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Cancer cell plasticity due to the dynamic architecture of interactome networks provides a vexing outlet for therapy evasion. Here, through chemical biology approaches for systems level exploration of protein connectivity changes applied to pancreatic cancer cell lines, patient biospecimens, and cell- and patient-derived xenografts in mice, we demonstrate interactomes can be re-engineered for vulnerability. By manipulating epichaperomes pharmacologically, we control and anticipate how thousands of proteins interact in real-time within tumours. Further, we can essentially force tumours into interactome hyperconnectivity and maximal protein-protein interaction capacity, a state whereby no rebound pathways can be deployed and where alternative signalling is supressed. This approach therefore primes interactomes to enhance vulnerability and improve treatment efficacy, enabling therapeutics with traditionally poor performance to become highly efficacious. These findings provide proof-of-principle for a paradigm to overcome drug resistance through pharmacologic manipulation of proteome-wide protein-protein interaction networks., Joshi, Gomes et al. employ a chemical modulation approach of the cellular interactome to a hyperconnectivity state and show association with the increased response of pancreatic cancer cell lines to specific drugs, including those that target the MAPK-pathways and PI3K-mTOR pathway. To achieve this, the authors employ chemical modulation of the interactome via epichaperome inhibition with the small molecule PU-H71.

Very-early-onset inflammatory bowel disease (VEO-IBD) is a heterogeneous phenotype associated with a spectrum of rare Mendelian disorders. Here, we perform whole-exome-sequencing and genome-wide genotyping in 145 patients (median age-at-diagnosis of 3.5 years), in whom no Mendelian disorders were clinically suspected. In five patients we detect a primary immunodeficiency or enteropathy, with clinical consequences (XIAP, CYBA, SH2D1A, PCSK1). We also present a case study of a VEO-IBD patient with a mosaic de novo, pathogenic allele in CYBB. The mutation is present in ~70% of phagocytes and sufficient to result in defective bacterial handling but not life-threatening infections. Finally, we show that VEO-IBD patients have, on average, higher IBD polygenic risk scores than population controls (99 patients and 18,780 controls; P, Adult forms of inflammatory bowel disease (IBD) are of a polygenic nature, but paediatric and very early onset (VEO) IBD also occur as monogenic forms. Here, using whole exome sequencing, the authors explore both the monogenic and polygenic contribution to VEO-IBD and characterize a rare somatic mosaic VEO-IBD patient.

The chaperome is a large family of proteins composed of chaperones, co-chaperones and a multitude of other factors. Elegant studies in yeast and other organisms have paved the road to how we currently understand the complex organization of this large family into protein networks. The goal of this chapter is to provide an overview of chaperome networks in cancer cells, with a focus on two cellular states defined by chaperome network organization. One state characterized by chaperome networks working in isolation and with little overlap, contains global chaperome networks resembling those of normal, non-transformed, cells. We propose that in this state, redundancy in chaperome networks results in a tumor type unamenable for single-agent chaperome therapy. The second state comprises chaperome networks interconnected in response to cellular stress, such as MYC hyperactivation. This is a state where no redundant pathways can be deployed, and is a state of vulnerability, amenable for chaperome therapy. We conclude by proposing a change in how we discover and implement chaperome inhibitor strategies, and suggest an approach to chaperome therapy where the properties of chaperome networks, rather than genetics or client proteins, are used in chaperome inhibitor implementation.

We explored the association between liver metastases, tumor CD8+ T-cell count, and response in patients with melanoma or lung cancer treated with the anti-PD-1 antibody, pembrolizumab. The melanoma discovery cohort was drawn from the phase I Keynote 001 trial, whereas the melanoma validation cohort was drawn from Keynote 002, 006, and EAP trials and the non–small cell lung cancer (NSCLC) cohort from Keynote 001. Liver metastasis was associated with reduced response and shortened progression-free survival [PFS; objective response rate (ORR), 30.6%; median PFS, 5.1 months] compared with patients without liver metastasis (ORR, 56.3%; median PFS, 20.1 months) P ≤ 0.0001, and confirmed in the validation cohort (P = 0.0006). The presence of liver metastasis significantly increased the likelihood of progression (OR, 1.852; P < 0.0001). In a subset of biopsied patients (n = 62), liver metastasis was associated with reduced CD8+ T-cell density at the invasive tumor margin (liver metastasis+ group, n = 547 ± 164.8; liver metastasis− group, n = 1,441 ± 250.7; P < 0.016). A reduced response rate and shortened PFS was also observed in NSCLC patients with liver metastasis [median PFS, 1.8 months; 95% confidence interval (CI), 1.4–2.0], compared with those without liver metastasis (n = 119, median PFS, 4.0 months; 95% CI, 2.1–5.1), P = 0.0094. Thus, liver metastatic patients with melanoma or NSCLC that had been treated with pembrolizumab were associated with reduced responses and PFS, and liver metastases were associated with reduced marginal CD8+ T-cell infiltration, providing a potential mechanism for this outcome. Cancer Immunol Res; 5(5); 417–24. ©2017 AACR.

