CD27/CD70 pathway activation in primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder.

Primary cutaneous CD4+ small or medium T-cell lymphoproliferative disorder (PCSM-LPD) is a clonal T-cell proliferation disease confined to the skin. PCSM-LPD shares expression of T follicular helper (Tfh) cell markers with various mature T-cell lymphomas. However, the benign presentation of PCSM-LPD contrasts the clinical behavior of other Tfh-lymphomas. The aim of our study was to delineate the molecular similarities and differences between PCSM-LPD and other Tfh-derived lymphomas to explain the clinical behavior and unravel possible pathological mechanisms. We performed targeted next-generation sequencing of 19 genes recurrently mutated in T-cell neoplasms in n = 17 PCSM-LPD with high and in n = 21 PCSM-LPD with low tumor cell content. Furthermore, gene expression profiling was used to identify genes potentially expressed in the PD1-positive (PD1+) neoplastic cells. Expression of some of these genes was confirmed in situ using multistain immunofluorescence. We found that PCSM-LPD rarely harbored mutations recurrently detected in other T-cell neoplasms. PCSM-LPD is characterized by the invariable expression of the T-cell-receptor-associated LCK protein. CD70 and its ligand CD27 are co-expressed on PD1+ PCSM-LPD cells, suggestive of autoactivation of the CD70 pathway. In conclusion, PCSM-LPD differs from disseminated lymphomas of Tfh origin by their mutation profile. Activation of CD70 signaling also found in cutaneous T-cell lymphoma represents a potential driver of neoplastic proliferation of this benign neoplasia of Tfh. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

MDM2 amplification is rare in gastric cancer.

The MDM2 proto-oncogene (MDM2) is a primary negative regulator of p53. The latter is frequently mutated in gastric cancer (GC). In the present study, we aimed to validate gene amplification, protein expression, and the putative tumor biological function of MDM2 in a well-characterized Western GC cohort. MDM2 amplification and protein expression were studied in a cohort of 327 GCs by fluorescence in situ hybridization (FISH) and immunohistochemistry. Gene amplification and protein expression were correlated with diverse clinicopathological patient characteristics including patient outcome. Immunohistochemically, 97 GCs (29.7%) were categorized as MDM2 positive and 230 GCs (70.3%) as negative. An amplification of MDM2 was found in 11 (3.4%) cases without evidence of intratumoral heterogeneity. Nine of these eleven (81.8%) cases showed MDM2 protein expression. MDM2 amplification correlated significantly with MDM2 protein expression (p < 0.001). On a case-by-case analysis, MDM2-amplified cases showed varied histological phenotypes and were most commonly microsatellite stable; EBV, HER2, and MET negative; and FGFR2 positive. A single case harbored both, MDM2 amplification and TP53 mutation. MDM2 amplification and MDM2 expression, respectively, did not correlate with overall or tumor-specific survival. Our targeted analysis of MDM2 in a well-characterized cohort of GC patients showed that MDM2 amplification is rare, of no specific histological phenotype, and may not be always mutually exclusive with TP53 mutations. Given the low number of cases, currently, no diagnostic or therapeutic recommendation related to MDM2 amplification can be given for GC of Western origin.

Sequential Treatment with Temozolomide Plus Naturally Derived AT101 as an Alternative Therapeutic Strategy: Insights into Chemoresistance Mechanisms of Surviving Glioblastoma Cells.

Glioblastoma (GBM) is a poorly treatable disease due to the fast development of tumor recurrences and high resistance to chemo- and radiotherapy. To overcome the highly adaptive behavior of GBMs, especially multimodal therapeutic approaches also including natural adjuvants have been investigated. However, despite increased efficiency, some GBM cells are still able to survive these advanced treatment regimens. Given this, the present study evaluates representative chemoresistance mechanisms of surviving human GBM primary cells in a complex in vitro co-culture model upon sequential application of temozolomide (TMZ) combined with AT101, the R(-) enantiomer of the naturally occurring cottonseed-derived gossypol. Treatment with TMZ+AT101/AT101, although highly efficient, yielded a predominance of phosphatidylserine-positive GBM cells over time. Analysis of the intracellular effects revealed phosphorylation of AKT, mTOR, and GSK3ß, resulting in the induction of various pro-tumorigenic genes in surviving GBM cells. A Torin2-mediated mTOR inhibition combined with TMZ+AT101/AT101 partly counteracted the observed TMZ+AT101/AT101-associated effects. Interestingly, treatment with TMZ+AT101/AT101 concomitantly changed the amount and composition of extracellular vesicles released from surviving GBM cells. Taken together, our analyses revealed that even when chemotherapeutic agents with different effector mechanisms are combined, a variety of chemoresistance mechanisms of surviving GBM cells must be taken into account.

