Interobserver Variation in the Assessment of Immunohistochemistry Expression Levels in HER2-Negative Breast Cancer: Can We Improve the Identification of Low Levels of HER2 Expression by Adjusting the Criteria? An International Interobserver Study.

The classification of human epidermal growth factor receptor 2 (HER2) expression is optimized to detect HER2-amplified breast cancer (BC). However, novel HER2-targeting agents are also effective for BCs with low levels of HER2. This raises the question whether the current guidelines for HER2 testing are sufficiently reproducible to identify HER2-low BC. The aim of this multicenter international study was to assess the interobserver agreement of specific HER2 immunohistochemistry scores in cases with negative HER2 results (0, 1+, or 2+/in situ hybridization negative) according to the current American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines. Furthermore, we evaluated whether the agreement improved by redefining immunohistochemistry (IHC) scoring criteria or by adding fluorescent in situ hybridization (FISH). We conducted a 2-round study of 105 nonamplified BCs. During the first assessment, 16 pathologists used the latest version of the ASCO/CAP guidelines. After a consensus meeting, the same pathologists scored the same digital slides using modified IHC scoring criteria based on the 2007 ASCO/CAP guidelines, and an extra "ultralow" category was added. Overall, the interobserver agreement was limited (4.7% of cases with 100% agreement) in the first round, but this was improved by clustering IHC categories. In the second round, the highest reproducibility was observed when comparing IHC 0 with the ultralow/1+/2+ grouped cluster (74.3% of cases with 100% agreement). The FISH results were not statistically different between HER2-0 and HER2-low cases, regardless of the IHC criteria used. In conclusion, our study suggests that the modified 2007 ASCO/CAP criteria were more reproducible in distinguishing HER2-0 from HER2-low cases than the 2018 ASCO/CAP criteria. However, the reproducibility was still moderate, which was not improved by adding FISH. This could lead to a suboptimal selection of patients eligible for novel HER2-targeting agents. If the threshold between HER2 IHC 0 and 1+ is to be clinically actionable, there is a need for clearer, more reproducible IHC definitions, training, and/or development of more accurate methods to detect this subtle difference in protein expression levels.

Progression of a solitary plasmacytoma to multiple myeloma. A population-based registry of the northern Netherlands.

Plasmacytoma is characterized by a local accumulation of monoclonal plasma cells without criteria for multiple myeloma (MM). The current treatment regimen is local radiotherapy. However, more than 50% of patients develop MM within 2 years after treatment. A population-based registry was consulted for the diagnosis of solitary plasmacytoma between 1988 and 2011. Progression to MM and prognostic features for progression to MM were scored, including hypoxia inducible factors (HIF), vascular endothelial growth factor (VEGF, also termed VEGFA) and micro-vessel density (MVD) expression in biopsy material. A total of 76 patients were included, 34% having extramedullary plasmacytoma (EMP) while 66% had a solitary plasmacytoma of the bone (SBP). Median follow-up was 89 months, (7-293 months). In Seventy per cent of SBP patients developed MM with a median time to progression of 19 months (5-293). Three patients (12%) with EMP developed MM. High expression of VEGF and HIF-2α (also termed EPAS1) was demonstrated in conjunction with an increased MVD in 66% of the patients. No association could be shown between angiogenesis parameters and progression to MM. In conclusion, this population-based study demonstrates that SBP patients have a higher risk of developing MM following local radiotherapy, indicating that this group might benefit from added systemic chemotherapy.

Promoter hypermethylation in male breast cancer: analysis by multiplex ligation-dependent probe amplification.

INTRODUCTION: Epigenetic events are, along with genetic alteration, important in the development and progression of cancer. Promoter hypermethylation causes gene silencing and is thought to be an early event in carcinogenesis. The role of promoter hypermethylation in male breast cancer has not yet been studied. METHODS: In a group of 108 male breast cancers, the methylation status of 25 genes was studied using methylation-specific multiplex ligation-dependent probe amplification. Methylation of more than 15% was regarded indicative for promoter hypermethylation. Methylation status was correlated with clinicopathological features, with patients' outcome and with 28 female breast cancer cases. RESULTS: Promoter hypermethylation of the genes MSH6, WT1, PAX5, CDH13, GATA5 and PAX6 was seen in more than 50% of the cases, but was uncommon or absent in normal male breast tissue. High overall methylation status was correlated with high grade (P = 0.003) and was an independent predictor of poor survival (P = 0.048; hazard ratio 2.5). ESR1 and GSTP1 hypermethylation were associated with high mitotic count (P = 0.037 and P = 0.002, respectively) and high grade (both P = 0.001). No correlation with survival was seen for individual genes. Compared with female breast cancers (logistic regression), promoter hypermethylation was less common in a variety of genes, particularly ESR1 (P = 0.005), BRCA1 (P = 0.010) and BRCA2 (P < 0.001). The most frequently hypermethylated genes (MSH6, CDH13, PAX5, PAX6 and WT1) were similar for male and female breast cancer. CONCLUSION: Promoter hypermethylation is common in male breast cancer and high methylation status correlates with aggressive phenotype and poor survival. ESR1 and GSTP1 promoter hypermethylation seem to be involved in development and/or progression of high-grade male breast cancer. Although female and male breast cancer share a set of commonly methylated genes, many of the studied genes are less frequently methylated in male breast cancer, pointing towards possible differences between male and female breast carcinogenesis.

