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Identifying patients with locally advanced head and neck carcinoma on high risk of recurrence after definitive concurrent radiochemotherapy is of key importance for the selection for consolidation therapy and for individualized treatment intensification. In this multicenter study we analyzed recurrence-associated single-nucleotide polymorphisms (SNPs) in DNA repair genes in tumor DNA from 132 patients with locally advanced head and neck carcinoma (LadHnSCC). Patients were treated with definitive radiotherapy and simultaneous cisplatin-based chemotherapy at six partner sites of the German Cancer Consortium (DKTK) Radiation Oncology Group from 2005 to 2011. For validation, a group of 20 patients was available. Score selection method using proportional hazard analysis and leave-one-out cross-validation were performed to identify markers associated with outcome. The SNPs rs1799793 and rs13181 were associated with survival and the same SNPs and in addition rs17655 with freedom from loco-regional relapse (ffLRR) in the trainings datasets from all patients. The homozygote major rs1799793 genotype at the ERCC2gene was associated with better (Hazard ratio (HR): 0.418 (0.234–0.744), p= 0.003) and the homozygote minor rs13181 genotype at ERCC2with worse survival (HR: 2.074, 95% CI (1.177–3.658), p= 0.017) in comparison to the other genotypes. At the ffLRR endpoint, rs1799793 and rs13181 had comparable prognostic value. The rs1799793 and rs13181 genotypes passed the leave-one-out cross-validation procedure and associated with survival and ffLRR in patients with LadHnSCC treated with definitive radiochemotherapy. While findings were confirmed in a small validation dataset, further validation is underway within a prospective biomarker study of the DKTK.

Immunohistochemical analysis of formalin-fixed paraffin-embedded (FFPE) tissues provides important diagnostic and prognostic information in pathology. Metal nanoparticles (NPs) and, in particular, surface-enhanced Raman scattering (SERS) nanotags as a new class of labeling reagents are promising to be used for multiplexed protein profiling on tissue sections. However, nonspecific binding of NPs onto the tissue specimens greatly hampers their clinical applications. In this study, we found that the antigen retrieval method strongly influences the extent of nonspecific binding of the antibody–SERS NP conjugates to the tissue. Our SERS labels comprised ca. 70 nm Au nanostars coated with ethylene glycol-modified Raman reporter molecules for hydrophilic stabilization and subsequent covalent bioconjugation to antibodies. We systematically investigated the influence of heat- and protease-induced epitope retrieval (HIER and PIER, respectively) on the immunostaining quality of prostate-specific antigen (PSA) on human prostate tissue sections. The best staining results were obtained with PIER. Pretreatment of the tissue sections by HIER led to selective but nonspecific adsorption of the antibody–Au nanostar conjugates onto epithelial cells, while enzymatic treatment within PIER did not. In addition to gold nanostars, also other types of metal NPs with different shapes and sizes (including ca. 20 nm quasi-spherical Au NPs and ca. 60 nm quasi-spherical Au/Ag nanoshells) as well as tissue sections from different organs (including prostate and breast) were tested; in each case the same tendency was observed, i.e., PIER yielded better results than HIER. Therefore, we recommend PIER for future NP-based tissue immunostaining such as immuno-SERS microscopy. Alternatively, for antigens that can only be unmasked by heating, PEGylation of the NPs is recommended to avoid nonspecific binding.

Human epidermal growth factor receptor 2 (HER2) is a central predictive biomarker in breast cancer. Inaccurate HER2 results in different laboratories could be as high as 20%. However, this statement is based on data generated more than 13 years ago and may not reflect the standards of modern diagnostic pathology. We compared central and local HER2 testing in a total of 1581 HER2-positive tumors from five clinical trials. We evaluated the clinical relevance for pathological complete response (pCR) and disease-free survival in a subgroup of 677 tumors, which received an anti-HER2 therapy. Over the period of 12 years, the discordance rate for HER2 decreased from 52.4 (GeparTrio) to 8.4% (GeparSepto). Discordance rates were significantly higher in hormone receptor (HR)-positive tumors (26.6%), compared to HR-negative tumors (16.3%, P<0.0001), which could be explained by a different distribution of HER2 mRNA levels in HR-positive and HR-negative tumors. pCR rates were significantly lower in discordant tumors (13.7%) compared to concordant tumors (32.2%, GeparQuattro and GeparQuinto, P<0.001). In survival analysis, tumors with discordant HER2 testing had a reduced overall survival (OS) in the HR-negative group (P=0.019) and a trend for improved OS in the HR-positive group (P=0.125). The performance of local HER2 testing was considerably improved over time and has reached a 92% concordance, which shows that quality initiatives in diagnostic pathology are working. Tumors with discordant HER2 testing had a reduced therapy response and different survival rates.