FGFR2 overexpression and compromised survival in diffuse-type gastric cancer in a large central European cohort.

The significance of fibroblast growth factor receptor 2 (FGFR2) in gastric cancer (GC) has been studied predominantly in Asian patient cohorts. Data on White patients are scarce. Here, we aimed to independently validate the expression and putative tumor biological significance of FGFR2 in a large non-Asian GC cohort. Immunohistochemistry (IHC) was performed on large-area tissue sections from 493 patients with GC and evaluated using the HScore. GCs with moderate and strong FGFR2 expression were studied for Fgfr2 amplification using chromogenic in situ hybridization (CISH). Median overall survival was determined using the Kaplan-Meier method. The majority [240 (99.1%)] of FGFR2-positive GCs showed a variable combination of staining intensities with marked intratumoral heterogeneity, including weak [198 (40.2%) cases], moderate [145 (29.4%)], and strong [108 (21.9%)] staining in diverse combinations. 250 (50.9%) GCs expressed no FGFR2. Fgfr2 gene amplification was found in 40% of selected cases with high protein expression and was also heterogeneous at the cell level. FGFR2 protein expression did not correlate with patient survival in the entire cohort However, using different cutoff values, a negative correlation between FGFR2-expression and patient outcome was found for diffuse-type GC. FGFR2 expression was associated with a lower tumor grade and intestinal phenotype (p≤0.0001). FGFR2-positive diffuse-type GCs classify a small subset of patients with a poor tumor specific survival (5.29±1.3 vs. 14.67±1.9 months; p = 0.004).

Multiscale heterogeneity in gastric adenocarcinoma evolution is an obstacle to precision medicine.

BACKGROUND: Cancer is a somatic evolutionary disease and adenocarcinomas of the stomach and gastroesophageal junction (GC) may serve as a two-dimensional model of cancer expansion, in which tumor subclones are not evenly mixed during tumor progression but rather spatially separated and diversified. We hypothesize that precision medicine efforts are compromised when clinical decisions are based on a single-sample analysis, which ignores the mechanisms of cancer evolution and resulting intratumoral heterogeneity. Using multiregional whole-exome sequencing, we investigated the effect of somatic evolution on intratumoral heterogeneity aiming to shed light on the evolutionary biology of GC. METHODS: The study comprised a prospective discovery cohort of 9 and a validation cohort of 463 GCs. Multiregional whole-exome sequencing was performed using samples form 45 primary tumors and 3 lymph node metastases (range 3-10 tumor samples/patient) of the discovery cohort. RESULTS: In total, the discovery cohort harbored 16,537 non-synonymous mutations. Intratumoral heterogeneity of somatic mutations and copy number variants were present in all tumors of the discovery cohort. Of the non-synonymous mutations, 53-91% were not present in each patient's sample; 399 genes harbored 2-4 different non-synonymous mutations in the same patient; 175 genes showed copy number variations, the majority being heterogeneous, including CD274 (PD-L1). Multi-sample tree-based analyses provided evidence for branched evolution being most complex in a microsatellite instable GC. The analysis of the mode of evolution showed a high degree of heterogeneity in deviation from neutrality within each tumor. We found evidence of parallel evolution and evolutionary trajectories: different mutations of SMAD4 aligned with different subclones and were found only in TP53 mutant GCs. CONCLUSIONS: Neutral and non-neutral somatic evolution shape the mutational landscape in GC along its lateral expansions. It leads to complex spatial intratumoral heterogeneity, where lymph node metastases may stem from different areas of the primary tumor, synchronously. Our findings may have profound effects on future patient management. They illustrate the risk of mis-interpreting tumor genetics based on single-sample analysis and open new avenues for an evolutionary classification of GC, i.e., the discovery of distinct evolutionary trajectories which can be utilized for precision medicine.

p53 immunostaining cannot be used to predict TP53 mutations in gastric cancer: results from a large Central European cohort.