Histological assessment of the sclerotic graft-versus-host response in the humanized RAG2-/-γc-/- mouse model.

Graft-versus-host disease (GVHD) remains a frequently occurring and difficult-to-treat complication in human allogeneic stem cell transplantation. Murine transplantation models are often used to study and understand the complex pathogenesis of GVHD and to explore new treatment strategies. Although GVHD kinetics may differ in murine and human models, adequate models are essential for identification of the crucial factors responsible for the major pathology in GVHD. We present a detailed description of the specific histological features of a graft-versus-host-induced fibrotic response in xenogeneic RAG2(-/-)γc(-/-) mice after total body irradiation and injection with human peripheral blood mononuclear cells. We describe the full morphological features of this reaction, including a detailed analysis of the specific tissue infiltration patterns of the human peripheral blood mononuclear cells. Our data show the development of fibrosis, predominantly near blood vessels, and reveal different cell populations and specific cell migration patterns in the affected organs. The combination of immunohistochemical cell characterization and mRNA expression analysis of both human (donor)- and murine (host)-derived cytokines reveals an interaction between host tissues and donor-derived cells in an entangled cytokine profile, in which both donor- and host-derived cytokines contribute to the formation of fibrosis.

Oncogene amplification in male breast cancer: analysis by multiplex ligation-dependent probe amplification.

Gene amplification is an important mechanism for oncogene activation, a crucial step in carcinogenesis. Compared to female breast cancer, little is known on the genetic makeup of male breast cancer, because large series are lacking. Copy number changes of 21 breast cancer related genes were studied in 110 male breast cancers using multiplex ligation-dependent probe amplification. A ratio of >1.3 was regarded indicative for gene copy number gain and a ratio >2.0 for gene amplification. Data were correlated with clinicopathological features, prognosis and 17 genes were compared with a group of female breast cancers. Gene copy number gain of CCND1, TRAF4, CDC6 and MTDH was seen in >40 % of the male breast cancer cases, with also frequent amplification. The number of genes with copy number gain and several single genes were associated with high grade, but only CCND1 amplification was an independent predictor of adverse survival in Cox regression (p = 0.015; hazard ratio 3.0). In unsupervised hierarchical clustering a distinctive group of male breast cancer with poor prognosis (p = 0.009; hazard ratio 3.4) was identified, characterized by frequent CCND1, MTDH, CDC6, ADAM9, TRAF4 and MYC copy number gain. Compared to female breast cancers, EGFR (p = 0.005) and CCND1 (p = 0.041) copy number gain was more often seen in male breast cancer, while copy number gain of EMSY (p = 0.004) and CPD (p = 0.001) and amplification in general was less frequent. In conclusion, several female breast cancer genes also seem to be important in male breast carcinogenesis. However, there are also clear differences in copy number changes between male and female breast cancers, pointing toward differences in carcinogenesis between male and female breast cancer and emphasizing the importance of identifying biomarkers and therapeutic agents based on research in male breast cancer. In addition CCND1 amplification seems to be an independent prognosticator in male breast cancer.

Fibrotic focus and hypoxia in male breast cancer.