Transient, multi-protein complexes are important facilitators of cellular functions. This includes the chaperome, an abundant protein family comprising chaperones, co-chaperones, adaptors, and folding enzymes—dynamic complexes of which regulate cellular homeostasis together with the protein degradation machinery1–6. Numerous studies have addressed the role of chaperome members in isolation, yet little is known about their relationships regarding how they interact and function together in malignancy7–17. As function is probably highly dependent on endogenous conditions found in native tumours, chaperomes have resisted investigation, mainly due to the limitations of methods needed to disrupt or engineer the cellular environment to facilitate analysis. Such limitations have led to a bottleneck in our understanding of chaperome-related disease biology and in the development of chaperome-targeted cancer treatment. Here we examined the chaperome complexes in a large set of tumour specimens. The methods used maintained the endogenous native state of tumours and we exploited this to investigate the molecular characteristics and composition of the chaperome in cancer, the molecular factors that drive chaperome networks to crosstalk in tumours, the distinguishing factors of the chaperome in tumours sensitive to pharmacologic inhibition, and the characteristics of tumours that may benefit from chaperome therapy. We find that under conditions of stress, such as malignant transformation fuelled by MYC, the chaperome becomes biochemically ‘rewired’ to form a network of stable, survival-facilitating, high-molecular-weight complexes. The chaperones heat shock protein 90 (HSP90) and heat shock cognate protein 70 (HSC70) are nucleating sites for these physically and functionally integrated complexes. The results indicate that these tightly integrated chaperome units, here termed the epichaperome, can function as a network to enhance cellular survival, irrespective of tissue of origin or genetic background. The epichaperome, present in over half of all cancers tested, has implications for diagnostics and also provides potential vulnerability as a target for drug intervention.

© 2016 Massachusetts Medical Society. BACKGROUND Approximately 75% of objective responses to anti-programmed death 1 (PD-1) therapy in patients with melanoma are durable, lasting for years, but delayed relapses have been noted long after initial objective tumor regression despite continuous therapy. Mechanisms of immune escape in this context are unknown. METHODS We analyzed biopsy samples from paired baseline and relapsing lesions in four patients with metastatic melanoma who had had an initial objective tumor regression in response to anti-PD-1 therapy (pembrolizumab) followed by disease progression months to years later. RESULTS Whole-exome sequencing detected clonal selection and outgrowth of the acquired resistant tumors and, in two of the four patients, revealed resistance-associated lossof- function mutations in the genes encoding interferon-receptor-associated Janus kinase 1 (JAK1) or Janus kinase 2 (JAK2), concurrent with deletion of the wild-type allele. A truncating mutation in the gene encoding the antigen-presenting protein beta-2-microglobulin (B2M) was identified in a third patient. JAK1 and JAK2 truncating mutations resulted in a lack of response to interferon gamma, including insensitivity to its antiproliferative effects on cancer cells. The B2M truncating mutation led to loss of surface expression of major histocompatibility complex class I. CONCLUSIONS In this study, acquired resistance to PD-1 blockade immunotherapy in patients with melanoma was associated with defects in the pathways involved in interferon-receptor signaling and in antigen presentation. (Funded by the National Institutes of Health and others.)

Purpose: Several biomarkers have been individually associated with response to PD-1 blockade, including PD-L1 and tumor mutational burden (TMB) in non–small cell lung cancer (NSCLC), and CD8 cells in melanoma. We sought to examine the relationship between these distinct variables with response to PD-1 blockade and long-term benefit. Experimental Design: We assessed the association between baseline tumor characteristics (TMB, PD-L1, CD4, and CD8) and clinical features and outcome in 38 patients with advanced NSCLC treated with pembrolizumab (median follow-up of 4.5 years, range 3.8–5.5 years). Results: PD-L1 expression and CD8 infiltration correlated with each other and each significantly associated with objective response rate (ORR) and progression-free survival (PFS). TMB was independent of PD-L1 and CD8 expression, and trended towards association with ORR and PFS. There was no association between CD4 infiltration and outcomes. Only PD-L1 expression was correlated with overall survival (OS). Among 5 patients with long-term survival >3 years with no additional systemic therapy, PD-L1 expression was the only discriminating feature. The increased predictive value for PFS and OS of composite biomarker inclusive of PD-L1, CD8, CD4, and TMB was limited. Conclusions: In patients with NSCLC treated with PD-1 blockade with long-term follow up, TMB, PD-L1, and CD8 were each associated with benefit from PD-1 blockade. Pretreatment PD-L1 expression was correlated with T lymphocyte infiltration and OS, whereas models incorporating TMB and infiltrating CD4 and CD8 lymphocytes did not substantially add to the predictive value of PD-L1 alone for OS.