Four molecular subgroups of gastric cancer (GC) have been proposed, ie, Epstein-Barr virus (EBV)-positive, microsatellite instable, chromosomal instable (CIN), and genomically stable GC. Based on the complex relationship between chromosomal instability and TP53 mutational status, we hypothesized that the typical clinicopathological characteristics caused by chromosomal instability are correlated with the p53 expression that is detected by immunohistochemistry. Four hundred sixty-seven whole-tissue sections of patients with therapy-naive GC were stained with anti-p53 antibody. The histoscore and staining pattern were analyzed for each slide. Different algorithms of immunohistochemistry evaluation were formed and correlated with clinicopathological characteristics. The algorithms were validated by assessing the mutational status of TP53 in 111 cases. Four hundred forty-two GCs were p53 positive, and 25 were negative, including 414 GCs with a homogeneous pattern and 53 GCs with a heterogeneous staining pattern. There was no correlation with overall or tumor-specific survival. In comparison with clinicopathological characteristics, the algorithm high versus low showed correlations with microsatellite instability, hepatocyte growth factor receptor (MET), and TP53 mutational status. The algorithm Q1/Q4 versus Q2/Q3 appeared to be correlated with the phenotype as per the Laurén classification, microsatellite instability, EBV status, and p53 expression pattern. The algorithm <90% = 0 and <50% = 3+ versus ≥90% = 0 or ≥50% = 3+ showed correlations with the EBV status, microsatellite instability, grading, and p53 expression pattern. The algorithm homogeneous versus heterogeneous did not correlate with any clinicopathological characteristic. Our results showed that the immunohistochemistry of p53, TP53 mutational status, and CIN subtype were connected. However, different algorithms for p53 immunohistochemical evaluation cannot be used to predict TP53 mutations in CIN GCs in individual cases.

miRNA-expression in tonsillar squamous cell carcinomas in relation to HPV infection and expression of the antileukoproteinase SLPI.

The aim of this study was to determine if micro-(mi-)RNAs are involved in the previously reported inverse correlation between the antileukoproteinase SLPI, HPV, and smoking habit of head and neck squamous cells carcinoma (HNSCC) patients. HPV-status and SLPI-protein expression were determined in tonsillar SCC (TSCC; n=126). Differentially expressed miRNAs dependent on HPV-status and SLPI-expression were detected by microarray; possible binding-sites in SLPI- and HPVE6-mRNAs were determined in silico. Survival rates were estimated testing prognostic values of HPV-status, SLPI- and miRNA-expression. miRNA-array identified 24 up-regulated and 10 down-regulated miRNAs in HPV-positive versus HPV-negative TSCC (p<0.01; HPV-positivity: 42.1%). HPV-positivity resulted in two up-regulated miRNAs in SLPI-positive TSCC. Of 16 further miRNAs, eight miRNAs were up- and eight were down-regulated in SLPI-negative TSCC. RT-q-PCR-validation of the four most differentially expressed miRNAs showed that miR-363 is expressed strongest in SLPI-negative/HPV-positive TSSC. In silico-analysis of all differentially expressed miRNAs identified miR-363, miR-210, miR-130a, and miR-181a with possible binding sites in the HPV16-E6-mRNA, but none were predicted in the SLPI-mRNA. HPV-positivity, low SLPI-levels and high miR-363-levels are significantly associated with better survival rates. The data presented here show that miR-363 is associated with HPV-positive/SPLI-negative TSCC. The prognostic value of miR-363 suggests a role in the assumed inverse correlation of smoking and SPLI-expression in the mode of HPV-infections in tonsillar but possibly also other HNSCC.

Interpreting whole genome and exome sequencing data of individual gastric cancer samples.