Human epidermal growth factor receptor 2 (HER2) is a central predictive biomarker in breast cancer. Inaccurate HER2 results in different laboratories could be as high as 20%. However, this statement is based on data generated more than 13 years ago and may not reflect the standards of modern diagnostic pathology. We compared central and local HER2 testing in a total of 1581 HER2-positive tumors from five clinical trials. We evaluated the clinical relevance for pathological complete response (pCR) and disease-free survival in a subgroup of 677 tumors, which received an anti-HER2 therapy. Over the period of 12 years, the discordance rate for HER2 decreased from 52.4 (GeparTrio) to 8.4% (GeparSepto). Discordance rates were significantly higher in hormone receptor (HR)-positive tumors (26.6%), compared to HR-negative tumors (16.3%, P<0.0001), which could be explained by a different distribution of HER2 mRNA levels in HR-positive and HR-negative tumors. pCR rates were significantly lower in discordant tumors (13.7%) compared to concordant tumors (32.2%, GeparQuattro and GeparQuinto, P<0.001). In survival analysis, tumors with discordant HER2 testing had a reduced overall survival (OS) in the HR-negative group (P=0.019) and a trend for improved OS in the HR-positive group (P=0.125). The performance of local HER2 testing was considerably improved over time and has reached a 92% concordance, which shows that quality initiatives in diagnostic pathology are working. Tumors with discordant HER2 testing had a reduced therapy response and different survival rates.

Tumor-draining lymph nodes (LNs) play a crucial role during cancer spread and in initiation of anti-cancer adaptive immunity. Neutrophils form a substantial population of cells in LNs with poorly understood functions. Here, we demonstrate that, during head and neck cancer (HNC) progression, tumor-associated neutrophils transmigrate to LNs and shape anti-tumor responses in a stage-dependent manner. In metastasis-free stages (N0), neutrophils develop an antigen-presenting phenotype (HLA-DR+CD80+CD86+ICAM1+PD-L1−) and stimulate T cells (CD27+Ki67highPD-1−). LN metastases release GM-CSF and via STAT3 trigger development of PD-L1+immunosuppressive neutrophils, which repress T cell responses. The accumulation of neutrophils in T cell-rich zones of LNs in N0 constitutes a positive predictor for 5-year survival, while increased numbers of neutrophils in LNs of N1—3 stages predict poor prognosis in HNC. These results suggest a dual role of neutrophils as essential regulators of anti-cancer immunity in LNs and argue for approaches fostering immunostimulatory activity of these cells during cancer therapy.

Salivary gland-like tumors of the breast show a great variety of architectural patterns and cellular differentiations such as glandular, myoepithelial, squamous, and even mesenchymal phenotypes. However, currently little is known about the evolution and cellular differentiation of these tumors. For that reason, we performed an in situtriple immunofluorescence lineage/differentiation tracing (isTILT) and qRT-PCR study of basal (K5/K14), glandular (K7/K8/18), and epidermal-specific squamous (K10) keratins, p63, and smooth muscle actin (SMA; myoepithelial marker) with the aim to construct and trace different cell lineages and define their cellular hierarchy in tumors with myoepithelial differentiation. isTILT analysis of a series of 28 breast, salivary, and lacrimal gland tumors, including pleomorphic adenomas (n=8), epithelial-myoepithelial tumors (n=9), and adenoid cystic carcinomas (n=11) revealed that all tumor types contained K5/K14-positive progenitor cells in varying frequencies from a few percent up to 15%. These K5/K14-positive tumor cells were found to differentiate to glandular- (K8/18-positive) and myoepithelial-lineage (SMA-positive)-specific cells and were also shown to generate various heterologeous cell differentiations such as squamous and mesenchymal progenies. p63 was co-expressed with K5/K14 in basal-like progenitor cells, myoepithelial, and squamous cells but not in glandular cells. Our results show that the corresponding counterpart tumors of breast and salivary/lacrimal glands have identical cellular compositions. Taken together, our isTILT and RNA-expression data indicate that look-alike tumors of the breast represent a special subgroup of basal-type tumors with benign or usually low malignant potential.

Salivary gland-like tumors of the breast show a great variety of architectural patterns and cellular differentiations such as glandular, myoepithelial, squamous, and even mesenchymal phenotypes. However, currently little is known about the evolution and cellular differentiation of these tumors. For that reason, we performed an in situ triple immunofluorescence lineage/differentiation tracing (isTILT) and qRT-PCR study of basal (K5/K14), glandular (K7/K8/18), and epidermal-specific squamous (K10) keratins, p63, and smooth muscle actin (SMA; myoepithelial marker) with the aim to construct and trace different cell lineages and define their cellular hierarchy in tumors with myoepithelial differentiation. isTILT analysis of a series of 28 breast, salivary, and lacrimal gland tumors, including pleomorphic adenomas (n=8), epithelial-myoepithelial tumors (n=9), and adenoid cystic carcinomas (n=11) revealed that all tumor types contained K5/K14-positive progenitor cells in varying frequencies from a few percent up to 15%. These K5/K14-positive tumor cells were found to differentiate to glandular- (K8/18-positive) and myoepithelial-lineage (SMA-positive)-specific cells and were also shown to generate various heterologeous cell differentiations such as squamous and mesenchymal progenies. p63 was co-expressed with K5/K14 in basal-like progenitor cells, myoepithelial, and squamous cells but not in glandular cells. Our results show that the corresponding counterpart tumors of breast and salivary/lacrimal glands have identical cellular compositions. Taken together, our isTILT and RNA-expression data indicate that look-alike tumors of the breast represent a special subgroup of basal-type tumors with benign or usually low malignant potential.