Desmoplastic melanoma is a rare subtype of melanoma characterized by dense fibrous stroma, resistance to chemotherapy and a lack of actionable driver mutations, and is highly associated with ultraviolet light-induced DNA damage. We analysed sixty patients with advanced desmoplastic melanoma who had been treated with antibodies to block programmed cell death 1 (PD-1) or PD-1 ligand (PD-L1). Objective tumour responses were observed in forty-two of the sixty patients (70%; 95% confidence interval 57-81%), including nineteen patients (32%) with a complete response. Whole-exome sequencing revealed a high mutational load and frequent NF1 mutations (fourteen out of seventeen cases) in these tumours. Immunohistochemistry analysis from nineteen desmoplastic melanomas and thirteen non-desmoplastic melanomas revealed a higher percentage of PD-L1-positive cells in the tumour parenchyma in desmoplastic melanomas (P = 0.04); these cells were highly associated with increased CD8 density and PD-L1 expression in the tumour invasive margin. Therefore, patients with advanced desmoplastic melanoma derive substantial clinical benefit from PD-1 or PD-L1 immune checkpoint blockade therapy, even though desmoplastic melanoma is defined by its dense desmoplastic fibrous stroma. The benefit is likely to result from the high mutational burden and a frequent pre-existing adaptive immune response limited by PD-L1 expression.

Loss-of-function mutations in JAK1/2 can lead to acquired resistance to anti-programmed death protein 1 (PD-1) therapy. We reasoned that they may also be involved in primary resistance to anti–PD-1 therapy. JAK1/2-inactivating mutations were noted in tumor biopsies of 1 of 23 patients with melanoma and in 1 of 16 patients with mismatch repair–deficient colon cancer treated with PD-1 blockade. Both cases had a high mutational load but did not respond to anti–PD-1 therapy. Two out of 48 human melanoma cell lines had JAK1/2 mutations, which led to a lack of PD-L1 expression upon interferon gamma exposure mediated by an inability to signal through the interferon gamma receptor pathway. JAK1/2 loss-of-function alterations in The Cancer Genome Atlas confer adverse outcomes in patients. We propose that JAK1/2 loss-of-function mutations are a genetic mechanism of lack of reactive PD-L1 expression and response to interferon gamma, leading to primary resistance to PD-1 blockade therapy. Significance: A key functional result from somatic JAK1/2 mutations in a cancer cell is the inability to respond to interferon gamma by expressing PD-L1 and many other interferon-stimulated genes. These mutations result in a genetic mechanism for the absence of reactive PD-L1 expression, and patients harboring such tumors would be unlikely to respond to PD-1 blockade therapy. Cancer Discov; 7(2); 188–201. ©2016 AACR. See related commentary by Marabelle et al., p. 128. This article is highlighted in the In This Issue feature, p. 115

Tumors expressing programmed death ligand 1 (PD-L1) interact with the corresponding negative-signal generating immune receptor on the surface of CD8 T cells, PD-1, thereby suppressing antitumor activity. Therapeutics blocking this interaction have shown promise in various cancers by restoring functional antitumor T-cell activity. We explored the degree of PD-L1, PD-1, and CD8 expression in a retrospective analysis of 29 clinical synovial sarcoma samples. Quantitative immunohistochemistry and multiplex immunofluorescence were used to determine relative quantification of CD8+ and PD-1+ T cells and PD-L1 expression within the intratumor area and the interface between the tumor and the surrounding nontumor tissue (i.e., invasive margin), and colocalization of these factors, respectively. PD-L1, PD-1, and CD8 cell densities in the tumor-invasive margins were significantly higher in the metastatic tumors than the primary tumors (P < 0.01), and PD-L1, PD-1, and CD8 cell densities were all significantly positively correlated with one other (P < 0.0001). PD-1 cell density in the tumor-invasive margin was significantly associated with worse progression-free survival. Multiplex immunofluorescence demonstrated coexpression of PD-1 and CD8 on lymphocytes within the invasive margin, as well as relative proximity between PD-1+ CD8 cells and PD-L1+ tumor cells. Our results provide a preclinical rationale for screening of patients with synovial sarcoma for the colocalization of CD8, PD-1, and PD-L1, which may be a marker for response to PD-1 blockade therapy. Cancer Immunol Res; 5(2); 118–26. ©2016 AACR.