BACKGROUND: Gastric cancer is the fourth most common cancer and the second leading cause of cancer death worldwide. In order to understand the genetic background, we sequenced the whole exome and the whole genome of one microsatellite stable as well as one microsatellite unstable tumor and the matched healthy tissue on two different NGS platforms. We here aimed to provide a comparative approach for individual clinical tumor sequencing and annotation using different sequencing technologies and mutation calling algorithms. RESULTS: We applied a population-based whole genome resource as a novel pathway-based filter for interpretation of genomic alterations from single nucleotide variations (SNV), indels, and large structural variations. In addition to a comparison with tumor genome database resources and a filtering approach using data from the 1000 Genomes Project, we performed pyrosequencing analysis and immunohistochemistry in a large cohort of 428 independent gastric cancer cases. CONCLUSION: We here provide an example comparing the usefulness and potential pitfalls of different technologies for a clinical interpretation of genomic sequence data of individual gastric cancer samples. Using different filtering approaches, we identified a multitude of novel potentially damaging mutations and could show a validated association between a mutation in GNAS and gastric cancer.

Clinicopathologic Characteristics of Microsatellite Instable Gastric Carcinomas Revisited: Urgent Need for Standardization.

Microsatellite instable gastric cancer (MSI-GC) is a specific molecular subtype of GC. We studied the phenotypes, genotypes, and clinicopathologic characteristics of MSI-GC in a white GC cohort and compared our findings with an extended literature review. The study cohort consisted of 482 patients. Specimens were available from 452 cases and were used for immunostaining (MLH1, PMS2, MSH2, MSH6) and molecular biological analyses (BAT-25, BAT-26, NR-21, NR-24, NR-27; Epstein-Barr virus in situ hybridization). Thirty-four (7.5%) GCs were MSI. Loss of MLH1 and/or PMS2 was found in 30 (88%) MSI-GC, 3 (9%) showed loss of MSH2 and/or MSH6. One (3%) MSI-GC was identified only by molecular biological testing. A single case was heterogeneous and contained microsatellite-stable and instable tumor areas. Twenty-one (62%) MSI-GCs showed unusual histologic features. MSI-GC was not found in diffuse-type or Epstein-Barr virus-positive GC. MSI-GC was significantly more prevalent in elderly patients, distal stomach, and was associated with a significantly lower number of lymph node metastases and a significantly better overall and tumor-specific survival. MSI-GC constitutes a small but relevant subgroup of GC with distinct clinicopathologic characteristics. Our literature review illustrates the shortcomings of missing standardized testing algorithms with prevalences of MSI-GC ranging from 0% to 44.5%. Future studies should test the hypothesis that patients with MSI-GCs may not need adjuvant/perioperative chemotherapy. However, this will require a standardized, quality-controlled diagnostic algorithm of MSI for GC.

Lysyl oxidase like-4 monoclonal antibody demonstrates therapeutic effect against head and neck squamous cell carcinoma cells and xenografts.

A new member of the lysyl oxidase (LOX) family, lysyl oxidase-like 4 (LOXL4), is overexpressed in head and neck squamous cell carcinoma (HNSCC) compared to normal squamous epithelium. A monoclonal antibody (mAb) derived from fusion of Balb/c mouse splenocytes immunized with LOXL4 specific peptide was used to evaluate its therapeutic efficacy in 15 HNSCC cell lines associated with LOXL4 overexpression. For xenograft experiments 41 severe combined immunodeficient (SCID) mice were used to analyze LOXL4-mAb mediated tumor regression. Cell viability was analyzed using cytotoxicity-, and clonogenic-assays. Significant suppression of tumor cell growth was observed in 12 out of 15 (80%) tumor cell lines after 48 hr exposure to the mAb (LD50 of 15 µg/ml to 45 µg/ml). The effect induced by the antibody could be blocked by pre-incubation of the antibody with the peptide used for immunization of the mice and antibody generation, indicating that the effect of the antibody is specific. In mice inoculated with HNSCC cells, i.v. injections of the LOXL4-mAb resulted within 70 days in extensive tumor destruction in all treated animals whereas no tumor regression occurred in control animals. In mice pre-immunized i.v. with LOXL4-mAb and subsequently injected with HNSCC cells, tumor development was considerably delayed in contrast to non LOXL4-mAb pre-immunized animals. These results demonstrate that the LOXL4-mAb has potent antitumor activity and suggest its suitability as a therapeutic immune agent applicable to HNSCC exhibiting tumor specific upregulation of LOXL4.

Varying Mutational Alterations in Multiple Primary Melanomas.

