NOTCH1 and PIK3CA mutation are related to HPV-associated vulvar squamous cell carcinoma.

NOTCH1 and PIK3CA are members of important cell signalling pathways that are deregulated in squamous cell carcinomas of various organs. Vulvar squamous cell carcinomas (vulvSCC) are classically divided into two pathways, HPV-associated or HPV-independent, but the effect of NOTCH1 and PIK3CA mutations in both groups is unclear. We analysed two different cohorts of vulvSCC using Hybrid Capture-based Comprehensive Genomic Profiling and identified NOTCH1 and PIK3CA mutations in 35% and 31% of 48 primary vulvSCC. In this first cohort, PIK3CA and NOTCH1 mutations were significantly correlated with HPV infection (p < 0.01). Furthermore, mutations in both genes were associated with an advanced tumor stage and poorly differentiated status (p < 0.05). PIK3CA and NOTCH1 mutations were also associated with shorter patient survival which did not reach significance. In the second cohort of 735 advanced vulvSCC from metastatic site biopsies or from sites of unresectable loco-regional disease, NOTCH1 and PIK3CA mutations were reported in 14% and 20.3%, respectively. 4 of 48 (8%) and 22 of 735 vulvSCC (3.0%) featured genomic alterations (short variants and/or copy number changes and/or rearrangements) in both NOTCH1 and PIK3CA. NOTCH1 mutations were mostly located in the extracellular EGF-like domains, were inactivating and indicated that NOTCH1 functions predominantly as a tumor suppressor gene in vulvSCC. In contrast, PIK3CA mutations favored hotspot codons 1624 and 1633 of the gene, indicating that PIK3CA acts as an oncogene in vulvar carcinogenesis. In conclusion, NOTCH1 and PIK3CA mutations are detectable in a substantial proportion of vulvSCC and are related to HPV infection and more aggressive tumor behaviour.

PD-L1 receptor expression in vulvar carcinomas is HPV-independent.

PD-L1 (programmed cell death 1 ligand) is expressed on many cancer cells and prevents tumor cell death by blocking T cell activity. PD-L1 overexpression has been reported in squamous cell carcinomas of head and neck and lung cancer. To better understand the role of PD-L1 expression in vulvar cancer, we analyzed PD-L1 expression by immunohistochemistry in 55 well-characterized squamous cell carcinomas of vulva. PD-L1 was found in 72.7% of tumors. 27.3% of vulvar carcinomas showed moderate or strong PD-L1 expression. PD-L1 expression was correlated with low tumor stage (p < 0.05). There was no association to other clinicopathological parameters, HPV status, and overall survival of vulvar carcinoma patients. In conclusion, PD-L1 overexpression is detectable in a substantial proportion of vulvar carcinomas in all stages independent of HPV and may be a suitable therapeutic target in these cancers.

Prognostic relevance of AIB1 (NCoA3) amplification and overexpression in breast cancer.

UNLABELLED: AIB1 (amplified in breast cancer 1) is an estrogen receptorα (ERα) co-activator, known to be amplified and overexpressed in a fraction of breast cancers. It has been linked to prognosis and tamoxifen resistance. However, results have been ambiguous. The different functions of AIB1 in ERα-positive and -negative disease are poorly understood. Therefore, we analyzed the clinical significance of AIB1 in breast cancer with respect to ERα-status and characterized the subgroups. 2,197 breast carcinomas sampled on a pre-existing tissue microarray (TMA) were analyzed for AIB1 expression and amplification by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). RESULTS: AIB1 expression was detected in 60 % of the tumors. It was associated with tumor size (p = 0.003), high histological grade (p < 0.0001), poor disease-specific, and overall survival (p = 0.0018 and p = 0.003). There was a strong inverse relationship between AIB1 and ERα expression (p < 0.0001). AIB1 overexpression was associated with increased Ki67 labeling index (p < 0.0001), even if analyzed for different ER expression levels. AIB1 amplification was found in 11 % of the carcinomas. It was associated with high histological grade (p = 0.0012), lymph node involvement (p = 0.0163), and poor disease-specific survival (p = 0.0032) but not with overall survival (p = 0.1672) or ER status (p = 0.4456). If ER-positive tumors were stratified according to their AIB1 amplification status, there was a significant worse disease-specific survival in cases showing AIB1 amplification (p = 0.0017). AIB1 expression is associated with unfavorable prognosis and tumor phenotype. It seems to unfold its oncogenic potential at least in part independent from its role as an ERα co-activator. AIB1 has an impact on cell cycle regulation in ERα-positive as well as ERα-negative tumors. Furthermore, AIB1 amplification characterizes a subgroup of ERα-positive breast cancer with worse outcome. Therefore, AIB1 might be helpful to identify those ERα-positive breast cancers patients who are candidates for adjuvant chemotherapy.

BIRC2 amplification in squamous cell carcinomas of the uterine cervix.