Beside its role in motor coordination, the cerebellum is involved in cognitive function such as attention, working memory, verbal learning, and sensory discrimination. In schizophrenia, a disturbed prefronto-thalamo-cerebellar circuit has been proposed to play a role in the pathophysiology. In addition, a deficit in the glutamatergic N-methyl-D-aspartate (NMDAf) receptor has been hypothesized. The risk gene neuregulin 1 may play a major role in this process. We demonstrated a higher expression of the NMDA receptor subunit 2D in the right cerebellar regions of schizophrenia patients, which may be a secondary upregulation due to a dysfunctional receptor. In contrast, the neuregulin 1 risk variant containing at least one C-allele was associated with decreased expression of NMDA receptor subunit 2C, leading to a dysfunction of the NMDA receptor, which in turn may lead to a dysfunction of the gamma amino butyric acid (GABA) system. Accordingly, from post-mortem studies, there is accumulating evidence that GABAergic signaling is decreased in the cerebellum of schizophrenia patients. As patients in these studies are treated with antipsychotics long term, we evaluated the effect of long-term haloperidol and clozapine treatment in an animal model. We showed that clozapine may be superior to haloperidol in restoring a deficit in NMDA receptor subunit 2C expression in the cerebellum. We discuss the molecular findings in the light of the role of the cerebellum in attention and cognitive deficits in schizophrenia. peerReviewed

HER-2/neu status is critical for the therapy for breast carcinoma. Fluorescent in situ hybridization for gene amplification and immunohistochemical stains for protein expression are widely used methods to detect HER-2/neu status. Multiple studies have shown fluorescent in situ hybridization and immunohistochemical stain results to have high concordance rates. To our knowledge, a comparison between fluorescent in situ hybridization results for core needle biopsy and the subsequent excisional biopsy specimens has not yet been studied. We retrospectively evaluated the fluorescence in situ hybridization and immunohistochemical results in both the breast core needle and the excisional biopsy of 125 patients with invasive breast carcinoma from 2002 to 2005. There was complete concordance with respect to both immunohistochemical and fluorescence in situ hybridization results for core needle biopsy and excisional biopsy specimens in 87% of the patients evaluated. Comparison of fluorescent in situ hybridization results of the 129 core needle biopsies to the 131 excisional biopsies of all 125 patients showed a concordance rate of 92%. The immunohistochemical stain results of the same core needle and excisional biopsies showed a concordance rate of 98%. Comparison of the immunohistochemical stain results with the fluorescent in situ hybridization results for all 260 cases examined showed 95% concordance. On the basis of our study, we observed that repeating HER-2/neu testing by immunohistochemical stain and/or fluorescent in situ hybridization methods on excisional biopsy is not unreasonable, in particular in cases of intratumoral heterogeneity, indeterminate/borderline HER-2/neu results and after neoadjuvant chemotherapy.

OBJECTIVE. The aim of this study was to investigate the effect of catch-up growth occurring at different stages of childhood on glucose levels and β-cell function at 7 years of age.METHODS. Oral glucose tolerance tests were performed on 152 7-year-old children. Anthropometric data were available from birth to 7 years of age. Children were split into catch-up, catch-down, and normal-growth groups on the basis of growth rates between birth and 1 year, birth and 5 years, and birth and 7 years. Fasting and 30- and 120-minute blood samples collected during the oral glucose tolerance tests were assayed for glucose, insulin, proinsulin, and des-31,32-proinsulin levels, and area-under-the-curve values were calculated.RESULTS. Children with catch-up growth between birth and 5 years or birth and 7 years had greater area-under-the-curve insulin levels than the children with catch-down growth. Children with catch-up growth only between birth and 7 years exhibited higher proinsulin levels and a greater insulin secretory response to glucose than those who experienced catch-up growth between both birth and 1 year and birth and 7 years of age. Low birth weight children with no catch-up growth between birth and 7 years had the highest glucose and lowest insulinogenic index levels, whereas children with high birth weight and catch-up growth had the highest insulin levels.CONCLUSIONS. Extremes of birth weight in conjunction with extremes of postnatal growth are all detrimental to childhood metabolism. The negative metabolic effects of catch-up growth between birth and 7 years may be attenuated if catch-up growth also occurs between birth and 1 year of age.

Objective To examine the antigenic distribution of human leukocyte antigens (HLA) of the human larynx. Study design and setting Twelve human larynges were examined for Class I (HLA-A, -B, -C) and Class II (HLA-DR) histocompatibility antigens using mouse monoclonal antibodies in an indirect immunoperoxidase assay. Structures of the larynx and surrounding tissues were examined and given a semiquantitative score based on HLA Class I and II expression. Results The mucosal surface epithelium of the larynx stains 2+ or stronger for HLA Class I antigens and 1+ for Class II antigens. The deeper submucosal glands stain 1+ for Class I antigens and 2+ or stronger for Class II antigens. Thyroid cartilage showed 2+ or stronger staining of the chondrocytes for Class I antigens only. Thyroid follicular cells also stain only for Class I antigens. Perichondrium and Schwann cells of nerves stain stronger for Class I antigens than Class II antigens. Cartilage matrix, muscle cells, and axons of nerves do not stain for either class of antigens. Endothelium stains 3+ for both classes of antigens. Conclusions The detailed distribution of major transplantation antigens in the human larynx is elucidated. Class II antigens implicated as initiators of organ transplant rejection were primarily found in 6 areas: mucosal surface epithelium, submucosal glands, ducts, vascular endothelium, perichondrium, and Schwann cells of nerves. The relevance of these findings to the initiation and detection of laryngeal allograft graft rejection is discussed.