In melanoma, the mitogen-activated protein (MAP) kinase pathway plays a crucial oncogenic role. Recent studies identified additional genetic alterations, eg, TERT-promoter mutations. Up to 8% of melanoma patients present with multiple primary melanomas (MPMs). The pathogenesis is not fully understood, and data on the genetic diversity of MPMs are limited. To identify putative diagnostic and therapeutic consequences, we assessed the mutational status of the BRAF and NRAS genes and TERT promoter in patients with MPMs. The study cohort consisted of 96 patients with 237 malignant melanomas. The BRAF, NRAS, and TERT-promoter genotypes were assessed in all MPMs and were correlated with patients' clinicopathological characteristics. BRAF mutations were found in 84 melanomas (35.4%), NRAS mutations, in 33 (14.0%); and TERT-promoter mutations, in 112 (47.3%). Mutation patterns were concordant between first and subsequent primary tumors in 23.9% of patients and were discordant in 61.4% of patients. The genetic alterations were partially different in 14.7% of patients. By Cox regression analysis, only the NRAS mutation had a significant negative prognostic impact on time to progression to stage III (P = 0.016) and on distant metastasis-free survival (P = 0.032). In the majority of primary melanomas in patients with MPMs, BRAF, NRAS, and TERT-promoter genotypes were discordant. Thus, molecular testing for targeted therapy should be performed on metastatic tissue and not on primary tumors.

Complex APC germline mutation associated metaplasia and intraepithelial neoplasia (CAM-IEN) of the gallbladder.

Preneoplasic and neoplastic changes of the gallbladder of patients with a familial adenomatous polyposis (FAP) are rare, and very little is known about their incidence in patients with an attenuated FAP. We herein report on a unique case of a woman with an attenuated FAP who shows eight distinct, partially preneoplastic differentiation patterns within the gallbladder mucosa, which are: (1) regular gallbladder epithelium, (2) low grade biliary intraepithelial neoplasia, (3) papillary adenoma, (4) Paneth cell metaplasia, (5) goblet cell metaplasia, (6) pancreatic metaplasia, (7) pseudopyloric metaplasia, and (8) neuroendocrine differentiation. Moreover, this is the first case of a KRAS mutation in a gallbladder adenoma of a patient with an APC germline mutation, which is highly suggestive of an early event of malignant transformation. As a consequence of our findings, clinicians should draw special attention to the gallbladder of FAP patients, and a simultaneous protective cholecystectomy of FAP patients, which undergo colectomy and show conspicuous changes of the gallbladder mucosa, should be performed in these patients in order to eliminate the risk of a synchronous or metachronous gallbladder neoplasia.

MET in gastric cancer--discarding a 10% cutoff rule.

AIMS: We aimed to develop a putative predictive biomarker score for future hepatocyte growth factor receptor (MET)-targeted therapy of gastric cancer (GC). METHODS AND RESULTS: MET expression and MET amplification were analysed by immunohistochemistry (IHC) and chromogenic in-situ hybridization (CISH) in 470 GC patients. Immunostaining was documented with the HistoScore. The percentage area of MET-amplified tumour cell clones was assessed by virtual microscopy. The expression of MET was heterogeneous in primary and metastatic GC. Immunostaining intensity (MET-IHC 2+/3+) correlated with MET amplification and a positive MET status was defined by a combination of MET-IHC 2+ or 3+ with MET amplification, or MET-IHC 3+ without MET amplification. The prognostic significance of the MET status was independent from the percentage area of positive tumour cells (e.g. <10 versus ≥10%). MET-positive GCs were microsatellite stable and of KRAS/PIK3CA wild-type. MET-positive GCs had a very poor prognosis, with a median survival of 5.4 months and a hazard ratio of 2.126. CONCLUSIONS: A combination of immunohistochemistry and CISH is suitable to assess MET status. If MET status is used as a predictive biomarker, prospective studies should pay specific attention to adequate tissue sampling, should ignore cutoff values for tumour areas, may consider the KRAS and PIK3CA genotype as negative predictive markers and should carry out the analysis expeditiously.

Geographical and anatomical influences on human papillomavirus prevalence diversity in head and neck squamous cell carcinoma in Germany.

