In subset analysis, this was only significant for triple-negative tumors (P < .001) and HER2-positive tumors (P < .05). Residual tumor after NAC in the triple-negative and HER2-positive group is significantly associated with the change in largest diameter of late enhancement during NAC (AUC, 0.76; P < .001). No associations were found for ER-positive/HER2-negative tumors. CONCLUSION MRI during NAC to monitor response is effective in triple-negative or HER2-positive disease but is inaccurate in ER-positive/HER2-negative breast cancer.

Somatic mutations in the chromatin remodeling gene ARID1A occur in several tumor types.

Mutations in the chromatin remodeling gene ARID1A have recently been identified in the majority of ovarian clear cell carcinomas (OCCCs). To determine the prevalence of mutations in other tumor types, we evaluated 759 malignant neoplasms including those of the pancreas, breast, colon, stomach, lung, prostate, brain, and blood (leukemias). We identified truncating mutations in 6% of the neoplasms studied; nontruncating somatic mutations were identified in an additional 0.4% of neoplasms. Mutations were most commonly found in gastrointestinal samples with 12 of 119 (10%) colorectal and 10 of 100 (10%) gastric neoplasms, respectively, harboring changes. More than half of the mutated colorectal and gastric cancers displayed microsatellite instability (MSI) and the mutations in these tumors were out‐of‐frame insertions or deletions at mononucleotide repeats. Mutations were also identified in 2–8% of tumors of the pancreas, breast, brain (medulloblastomas), prostate, and lung, and none of these tumors displayed MSI. These findings suggest that the aberrant chromatin remodeling consequent to ARID1A inactivation contributes to a variety of different types of neoplasms. Hum Mutat 33:100–103, 2012.

Increased risk of fundic gland polyps during long-term proton pump inhibitor therapy

It is controversial whether proton pump inhibitor use leads to fundic gland polyp development.

Tissue Distribution of the Death Ligand TRAIL and Its Receptors

Recombinant human (rh) TNF-related apoptosis-inducing ligand (TRAIL) harbors potential as an anticancer agent. RhTRAIL induces apoptosis via the TRAIL receptors TRAIL-R1 and TRAIL-R2 in tumors and is non-toxic to nonhuman primates. Because limited data are available about TRAIL receptor distribution, we performed an immunohistochemical (IHC) analysis of the expression of TRAIL-R1, TRAIL-R2, the anti-apoptotic TRAIL receptor TRAIL-R3, and TRAIL in normal human and chimpanzee tissues. In humans, hepatocytes stained positive for TRAIL and TRAIL receptors and bile duct epithelium for TRAIL, TRAIL-R1, and TRAIL-R3. In brains, neurons expressed TRAIL-R1, TRAIL-R2, TRAIL-R3 but no TRAIL. In kidneys, TRAIL-R3 was negative, tubuli contorti expressed TRAIL-R1, TRAIL-R2, and TRAIL, and cells in Henles loop expressed only TRAIL-R2. Heart myocytes showed positivity for all proteins studied. In colon, TRAIL-R1, TRAIL-R2, and TRAIL were present. Germ and Leydig cells were positive for all proteins studied. Endothelium in liver, heart, kidney, and testis lacked TRAIL-R1 and TRAIL-R2. In alveolar septa and bronchial epithelium TRAIL-R2 was expressed, brain vascular endothelium expressed TRAIL-R2 and TRAIL-R3, and in heart vascular endothelium only TRAIL-R3 was present. Only a few differences were observed between human and chimpanzee liver, brain, and kidney. In contrast to human, chimpanzee bile duct epithelium lacked TRAIL, TRAIL-R1, and TRAIL-R3, lung and colon showed no TRAIL or its receptors, TRAIL-R3 was absent in germ and Leydig cells, and vascular endothelium showed only TRAIL-R2 expression in the brain. In conclusion, comparable expression of TRAIL and TRAIL receptors was observed in human and chimpanzee tissues. Lack of liver toxicity in chimpanzees after rhTRAIL administration despite TRAIL-R1 and TRAIL-R2 expression is reassuring for rhTRAIL application in humans. (J Histochem Cytochem 52:821–831, 2004)