Therapies that target the programmed death-1 (PD-1) receptor have shown unprecedented rates of durable clinical responses in patients with various cancer types. One mechanism by which cancer tissues limit the host immune response is via upregulation of PD-1 ligand (PD-L1) and its ligation to PD-1 on antigen-specific CD8(+) T cells (termed adaptive immune resistance). Here we show that pre-existing CD8(+) T cells distinctly located at the invasive tumour margin are associated with expression of the PD-1/PD-L1 immune inhibitory axis and may predict response to therapy. We analysed samples from 46 patients with metastatic melanoma obtained before and during anti-PD-1 therapy (pembrolizumab) using quantitative immunohistochemistry, quantitative multiplex immunofluorescence, and next-generation sequencing for T-cell antigen receptors (TCRs). In serially sampled tumours, patients responding to treatment showed proliferation of intratumoral CD8(+) T cells that directly correlated with radiographic reduction in tumour size. Pre-treatment samples obtained from responding patients showed higher numbers of CD8-, PD-1- and PD-L1-expressing cells at the invasive tumour margin and inside tumours, with close proximity between PD-1 and PD-L1, and a more clonal TCR repertoire. Using multivariate analysis, we established a predictive model based on CD8 expression at the invasive margin and validated the model in an independent cohort of 15 patients. Our findings indicate that tumour regression after therapeutic PD-1 blockade requires pre-existing CD8(+) T cells that are negatively regulated by PD-1/PD-L1-mediated adaptive immune resistance.

Heat shock protein 90 (HSP90) serves as a critical facilitator for oncogene addiction. There has been augmenting enthusiasm in pursuing HSP90 as an anticancer strategy. In fact, since the initial serendipitous discovery that geldanamycin (GM) inhibits HSP90, the field has rapidly moved from proof-of-concept clinical studies with GM derivatives to novel second-generation inhibitors.The authors highlight the current status of the second-generation HSP90 inhibitors in clinical development. Herein, the authors note the lessons learned from the completed clinical trials of first- and second-generation inhibitors and describe various assays attempting to serve for a more rational implementation of these agents to cancer treatment. Finally, the authors discuss the future perspectives for this promising class of agents.The knowledge gained thus far provides perhaps only a glimpse at the potential of HSP90 for which there is still much work to be done. Lessons from the clinical trials suggest that HSP90 therapy would advance at a faster pace if patient selection and tumor pharmacokinetics of these drugs were better understood and applied to their clinical development. It is also evident that combining HSP90 inhibitors with other potent anticancer therapies holds great promise not only due to synergistic antitumor activity but also due to the potential of prolonging or preventing the development of drug resistance.

Background —Sympathetic nerve activity is known to be important in ventricular arrhythmogenesis, but there is little information on the relation between the distribution of cardiac sympathetic nerves and the occurrence of spontaneous ventricular arrhythmias in humans. Methods and Results —We studied 53 native hearts of transplant recipients, 5 hearts obtained at autopsy of patients who died of noncardiac causes, and 7 ventricular tissues that had been surgically resected from the origin of ventricular tachycardia. The history was reviewed to determine the presence (group 1A) or absence (group 1B) of spontaneous ventricular arrhythmias. Immunocytochemical staining for S100 protein, neurofilament protein, tyrosine hydroxylase, and protein gene product 9.5 was performed to study the distribution and the density of sympathetic nerves. The average left ventricular ejection fraction was 0.22±0.07. A total of 30 patients had documented ventricular arrhythmias, including ventricular tachycardia and sudden cardiac death. A regional increase in sympathetic nerves was observed around the diseased myocardium and blood vessels in all 30 hearts. The density of nerve fibers as determined morphometrically was significantly higher in group 1A patients (total nerve number 19.6±11.2/mm 2 , total nerve length 3.3±3.0 mm/mm 2 ) than in group 1B patients (total nerve number 13.5±6.1/mm 2 , total nerve length 2.0±1.1 mm/mm 2 , P P Conclusions —There is an association between a history of spontaneous ventricular arrhythmia and an increased density of sympathetic nerves in patients with severe heart failure. These findings suggest that abnormally increased postinjury sympathetic nerve density may be in part responsible for the occurrence of ventricular arrhythmia and sudden cardiac death in these patients.