The increased knowledge regarding HPV-infections in head and neck squamous cell carcinoma (HNSCC) has unexpectedly contributed to several uncertainties related to i) prevalence diversities depending on tumour site and geographical origin of the patients, ii) proportion of HPV-driven tumours among HPV-DNA-positive cases, and iii) identification of patients with HPV-attributed survival benefit. To investigate this heterogeneity, we analysed 307 HNSCC cases (tonsillar, n=135; non-tonsillar, n=172) from eight health care centers mostly from Northern Germany and determined HPV-DNA/mRNA and p16INK4A-status and combined results with the patient outcome. Overall HPV-DNA prevalence rate was 23.5% (72/307); attributed to: 43.7% (59/135) and 7.6% (13/172) tonsillar and non-tonsillar cases, respectively. Among these, 96.6% tonsillar and 38.5% non-tonsillar SCC were HPV-mRNA-positive. Although the study cohort was composed of patients from regions of rather close proximity, prevalence rates showed diversities of up to 40% in HNSCC subsite analysis with the lowest prevalence for tonsillar SCC in metropolitan areas (22.2%) vs. 50.9% in rural areas. Survival analysis identified p16INK4A alone as strongest predictor, followed by HPV-DNA-status alone or in combination with p16INK4A. This survival benefit was shown for tonsillar and non-tonsillar cases. Smoking significantly correlated with HPV-status, however, it does not influence survival when stratified for HPV. In conclusion, the data emphasize the urge for further data on HPV-infection in HNSCC to, e.g. clarify to what extent survival benefits of p16INK4A-positive patients are truly attributed to HPV-infections.

Melanomas of unknown primary frequently harbor TERT-promoter mutations.

Commonly, in patients with melanoma metastases of an unknown primary tumor (MUP), an extensive search for the primary tumor is carried out. Recently, highly recurrent telomerase reverse transcriptase (TERT)-promoter mutations were found in malignant melanomas, which may function as driver mutations of skin cancer. The aim of this study was to test the hypothesis that MUP and mucosal melanomas harbor different prevalences of TERT-promoter mutations. Thirty-nine patients with MUP and 53 patients with mucosal melanomas were retrieved. In total, 152 paraffin samples of 92 patients were analyzed, and in 38 patients, multiple samples were tested. Mutational analysis of the TERT-promoter, BRAF, NRAS, and KIT genes was carried out. In total, 92 patients were eligible for mutational analysis. TERT-promoter mutations were found in 33 patients (35.9%): chr5, 1,295,228 C>T (18 patients); chr5, 1,295,250 C>T (11 patients); chr5, 1,295,228-229 CC>TT (three patients); chr5, 1,295,242-243 CC>TT (one patient). The mutations were significantly more prevalent in MUP [26 (66.7%)] than in mucosal melanomas [seven patients (13.2%); P<0.001]. In MUP, BRAF mutations were found in 46.2% of patients (18 patients) and NRAS mutations in 28.2% of patients (11 patients). In mucosal melanoma, NRAS mutations were found in 18.9% of patients (10), and BRAF and KIT mutations in 7.5% of patients (four patients), respectively. The prevalence of TERT-promoter mutations was associated with the patient's sex [23 (51.1%) men, 10 (21.3%) women; P=0.004]. No significant correlation was found between TERT-mutation and patient survival. The TERT-promoter genotype of MUP points toward a cutaneous and not mucosal origin. The significant sex differences merit further attention in having putative therapeutic implications.

The role of the antileukoprotease SLPI in smoking-induced human papillomavirus-independent head and neck squamous cell carcinomas.