Oncogene amplification is a key step in cell transformation towards malignancy. Chromosomal aberrations involving the long arm of chromosome 11, including amplifications at 11q13 and 11q22, have been previously reported in cervical cancer. While the role of the CCND1 gene as the driver gene for 11q13 amplification is well established in different tumor types, the significance of the 11q22 amplicon is less clear. The 11q22 amplicon corresponds to several putative target genes including the apoptose inhibitor BIRC2, recently detected as amplified in cervical cancer cell lines. To better understand the distribution and frequency of 11q amplification sites in uterine cervical carcinomas, we analyzed BIRC2 and CCND1 copy number changes using fluorescence in situ hybridization in a tissue microarray containing 238 cervical cancers. High-level amplification of BIRC2 was found in 12.9 % of tumors. Amplification of BIRC2 in cervical carcinomas was homogeneous as shown in corresponding whole tissue sections of amplified tumors at the tissue microarray. BIRC2 amplification was significantly more frequent than CCND1 amplification (2.1 %) in our cohort (p < 0.01), and amplification of both genes were independent from each other. BIRC2 amplification was associated with younger-patient age (p < 0.05) and squamous cell differentiation (p = 0.025) of cervix carcinomas. However, BIRC2 copy number changes were not related to tumor stage, grading and nodal status of cervical cancers. In conclusion, BIRC2 is amplified in a subset of squamous cell carcinoma of the uterine cervix. Further studies are necessary to evaluate possible prognostic effects of BIRC2 copy number gains in cervical carcinomas.

EGFR gene copy number increase in vulvar carcinomas is linked with poor clinical outcome.

EGFR copy number increases have been frequently reported in cancer including vulvar carcinomas. Co-amplification of cancer genes plays an important role in the development of many tumour types. To better understand the effect of EGFR aberrations on vulvar cancer phenotype and patient prognosis, the authors analysed EGFR copy number changes using fluorescence in situ hybridisation and EGFR expression by immunohistochemistry in a tissue microarray containing 183 squamous cell carcinomas of vulva. Furthermore, the authors analysed the co-amplification frequency of EGFR with HER2, CCND1, MYC and PIK3CA, respectively. EGFR copy number increase was found in 39.3% of the tumours. Seventeen per cent of vulvar carcinomas showed EGFR high polysomy including 9% with amplification of the EGFR gene. Copy number gain of the EGFR locus was associated with non-basaloid phenotype (p=0.03), high-tumour stage (p<0.001), human papillomaviruse negativity of tumours (p=0.04) and the number of lymph node metastases (p=0.02). EGFR protein expression was statistically correlated to EGFR copy number increase (p<0.05). The observed co-amplification rate of EGFR with all four additionally examined oncogenes was much higher than statistically expected. There was a highly significant association between EGFR copy number increase and CCND1 amplifications (p<0.001) as well as the total number of gene amplifications (p=0.04). EGFR copy number gains were significantly related to unfavourable patient outcome in univariate analysis and multivariate Cox regression analysis. In conclusion, EGFR copy number increases are detectable in a substantial proportion of vulvar carcinomas with relationships to advanced tumour stages and the development of lymph node metastases. EGFR copy number aberrations are connected to other gene amplifications and probably define an human papillomaviruses-independent pathway in the development of vulvar carcinomas. These data support the potential utility of EGFR inhibitors as a therapeutic alternative in a subset of vulvar carcinomas.

Gene amplification in ductal carcinoma in situ of the breast.

Multiple different biologically and clinically relevant genes are often amplified in invasive breast cancer, including HER2, ESR1, CCND1, and MYC. So far, little is known about their role in tumor progression. To investigate their significance for tumor invasion, we compared pure ductal carcinoma in situ (DCIS) and DCIS associated with invasive cancer with regard to the amplification of these genes. Fluorescence in situ hybridization (FISH) was performed on a tissue microarray containing samples from 130 pure DCIS and 159 DCIS associated with invasive breast cancer. Of the latter patients, we analyzed the intraductal and invasive components separately. In addition, lymph node metastases of 23 patients with invasive carcinoma were included. Amplification rates of pure DCIS and DCIS associated with invasive cancer did not differ significantly (pure DCIS vs. DCIS associated with invasive cancer: HER2 22.7 vs. 24.2%, ESR1 19.0 vs. 24.1%, CCND1 10.0 vs. 14.8%, MYC 11.8 vs. 6.5%; P > 0.05). Furthermore, we observed a high concordance of the amplification status for all genes if in situ and invasive carcinoma of individual patients were compared. This applied also to the corresponding lymph node metastases. Our results indicate no significant differences between the gene amplification status of DCIS and invasive breast cancer concerning HER2, ESR1, CCND1, and MYC. Therefore, our data suggest an early role of all analyzed gene amplifications in breast cancer development but not in the initiation of invasive tumor growth.

Oestrogen receptor gene (ESR1) amplification is frequent in endometrial carcinoma and its precursor lesions.