Accurate determination of HER2/neu status in breast carcinoma is essential. Alteration of preanalytic variables is known to affect HER2/neu results. American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) issued guidelines to standardize fixation for increased HER2/neu accuracy. We studied the effects of changing preanalytic variables on HER2/neu immunohistochemical and fluorescence in situ hybridization (FISH) results in a known HER2/neu+ invasive carcinoma. The clinical specimen was processed according to ASCO/CAP guidelines, with remaining tumor stored fresh without any fixatives for 4 days at 4°C and cut into core biopsy-sized pieces. Each was fixed in 10% formalin, 15% formalin, Pen-Fix (Richard-Allan Scientific, Kalamazoo, MI), Bouin solution, Sakura molecular fixative (Sakura Tissue-Tek Xpress, Torrance, CA), or zinc formalin for 0 to 168 hours. Immunohistochemical studies and FISH were performed. Compared with the clinical specimen, the samples showed no tumor degradation or marked difference by immunohistochemical studies, except the 1-hour 10% formalin and Bouin samples, or FISH, except the Bouin-fixed samples. Our study demonstrates that HER2/neu results remain accurate beyond ASCO/CAP-recommended preanalytic variables, with the exception of Bouin solution for FISH analysis.

Analysis of the effect of psychosocial factors and co-morbidities on the health status of patients with chronic nonspecific low back pain and patients with surgical intervention because of disk herniation was performed. One hundred and two nonselected consecutive inpatients with chronic nonspecific low back pain were included in the study. Their average age was 56.7 (SD = 10.9) years. The control group consisted of 199 subjects matched according to age and sex, chosen from the database of the national representative health survey Hungarostudy 2006, which involved 4,527 subjects. We measured quality of life including mental health with the SF-36 questionnaire validated for use in Hungary, the short 9-item version of the Beck Depression Inventory, the WHO-Five Well-Being Index, and the Hospital Anxiety-Depression Scale. We characterized the socio-demographic status with variables on age, sex, marital status, and education. Data on symptoms and signs of low back pain, other musculoskeletal diseases, and their treatments including spinal surgery were recorded. Co-morbidity and body mass index were considered as independent indicators of health. Depression as measured by Beck Depression Inventory and severity of depression did not vary significantly according to marital status, education, hypertension, diabetes, and gastrointestinal disease. Only half of the patients (52 %) were in the normal range of the scale; 22 % suffered from mild, 16 % from moderate, and 12 % from severe depression. Average values for anxiety and depression as measured by Hospital Anxiety-Depression Scale and Beck Depression Inventory were both significantly higher in the patient than in the control group (Hospital Anxiety Scale: p = 0.0001; Beck Depression Inventory: p = 0.0001). According to the WHO Well-Being Index-5 scale, the difference between patients and the control group was significant (p = 0.0001). Furthermore, correlation was found between the incidence of depression and surgery. Depression was demonstrated in 47.4 % of those patients who had no surgery, in 50 % of patients who had one round of surgery, and in 62.5 % of those who had undergone surgery more than once; the contingence coefficient was 0.211. According to different measurements, the psychological state of patients with chronic nonspecific low back pain was significantly altered as compared to the matched Hungarian population. Higher anxiety and depression markers occurred in 48 % of the patients. There was no correlation between the depression of patients with low back pain and variables such as marital status, education, and co-morbidities. Our study is the first to demonstrate that depression runs parallel with the number of surgical procedures. Therefore, if there is a relative indication for surgery, depression and severity of depression should be assessed and considered when deciding on the intervention.

Objective: Human papilloma virus (HPV) and p16INKa (p16) positivity in head and neck squamous cell carcinomas (HNSCCs) is currently thought to be an encouraging prognostic indicator. However, the histopathologic changes responsible for this behavior are poorly understood. It is our objective to elucidate these histopathologic characteristics to help define the clinical utility of these markers. Design: Retrospective cohort study. Methods: 71 HNSCC tumors between July 1, 2008 and August 30, 2009 were examined for HPV, p16, and epidermal growth factor receptor (EGFR). Specified pathologic features were examined: perivascular invasion (PVI), perineural invasion (PNI), grade of squamous differentiation, basaloid classification. Results: HPV and p16 had no direct impact on perineural or perivascular invasion. However, HPV and p16 were strongly predictive of poorly differentiated tumors, as well as basaloid squamous cell carcinoma (SCCA) (P < .001). Additionally, upon multivariate analysis, HPV(+) and p16(+) tumors had an increased risk of nodal metastasis (HPV: odds ratio [OR] = 23.9 (2.2, 265.1) p = .01; p16: OR = 6.5 (1.4, 31.2) p = .02; PVI: OR = 6.0 (1.6, 22.8) p < .01). The area under the curve (AUC) of receiver operating characteristic (ROC) curves demonstrated improved predictive value for lymph node metastasis above standard H&E histopathologic features (76.7%) for both HPV (83.2%) and p16 (81.3%) individually. Conclusions: HPV(+) and p16(+) are highly predictive for poorly differentiated tumors and basaloid SCCA. Additionally, HPV and p16 positivity demonstrate superior predictive value for lymph node metastasis above standard H&E histopathologic features. Although exact recommendations should be tempered by considerations of primary tumor subsite, T-stage, and depth of invasion, head and neck multidisciplinary teams should strongly consider aggressive lymph node treatment for any HPV(+) or p16(+) tumor. Laryngoscope, 2010