Recently, we showed that increased SLPI levels prevent human papillomavirus (HPV) infections and metastasis in smoking-induced, non-HPV-driven head and neck squamous cell carcinoma (HNSCC). Here, we focus on the role of SLPI in non-HPV-driven HNSCC, investigating tumor tissue and non-neoplastic mucosa from the same patients and from non-HNSCC patients. Gene and protein expression of SLPI and gene expression of annexin 2 (a SLPI receptor), nicotine receptor (α7AChR) and arylhydrocarbon receptor (AhR) were analyzed in HNSCC patients (20 smokers; 16 nonsmokers). SLPI-results were correlated with the patients' HPV status. Non-neoplastic mucosa of HNSCC patients and normal mucosa from non-HNSCC individuals (18 smokers; 20 nonsmokers) was analyzed for the same parameters. Tissue of the inferior turbinate (n = 10) was incubated with nicotine for analysis of the same genes. SLPI gene expression in tumor tissue was 109.26 ± 23.08 times higher in smokers versus nonsmokers. Non-neoplastic mucosa of smokers showed also higher SLPI gene expression (10.49 ± 1.89-fold non-HNSCC; 18.02 ± 3.93-fold HNSCC patients). Annexin 2 gene expression was also increased in smokers. SLPI data were corroborated by immunohistochemistry. A nicotine dependent correlation between SLPI and annexin 2 gene expression (r(2) = 0.15, p < 0.001) was shown ex vivo. Nicotine and smoking increased α7AChR and AhR gene expression. Five patients, showing no/low SLPI expression, were HPV16-positive. A significant correlation between smoking and SLPI expression in tumors and to our knowledge for the first time in mucosa of HNSCC and non-HNSCC patients was established. Together with the finding that all patients with HPV infection showed no/low SLPI expression, these data support our intriguing hypothesis that smoking induced upregulated SLPI prevents HPV infections.

Prognostic and putative predictive biomarkers of gastric cancer for personalized medicine.

We investigated various phenotypic and genotypic biomarkers of gastric cancer (GC) testing the following hypotheses: are these biomarkers suitable for the identification of GC subtypes, are they of prognostic significance, and should any of these biomarkers be considered to tailor patient treatment in the future. The study cohort consisted of 482 patients. pTNM-stage was based on surgical pathologic examination. The Laurén and mucin phenotype was assessed. Helicobacter pylori and Epstein-Barr virus infections were documented. The following biomarkers were determined: BRAF, KRAS, NRAS, and PIK3CA genotype, microsatellite instability, mucin 1, mucin 2, mucin 5, and mucin 6, CD10, E-cadherin, ß-catenin, and lysozyme. The histologic phenotype correlated with 10/13 (77%) clinicopathologic patient characteristics and 6/13 (46%) immunohistochemical/molecular biological biomarkers. Inversely, immunohistochemical biomarkers (mucin phenotype, E-cadherin, ß-catenin, and lysozyme) were unsuitable for subclassification of GC. It showed too much overlap between the different subtypes. Among the genotypes, only microsatellite instability correlated with tumor type being more prevalent in intestinal and unclassified GCs. Patient survival correlated significantly with 8 (62%) clinicopathologic and 5 (36%) immunohistochemical/molecular biomarkers. Interestingly, in proximal GCs, KRAS mutation was associated with worse prognosis, as was persistent H. pylori infection in unclassified GCs. Mucin 2 (all patients, proximal GCs) and PIK3CA (exon 20; intestinal type GC) prognosticated independently patient survival. The biomarkers examined herein are unsuitable to aid histologic classification of GC. However, several of them show a correlation with either phenotype and/or prognosis and may be considered to tailor patient treatment in the future, such as KRAS, PIK3CA, MSI, and H. pylori status.

Human papillomavirus infection in head and neck cancer: the role of the secretory leukocyte protease inhibitor.

We previously showed that secretory leukocyte protease inhibitor (SLPI) gene and protein expression is significantly lower in metastatic versus non-metastatic head and neck squamous cell carcinoma (HNSCC). However, we did not assess the human papillomavirus (HPV) status of these cases. Since SLPI plays a role in HIV and herpes simplex virus (HSV) infections, we hypothesized that SLPI may be involved in HPV-infected HNSCC. In HNSCC tissue (n=54), HPV DNA was determined and correlated with SLPI expression. Additionally, to investigate a possible role of smoking on SLPI expression in clinically normal mucosa, 19 patients treated for non­malignant diseases (non-HNSCC) were analyzed for SLPI expression and correlated with smoking habits. In HNSCC patients, SLPI expression showed a significant inverse correlation with HPV status. In patients with moderate/strong SLPI expression (n=19), 10.5% were HPV-positive. By contrast, patients with absent/weak SLPI expression (n=35), 45.7% were HPV-positive. Low SLPI expression was correlated with metastasis (P=0.003) independent of HPV status. HPV-positivity was clearly associated with lymph node status (81.3% N1-3 cases). In smoking non-HNSCC patients (n=7), 42.9% showed absent/weak and 57.1% moderate/strong SLPI staining. In non-smoking non-HNSCC patients (n=10) 83.3% showed absent/weak and 16.7% moderate/strong SLPI expression. For the first time, a correlation between SLPI downregulation and HPV infection was demonstrated, suggesting that high levels of SLPI, possibly induced by environmental factors such as tobacco smoking, correlate with protective effects against HPV infection. SLPI may be a potential biomarker identifying head and neck cancer patients not at risk of developing metastases (SLPI-positive), and those at risk to be infected by HPV (SLPI-negative) and likely to develop metastases.