Objectives/Hypothesis:In previous studies, we have demonstrated that the T‐allele of a specific single nucleotide polymorphism (SNP) in the Gαs gene (T393C) correlates with increased Gαs expression and hence apoptosis. The T‐allele was associated with a favorable outcome in a variety of human cancers, for example, carcinoma of the urinary bladder, kidney, colorectal, oro‐ and hypopharynx.

Osteosarcomas are the most frequent bone sarcomas. The molecular chromosomal aberrations in osteosarcomas were analyzed by comparative genomic hybridization (CGH). We studied 47 frozen tumors (41 primary samples, 6 relapses) in osteosarcoma patients registered in the Cooperative Osteosarcoma Study (COSS) protocol. Genomic imbalances were detected in 40 of 41 primary tumors and 6 of 6 relapsed tumors. Gains were more frequent than losses (ratio of 1.3:1). The median number of changes was 16 and 12 in primary and relapsed osteosarcomas, respectively. The median number of aberrations in primary high-grade osteosarcomas (17.0) was significantly higher than in low- or intermediate-grade osteosarcoma subtypes (3.0) (p = 0.038). The most frequent gains included 8q, 1p21-p31 and 1q21-q24, and the most frequent losses were 10q, 5q and 13q. High-level gains were observed on 8q23-q24, 17p13 and 1q21-q24. A gain of 19p (p &lt; 0.001) or loss of 9p (p = 0.027) was more frequent in poor responders than in good responders. Univariate analysis revealed that patients with primary metastases (p = 0.002), poor histologic responses (p = 0.005), high-level gains of 19p (p = 0.012) or losses of 13q14 (p = 0.042) had significantly lower event-free survival (EFS), whereas patients with a loss of 5q (p = 0.007) or a loss of 10q21-22 (p = 0.017) had significantly higher EFS than patients without these aberrations. Multivariate analysis demonstrated that primary metastasis, loss of 13q14 and loss of 5q were independent prognostic factors. The findings of our study seem to be useful for evaluating the prognosis of patients and may finally lead to treatment strategies based on genetic background of osteosarcoma. © 2002 Wiley-Liss, Inc.

Endomucin is a typical sialomucin that we recently identified on the surface of mouse endothelial cells and on putative hematopoetic clusters of the dorsal aorta in the embryo. We have generated a panel of monoclonal antibodies (mAbs) against the extracellular part of human endomucin and polyclonal antibodies against the cytoplasmic part. Using immunohistochemistry endomucin was specifically detected on endothelial cells of blood and lymphatic vessels of all analyzed human tissues. In addition, the polyclonal antibodies stained the epithelium of the epidermis as well as epithelial and myoepithelial cells of the eccrine and apocrine glands in the skin. This nonendothelial staining could only be seen with a subset of mAbs if the staining procedure was amplified. Although high endothelial venules (HEVs) were not significantly stained with mAbs against endomucin, the polyclonal antibodies clearly detected endomucin on HEVs in lymphatic organs of the mouse and human, suggesting HEV-specific glycosylation affecting recognition by the mAbs. Indeed, endomucin isolated from human and mouse lymphoid organs carried the MECA-79 epitope that defines a set of L-selectin ligands on HEVs called peripheral node addressins. We conclude that human and mouse endomucin are endothelial sialomucins with the potential to function as L-selectin ligands.

Summary A family of mono- and polyclonal antibodies raised against proteoglycans or their “subcomponents” served as novel markers to characterize the phenotypes of three non-Hodgkin lymphoma xenograft lines (HT 58 lymphoblastic, HT 117 centroblastic, HT 130 centrocytic) together with normal, human peripheral blood B lymphocytes. These xenografted NHL lines, maintained by serial transplantations on artificially immunosuppressed mice, expressed very similar B-cell-related antigens and differences on the cell surface (HT 58 > HT 117 > HT 130 > B cells) when they were exposed to monoclonal antibodies (mAb) to cartilage proteoglycans. Anti-proteoglycan antibodies used in this study recognize complex epitopes of core protein segment associated with carbohydrate, shared by human cartilage proteoglycans and certain lymphoma cells. The binding of these antibodies was independent of cell-cycle phase. The results suggest that the anti-proteoglycan mAbs could be used as new phenotypic markers to individualize non-Hodgkin lymphomas.

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