Previous studies have shown Snail expression integral to the epithelial-mesenchymal transition during tumor progression. However, its behavior in clinical head and neck squamous cell carcinomas (HNSCCs) is yet undefined. We therefore sought to (1) investigate clinical and histopathologic characteristics of Snail-positive HNSCC and (2) understand the link between Snail and other commonly used HNSCC tumor markers.A retrospective case-control study was conducted.This study was conducted in a large-scale academic center.Of 51 consecutive HNSCC, 42 surgical resections were included.Two separate pathologists performed standard histopathologic reviews along with immunohistochemistries (Snail, E-cadherin, p16, epidermal growth factor receptor [EGFR]) and in situ hybridization (human papilloma virus [HPV]). Medical review for all cases was performed.Twenty-two (52%) of 42 cases stained 4+ Snail (75% staining). The remaining 20 cases were considered negative. Snail was strongly inversely related to E-cadherin expression (ρ = -0.69, P.001), but statistically independent from HPV, p16, or EGFR expression. Snail(+) tumors were equally represented from each anatomic subsite. Snail(+) tumors were strongly associated with poor differentiation (P.001) and basaloid classification (P = .004). Snail(+) tumors were also strongly associated with lymphovascular invasion (P = .02), but not perineural invasion. Ultimately, 11 (50%) of 22 of Snail(+) tumors demonstrated positive nodal metastasis and 11 (79%) of 14 node-positive cases were Snail(+) (P = .02).This pilot study provides promising evidence of Snail' role as a molecular prognostic marker for HNSCC. Snail positivity is significantly predictive of poorly differentiated, lymphovascular invasive, as well as regionally metastatic tumors. Because Snail positivity appears independent of HPV, p16, and EGFR expression, Snail may prove to improve upon these markers' predictive limitations.

The purpose of the present study was to assess the efficacy and tolerability of diuretic-free antihypertensive therapy with a calcium antagonist and/or an angiotensin converting enzyme (ACE) inhibitor in patients with diabetes mellitus. 54 hypertensive [blood pressure (BP) above 140/90mm Hg] patients with diabetes mellitus type 1 (n = 7) or 2 (n = 47) and normal serum creatinine levels (mean 82 +/- 6 mumol/L) received either verapamil or enalapril after a 2-week washout and a 4-week placebo phase. If BP remained elevated, both agents were combined. Verapamil or enalapril alone normalised diastolic BP (to less than 90mm Hg) in 36 patients; verapamil decreased BP from 159/98 to 147/87mm Hg (n = 19, p < 0.001) and enalapril decreased BP from 166/99 to 146/88mm Hg (n = 17, p < 0.001). In 18 patients who remained hypertensive after 10 weeks of monotherapy, a combination of both drugs decreased BP from 169/104 to 151/90mm Hg (p < 0.001). Overall, 87% of patients achieved a target BP response at 30 weeks. Urinary albumin as related to creatinine excretion (UAE; micrograms albumin:mg creatinine) was on average not significantly changed after verapamil or enalapril treatment, alone or combined. Nevertheless, in patients with initial microalbuminuria, UAE decreased (p < 0.05) during enalapril treatment. Serum potassium, total lipids, high density lipoprotein cholesterol, low density lipoprotein cholesterol, glycosylated haemoglobin, serum C peptide and fructosamine levels were not significantly modified by treatment. Subjective tolerability of the drugs was also generally good. Thus, in hypertensive patients with diabetes, a diuretic-free therapy based on the calcium antagonist verapamil or the ACE inhibitor enalapril, alone or combined, can effectively decrease BP without adversely affecting carbohydrate and lipid metabolism.

In the past two years there has been a resurgence of polio with 21 previously polio-free countries importing wild poliovirus. Wild poliovirus importations into polio-free areas will continue to occur until endemic transmission is interrupted globally. Australia's acute flaccid paralysis (AFP) surveillance falls well short of the target of more than 80 per cent of AFP cases having two adequate stool specimens taken at least 24 hours apart within 14 days of onset for poliovirus examination. As most AFP cases are hospitalised, AFP should be immediately notifiable by hospitals to public health units or state or territory public health authorities to ensure appropriate follow up, including stool specimens.