Fibrotic focus is a scar-like lesion near the center of a carcinoma and has been associated with high-grade, lymph node metastases and poor survival in female breast cancers. Hypoxia is suggested to be the crucial link between fibrotic focus and aggressive tumor phenotype and is also itself a poor prognostic marker. We here set out to study fibrotic focus and hypoxia in male breast cancer for the first time. In a group of 134 male breast cancer patients, the presence and size of a fibrotic focus and the expression of three hypoxia-related immunohistochemical stainings, hypoxia-inducible factor-1α, carbonic anhydrase IX and Glut-1 were studied in correlation with clinicopathological features and prognosis. Fibrotic focus was seen in 25% of the male breast cancer cases and was correlated with hypoxia-inducible factor-1α overexpression (P=0.023), high grade (P=0.005), high mitotic activity (P=0.005) and lymph node metastases (P=0.037). Hypoxia-inducible factor-1α-positive tumors were more often high grade (P=0.003) and HER2 amplified (P=0.005). Glut-1 expression was also more common in grade 3 tumors (P=0.038), but no association between carbonic anhydrase IX and any clinicopathological feature was found. Fibrotic focus >8 mm and hypoxia-inducible factor-1α overexpression were correlated with decreased patients' outcome (P=0.035 and 0.008, respectively). Hypoxia-inducible factor-1α overexpression was an independent and the most powerful predictor of survival in multivariate analysis (P=0.029; hazard ratio 2.5). In conclusion, the presence of a fibrotic focus is associated with hypoxia-inducible factor-1α overexpression, and both are associated with aggressive tumor phenotype and poor survival in male breast cancer. These markers seem to have similar clinical importance as previously reported in female breast cancer.

Molecular subtyping of male breast cancer by immunohistochemistry.

Molecular subtyping of breast cancer by gene expression has proven its significance in females. Immunohistochemical surrogates have been used for this classification, because gene expression profiling is not yet routinely feasible. Male breast cancer is rare and large series are lacking. In this study, we used immunohistochemistry for molecular subtyping of male breast cancer. A total of 134 cases of male breast cancer were immunohistochemically stained on tissue microarrays for estrogen receptor (ER), progesterone receptor (PR), HER2 and epidermal growth factor receptor (EGFR), as well as for CK5/6, CK14, and Ki67. HER2 was also assessed by chromogen in situ hybridization. Cases were classified as luminal A (ER+ and/or PR+, and HER2- and Ki67 low), luminal B (ER+ and/or PR+, and HER2+ or Ki67 high), HER2 driven (ER-, PR-, HER2+), basal-like (ER-, PR-, HER2-, CK5/6+ and/or CK14+ and/or EGFR+), or unclassifiable triple-negative (negative for all six markers). Luminal type A was by far the most encountered type of male breast cancers, representing 75% of the cases. Luminal type B was seen in 21% and the remaining 4% of cases were classified as basal-like (n=4) and unclassifiable triple-negative (n=1). No HER2 driven cases were identified. Patients with basal-like cancer were significantly younger (P=0.034). Luminal B type cancers showed significantly higher histological grade (P<0.001), mitotic index (P<0.001), and PR negativity (P=0.005) compared with luminal type A cancers. In conclusion, most male breast cancers are luminal A and luminal B types, whereas basal-like, unclassifiable triple-negative, and HER2 driven male breast cancers are rare. Luminal type B seem to represent a subtype with an aggressive phenotype. This distribution of molecular subtypes in male breast cancer is clearly different compared with female breast cancers, pointing to possible important differences in carcinogenesis.

Immunophenotyping of male breast cancer.

AIMS: Male breast cancer is a rare disease, and knowledge of carcinogenesis is limited. Conflicting results, based on small series, have been reported for clinically relevant biomarkers. METHODS AND RESULTS: One hundred and thirty-four cases of male breast cancer were immunohistochemically stained on tissue microarrays for oestrogen receptor (ER), progesterone receptor (PR), androgen receptor, human epidermal growth factor receptor 2 (HER2), BRST2, cyclin D1, bcl-2, p53, p16, p21, Ki67, cytokeratin (CK) 5/6, CK14, and epidermal growth factor receptor. Data were correlated with clinicopathological features and patient outcome. High mitotic count and high grade were correlated with high Ki67, HER2 amplification/overexpression, p53 accumulation, high p21 expression, low PR expression, and low bcl-2 expression. PR negativity (P=0.009) and p53 accumulation (P=0.042) were correlated with decreased 5-year survival and were independent markers for patient outcome in Cox regression. In unsupervised hierarchical clustering, four groups were identified that were correlated with distinctive clinicopathological features. The hormone negative/ER-positive/high-grade cluster was significantly associated with decreased survival (P=0.011) and was an independent prognostic factor in Cox regression. CONCLUSIONS: Several tissue biomarkers are associated with an aggressive phenotype in male breast cancer. PR and p53 are the most promising individual prognostic markers. On the basis of immunophenotype, four distinctive and prognostically relevant male breast cancer groups were identified, indicating that protein expression profiling may be clinically useful in male breast cancer.