Oestrogen receptor alpha (ER) plays a critical, diverse and not fully understood role in endometrial carcinoma. Most endometrial carcinomas express ER and some of these tumours respond favourably to anti-oestrogen therapy. On the other hand, tamoxifen therapy constitutes a major risk factor for endometrial carcinoma development. Amplification of the ESR1 gene encoding ER was recently shown to constitute a mechanism for ER over-expression in breast carcinoma. This study was designed to determine the potential role of ESR1 amplifications in endometrial carcinoma. Tissue microarrays of 368 endometrial carcinomas and large sections of 43 cases of endometrial hyperplasia were analysed for ESR1 gene amplification and ER protein expression by means of fluorescence in situ hybridization (FISH) and immunohistochemistry. FISH revealed ESR1 amplification in 40/176 (23%) cancers, 6/19 (32%) atypical complex hyperplasias, 3/10 (30%) complex hyperplasias without atypia and 2/14 (14%) simple hyperplasias without atypia. Strong ER protein expression was significantly linked to ESR1 amplification in endometrial carcinoma (p = 0.0036). These data indicate that ESR1 amplification might be one mechanism for ER over-expression in endometrial carcinoma, and suggest an early role for ESR1 amplification in the development of a significant fraction of endometrial carcinoma. Given the predictive role of ESR1 amplification for tamoxifen response in breast carcinoma, it will be interesting to investigate the response of ESR1-amplified endometrial cancers to anti-oestrogenic drugs.

Disseminated periportal fatty degeneration after allogeneic intraportal islet transplantation in a patient with type 1 diabetes mellitus: a case report.

Insulin independence after islet transplantation has been significantly improved by using new steroid-free immunosuppressive protocols and increased islet mass. Only little is known about the influence on the morphology of the liver of intraportally transplanted islets. We describe a case of disseminated periportal fatty degeneration after allogeneic intraportal islet transplantation (ITx). A 35-year-old patient with type-1 diabetes mellitus who was suffering from repeated severe hypoglycemic episodes received two sequential intraportal islet grafts. Liver structure was normal before the first ITx, based upon ultrasound and magnetic resonance imaging (MRI). One week after the first ITx, ultrasound demonstrated normal liver morphology. Four months later, at the second ITx, we detected small, disseminated, and hypodense hepatic lesions (1 to 3 mm) by ultrasound, which were confirmed by MRI and interpreted to be fatty degenerations. Histologically we found focal drop-shaped fatty degenerations with signs of mild periportal chronic inflammation. These liver alterations without clinical symptoms or pathological liver function tests matched the predicted distribution of infused islets. Glucose metabolism markedly improved after the first ITx, namely 58.6% reduction of daily insulin requirements, 1.4% decrease in HbA1c, basal C-peptide of 0.8 to 1.3 ng/dl with no severe hypoglycemia. We interpreted these benign changes in liver morphology as reactions to a local hyperinsulinemia in the neighborhood of the transplanted islets. We hypothesized that a steroid-free immunosuppression with rapamycin and tacrolimus may have contributed to changes in the portal microenvironment.

[Diagnostic importance of flow cytometry in staging malignant lymphoma]. / Diagnostische Wertigkeit der Durchflusszytometrie beim Staging maligner Lymphome.

Bone marrow biopsy plays an important role in the clinical diagnosis of malignant lymphoma. Further diagnostic methods need to be established to increase accuracy in the light of advances over recent years in the immunophenotypical characterization of discrete lymphatic bone marrow lesions and the continuing difficulty in classifying them. This study compared the diagnostic value of flow cytometry to that of conventional bone marrow biopsy in 156 patients with 191 marrow biopsy specimens or bone marrow aspirates. The most important findings were that up to one-third of lymphomas could not be diagnosed by flow cytometry, and that the degree of infiltration was estimated as less than two-thirds. However, flow cytometric results were more satisfactory in acute lymphoblastic leukemia, lymphoblastic lymphoma, hairy cell leukemia, and chronic lymphocytic leukemia. In summary, flow cytometry has a complementary role in the staging of lymphoma but cannot fully replace conventional trephine biopsy.

[Cystic adventitial degeneration of the popliteal artery. Rare cause of intermittent claudication in middle-aged adults]. / Die zystische Adventitiadegeneration der Arteria poplitea. Seltene Ursache für eine Claudicatio intermittens im mittleren Erwachsenenalter.

We report on a 36-year old male patient with clinically prolonged intermittent claudication of the right leg. An operative specimen measuring 8.0 cm of the A. poplitea was sent for examination, which was sheathed by an aneurysmal cyst having a maximal diameter of 1.5 cm. Examination under the light microscope showed an incomplete septated ganglion-like cystic formation with flat connective tissue-like cells or even larger histiocytoid cell elements as inner lining. Immunohistochemically, the cells on the inner lining of the cyst showed to be strongly positive to macrophage marker PG-M1, KP1 and Ki-Mp1. The endothelium of the vessels of the wall of the cyst showed a marked expression of cytokeratin 18. Immunohistochemically as well as in conventional histology, there are general parallels between cysts in cystic adventitial degeneration, ganglia and normal synovia as the two last mentioned tissues also show an expression of cytokeratin 18 at the endothelia of smaller vessels as well as an expression of macrophage-associated antigens on cells of the inner lining. Aetiopathologically, the cysts developing in the course of the disease are rather adventitial ganglia.