Gene Expression Profiling to Identify the Histogenetic Origin of Metastatic Adenocarcinomas of Unknown Primary

PURPOSE Patients with adenocarcinoma of unknown primary origin (ACUP) constitute approximately 4% of all malignancies. For effective treatment of these patients, it is considered optimal to identify the primary tumor origins. Currently, the success rate of the diagnostic work-up is only 20% to 30%. Our goal was to evaluate the contribution of gene expression profiling for routine clinical practice in patients with ACUP. PATIENTS AND METHODS Formalin-fixed, paraffin-embedded (FFPE) samples were obtained from 84 patients with a known primary adenocarcinoma and from 38 patients with ACUP. An extensive immunohistochemical panel classified 16 of the patients with ACUP, whereas 22 patients remained unclassified for their histogenetic origin. Information about staging procedures and clinical follow-up were available in all patient cases. The expression data were analyzed in relation to clinicopathologic variables and immunohistochemical results. RESULTS The gene expression-based assay classified the primary site correctly in 70 (83%) of 84 patient cases of primary and metastatic tumors of known origin, with good sensitivity for the majority of the tumor classes and relatively poor sensitivity for primary lung adenocarcinoma. Gene expression profiling identified 15 (94%) of 16 patients with initial ACUP who were classified by immunohistochemistry, and it made a valuable contribution to a potential site of origin in 14 of the 22 patients with ACUP. CONCLUSION The gene expression platform can classify correctly from FFPE samples the majority of tumors classes both in patients with known primary and in patients with ACUP. Therefore, gene expression profiling represents an additional analytic approach to assist with the histogenetic diagnosis of patients with ACUP.

An aCGH classifier derived from BRCA1-mutated breast cancer and benefit of high-dose platinum-based chemotherapy in HER2-negative breast cancer patients

Background: Breast cancer cells deficient for BRCA1 are hypersensitive to agents inducing DNA double-strand breaks (DSB), such as bifunctional alkylators and platinum agents. Earlier, we had developed a comparative genomic hybridisation (CGH) classifier based on BRCA1-mutated breast cancers. We hypothesised that this BRCA1-likeCGH classifier could also detect loss of function of BRCA1 due to other causes besides mutations and, consequently, might predict sensitivity to DSB-inducing agents. Patients and methods: We evaluated this classifier in stage III breast cancer patients, who had been randomly assigned between adjuvant high-dose platinum-based (HD-PB) chemotherapy, a DSB-inducing regimen, and conventional anthracycline-based chemotherapy. Additionally, we assessed BRCA1 loss through mutation or promoter methylation and immunohistochemical basal-like status in the triple-negative subgroup (TN subgroup). Results: We observed greater benefit from HD-PB chemotherapy versus conventional chemotherapy among patients with BRCA1-likeCGH tumours [41/230 = 18%, multivariate hazard ratio (HR) = 0.12, 95% confidence interval (CI) 0.04–0.43] compared with patients with non-BRCA1-likeCGH tumours (189/230 = 82%, HR = 0.78, 95% CI 0.50–1.20), with a significant difference (test for interaction P = 0.006). Similar results were obtained for overall survival (P interaction = 0.04) and when analyses were restricted to the TN subgroup. Sixty-three percent (20/32) of assessable BRCA1-likeCGH tumours harboured either a BRCA1 mutation (n = 8) or BRCA1 methylation (n = 12). Conclusion: BRCA1 loss as assessed by CGH analysis can identify patients with substantially improved outcome after adjuvant DSB-inducing chemotherapy when compared with standard anthracycline-based chemotherapy in our series.