ATTR amyloid in the carpal tunnel ligament is frequently of wildtype transthyretin origin.

The carpal tunnel ligament often encloses transthyretin-derived (ATTR) amyloid deposits. In this study we tested the hypothesis that ATTR amyloid in the carpal tunnel ligament is most commonly of wildtype origin in a Caucasian population without endemic background of familial amyloid polyneuropathy. All resection specimens from the carpal tunnel ligament were retrieved from the Amyloid Registry of the University of Kiel spanning the period of 2004-2011 and dichotomized into two study groups: the first study group of 25 patients was obtained from diverse referring pathologists. The second group comprised a patient cohort of 73 patients obtained from a single-referring department of pathology. The selection of biopsies was based on the histological identification of amyloid by Congo red staining and polarization microscopy between crossed polars and immunohistochemical classification as ATTR amyloid. A novel anti-TTR-peptide antibody was raised in rabbits using a recombinant peptide (FHEHAEVVFTANDSGPRRYT) spanning residues 87-106 of the TTR protein. Amplification of the TTR exons 1, 2, 3 and 4 was done by a nested polymerase chain reaction approach. Ninety-eight biopsies were available from 98 patients, including 51 women and 47 men. All amyloid deposits showed strong immunoreactions with the novel anti-TTR peptide antibody. In 81 of 98 patients, genomic DNA was available. In 10 (12%) patients non-amyloidogenic TTR gene mutations were found with the following amino acid substitutions: p.G6S (normal allelic variant). A single patient carried a p.G6S and a p.M13I-variant. The remaining patients all showed wildtype sequence of the TTR gene (70 patients). No significant difference was found between the two study groups. ATTR amyloid in the carpal tunnel ligament is commonly of wildtype origin and genetic counseling is not mandatory in these patients.

HPV status in patients with head and neck of carcinoma of unknown primary site: HPV, tobacco smoking, and outcome.

OBJECTIVES: Infection with human papillomavirus (HPV) is linked to oropharyngeal cancer. This analysis investigated possible associations between HPV status, smoking history and survival outcome in patients with neck metastasis and carcinoma of unknown primary (CUP). MATERIALS AND METHODS: Registries at the Universities of Hamburg and Kiel were searched for patients with CUP diagnosed from 2002 to 2011 who had formalin-fixed and paraffin-embedded metastatic lymph node samples available. All patients underwent routine diagnostic procedures to establish the primary site and received radiotherapy (60Gy using conventional fractionation) with or without concurrent cisplatin-based chemotherapy depending on disease extent. Genotyping was performed using polymerase chain reaction; p16([INK4a]) expression was assessed using immunohistochemistry. RESULTS: Sixty-three patients were included; 23 (37%) had HPV DNA/p16+ samples and 40 (63%) were negative for either/both markers. A high proportion of patients had a history of tobacco smoking; significantly fewer patients with HPV+/p16+ samples were smokers than those who were negative for either/both markers (61% vs. 90%, respectively; p = 0.0067). There were no statistically significant differences between overall or recurrence-free survival in HPV+/p16+ patients vs. those negative for either/both markers. Overall survival appeared to be superior in patients with <10 pack-years smoking history and HPV+/p16+ disease. CONCLUSIONS: This study, the largest to date investigating HPV status in head and neck CUP, identified HPV and p16 overexpression in over one-third of patients. Tobacco smoking history appeared to affect survival in HPV+/p16+ patients. Smoking status should be considered as a prognostic factor in patients with CUP, along with HPV DNA status.