Modifying Graft-versus-Host Disease in a Humanized Mouse Model by Targeting Macrophages or B-Cells.

Humanized mouse models can well be modified to study specific aspects of Graft-versus-Host Disease (GvHD). This paper shows the results of both macrophage depletion and (early) B-cell depletion in a humanized mouse model using RAG2-/- γc-/- mice injected with HuPBMCs. Macrophage depletion showed a significant decrease in survival and also lead to a change in the histomorphology of the xenogeneic reaction. Higher levels of infiltrating B-cells were observed in various organs of mice depleted for macrophages. With (early) B-cell depletion using Rituximab, a clear improvement on clinical symptoms was observed, even when probably only inactivated B-cells were deleted. However, the histological examinations only showed a significant morphological effect on liver fibrosis. This may be related to a difference in the mRNA levels of TGF-ß. Also, lower mRNA levels of Tregs in some organs were observed after Rituximab treatment, which contradicts that a higher number of Tregs would always be related to less severe GvHD. Our data show that both macrophage depletion and (early) B-cell depletion in a xenogeneic mouse model can influence the clinical, histological, and cytokine production of a GvHD response.

Humanized mouse models in transplantation research.

The interest in the use of humanized mouse models for research topics like Graft versus Host Disease (GvHD), allograft studies and other studies to the human immune system is growing. The design of these models is still improving and enables even more complicated studies to these topics. For researchers it can be difficult to choose the best option from the current pool of available models. The decision will depend on which hypothesis needs to be tested, in which field of interest, and therefore 'the best model' will differ from one to another. In this review, we provide a guide to the most common available humanized mouse models, with regards to different mouse strains, transplantation material, transplantation techniques, pre- and post-conditioning and references to advantages and disadvantages. Also, an evaluation of experiences with humanized mouse models in studies on GvHD and allograft rejection is provided.

The influence of genetic factors on the osteoinductive potential of calcium phosphate ceramics in mice.

The efficacy of calcium phosphate (CaP) ceramics in healing large bone defects is, in general, not as high as that of autologous bone grafting. Recently, we reported that CaP ceramics with osteoinductive properties were as efficient in healing an ilium defect of a sheep as autologous bone graft was, which makes this subclass of CaP ceramics a powerful alternative for bone regeneration. Although osteoinduction by CaP ceramics has been shown in several large animal models it is sporadically reported in mice. Because the lack of a robust mouse model has delayed understanding of the mechanism, we screened mice from 11 different inbred mouse strains for their responsiveness to subcutaneous implantation of osteoinductive tricalcium phosphate (TCP). In only two strains (FVB and 129S2) the ceramic induced bone formation, and in particularly, in FVB mice, bone was found in all the tested mice. We also demonstrated that other CaP ceramics induced bone formation at the same magnitude as that observed in other animal models. Furthermore, VEGF did not significantly increase TCP induced bone formation. The mouse model here described can accelerate research of osteoinductive mechanisms triggered by CaP ceramics and potentially the development of therapies for bone regeneration.

A new xenograft model for graft-versus-host disease by intravenous transfer of human peripheral blood mononuclear cells in RAG2-/- gammac-/- double-mutant mice.

The safe application of new strategies for the treatment of graft-versus-host disease (GVHD) is hampered by the lack of a clinically relevant model for preclinical testing. Current models are based on intraperitoneal transfer of human peripheral blood mononuclear cells (huPBMCs) into NOD-SCID (nonobese diabetic-severe combined immunodeficient)/SCID mice. Intravenous transfer would be preferred but this has always been ineffective. We developed a new model for xenogeneic GVHD (X-GVHD) by intravenous transfer of huPBMCs into RAG2-/- gammac-/-mice. Our results show a high human T-cell chimerism of more than 20% (up to 98%) in more than 90% of mice, associated with a consistent development of XGVHD within 14 to 28 days and a total mortality rate of 85% shorter than 2 months. After murine macrophage depletion, engraftment was earlier and equally high with lower doses of huPBMCs. Human macrophages were also absent in these mice. Purified huCD3+ cells showed a similar X-GVH effect with contribution of both CD4 and CD8 phenotypes. Human immunoglobulins and cytokines were produced in diseased mice. One of 30 mice developed chronic X-GVHD with skin histology similar to human GVHD. In conclusion, we present a new model for X-GVHD by intravenous transfer of huPBMCs in RAG2-/- gammac-/- mice. Murine and human macrophages do not seem to be necessary for acute X-GVHD in this model.