Concordance of clinical and molecular breast cancer subtyping in the context of preoperative chemotherapy response

ER, PR and HER2 status in breast cancer are important markers for the selection of drug therapy. By immunohistochemistry (IHC), three major breast cancer subtypes can be distinguished: Triple negative (TNIHC), HER2+IHC and LuminalIHC (ER+IHC/HER2−IHC). By using the intrinsic gene set defined by Hu et al. five molecular subtypes (BasalmRNA, HER2+mRNA, Luminal AmRNA, Luminal BmRNA and Normal-likemRNA) can be defined. We studied the concordance between analogous subtypes and their prediction of response to neoadjuvant chemotherapy. We classified 195 breast tumors by both IHC and mRNA expression analysis of patients who received neoadjuvant treatment at the Netherlands Cancer institute for Stage II–III breast cancer between 2000 and 2007. The pathological complete remission (pCR) rate was used to assess chemotherapy response. The IHC and molecular subtypes showed high concordance with the exception of the HER2+IHC group. 60% of the HER2+IHC tumors were not classified as HER2+mRNA. The HER2+IHC/Luminal A or BmRNA group had a low response rate to a trastuzumab-chemotherapy combination with a pCR rate of 8%, while the HER2+mRNA group had a pCR rate of 54%. The Luminal AmRNA and Luminal BmRNA groups showed similar degrees of response to chemotherapy. Neither the PR status nor the endocrine responsiveness index subdivided the ER+IHC tumors accurately into Luminal AmRNA and Luminal BmRNA groups. Molecular subtyping suggests the existence of a HER2+IHC/LuminalmRNA group that responds poorly to trastuzumab-based chemotherapy. For LuminalIHC and triple negativeIHC tumors, further subdivision into molecular subgroups does not offer a clear advantage in treatment selection.

Receptor conversion in distant breast cancer metastases

IntroductionWhen breast cancer patients develop distant metastases, the choice of systemic treatment is usually based on tissue characteristics of the primary tumor as determined by immunohistochemistry (IHC) and/or molecular analysis. Several previous studies have shown that the immunophenotype of distant breast cancer metastases may be different from that of the primary tumor (receptor conversion), leading to inappropriate choice of systemic treatment. The studies published so far are however small and/or methodologically suboptimal. Therefore, definite conclusions that may change clinical practice could not yet be drawn. We therefore aimed to study receptor conversion for estrogen receptor alpha (ERα), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) in a large group of distant (non-bone) breast cancer metastases by re-staining all primary tumors and metastases with current optimal immunohistochemical and in situ hybridization methods on full sections.MethodsA total of 233 distant breast cancer metastases from different sites (76 skin, 63 liver, 43 lung, 44 brain and 7 gastro-intestinal) were IHC stained for ERα, PR and HER2, and expression was compared to that of the primary tumor. HER2 in situ hybridization (ISH) was done in cases of IHC conversion or when primary tumors or metastases showed an IHC 2+ result.ResultsUsing a 10% threshold, receptor conversion by IHC for ERα, PR occurred in 10.3%, 30.0% of patients, respectively. In 10.7% of patients, conversion from ER+ or PR+ to ER-/PR- and in 3.4% from ER-/PR- to ER+ or PR+ was found. Using a 1% threshold, ERα and PR conversion rates were 15.1% and 32.6%. In 12.4% of patients conversion from ER+ or PR+ to ER-/PR-, and 8.2% from ER-/PR- to ER+ or PR+ occurred. HER2 conversion occurred in 5.2%. Of the 12 cases that showed HER2 conversion by IHC, 5 showed also conversion by ISH. One further case showed conversion by ISH, but not by IHC. Conversion was mainly from positive in the primary tumor to negative in the metastases for ERα and PR, while HER2 conversion occurred equally both ways. PR conversion occurred significantly more often in liver, brain and gastro-intestinal metastases.ConclusionsReceptor conversion by immunohistochemistry in (non-bone) distant breast cancer metastases does occur, is relatively uncommon for ERα and HER2, and is more frequent for PR, especially in brain, liver and gastro-intestinal metastases.