Increased resistance to apoptosis promotes lymphomagenesis with aberrant expression of cell survival proteins such as BCL-2 and c-MYC occurring in distinct lymphoma subtypes. Galectin-3 is an anti-apoptotic protein that protects T cells, macrophages, and breast carcinoma cells from death triggered by a variety of agents. We have found high levels of galectin-3 protein expression in a subset of B-cell neoplasms including diffuse large B-cell lymphoma (DLBCL), primary effusion lymphoma (PEL), and multiple myeloma (MM), in both cell lines and patient samples. However, we failed to detect galectin-3 in Burkitt lymphoma (BL), follicular lymphoma (FL), marginal zone lymphoma (MZL), MALT lymphoma or B-small lymphocytic lymphoma (B-SLL) cell lines or patient samples. To determine whether galectin-3 expression protects B cells from apoptosis, galectin-3-negative BL cells were transfected with a galectin-3 expressing plasmid, which resulted in markedly increased resistance to anti-Fas-induced cell death. In contrast, galectin-3-positive PEL cells transfected with an amino-terminal truncated galectin-3 vector showed increased sensitivity to anti-Fas induced apoptosis. During normal B-cell development, galectin-3 expression was lowest in germinal center and plasma B cells, from which DLBCL, PEL, and MM derive, and highest in long-lived naive and memory B cells. This pattern of expression suggests that aberrantly increased galectin-3 levels in specific B-cell populations may yield a protective advantage during transformation and/or progression of certain B-cell neoplasms.

Preventive home visits with multidimensional geriatric assessment have been shown to delay or prevent the onset of disability and reduce nursing home admissions in older people. The purpose of the present study was to develop and test a multidimensional instrument for in-home preventive assessments in older persons. In developing the instrument, we conducted a systematic literature review of risk factors for functional status decline and of appropriate instruments for measuring these risk factors. Based on an Expert Panel using a modified Delphi process [1] the risk factor domains for functional status decline were chosen, [2] the instruments for evaluating each of the included risk factor domains were selected, and [3] the individual instruments were combined into one comprehensive assessment instrument. A German language version of the original English version of the instrument was developed based on translation, backtranslation, and cultural adaptation. The feasibility of use of the new instrument was evaluated in a field test in 150 people aged 75 years and older in Hamburg, Ulm, Germany, and Bern, Switzerland. The instrument was well accepted by the older persons. The prevalence of risk factors for functional status decline in these populations (e.g., physical inactivity, urinary incontinence, vision impairment) was high. There was also a high prevalence of underuse of preventive care measures (e.g., no pneumococcal vaccination in over 95 percent of persons). These preliminary results support the possible usefulness of this instrument for conducting preventive home visits or for epidemiological purposes (e.g., prevention surveillance). In a next phase, the test-retest reliability of the instrument, and the feasibility and reliability of self-administration as compared to interviewer administration will be described in a separate paper.

BUTHELEZI, ERNEST P., MARIA-TERESA VAN DER MERWE, PETER N. LONNROTH, I. PETER GRAY, AND NIGEL J. CROWTHER. Ethnic differences in the responsiveness of adipocyte lipolytic activity to insulin. Obes Res. 2000;8:171-178. Objective: The goal of this study was to quantify differences in lipid metabolism and insulin sensitivity in black and white subjects to explain ethnic clinicopathological differ- ences in type 2 diabetes. Research Methods and Procedures: The in vitro lipolytic activity of adipocytes isolated from obese black and white women was measured in the presence of insulin and iso- proterenol. Insulin resistance was assessed in vivo using the euglycemic hyperinsulinemic clamp technique. Results: Fasting plasma levels of insulin and nonesterified fatty acid (NEFA) in black and white women were 67 6 5p M vs. 152 6 20 pM (p , 0.01) and 863 6 93 mM vs. 412 6 34 m M( p , 0.01), respectively. Euglycemic hyperinsulinemic clamp studies showed that obese black subjects were more insulin-resistant than their white counterparts (glucose infusion rates: 1.3 6 0.2 vs. 2.2 6 0.3 mg/kg per min; p , 0.05). Isolated adipocytes from white women were more responsive to insulin than those from black women with 0.7 nM insulin causing a 55 6 4% inhibition of isoproterenol-stimulated li- polysis compared with 27 6 10% in black women (p , 0.05). Discussion: The low responsiveness of adipocyte lipolytic activity to insulin in black women in the presence of a relative insulinopenia may account for the high plasma NEFA levels seen in these women, which may, in turn, account for their higher in vivo insulin resistance. High NEFA levels may also contribute to the low insulin secre- tory activity observed in the obese black females. These data suggest that the pathogenesis of insulin resistance and type 2 diabetes within the black obese community is strongly influenced by their adipocyte metabolism.

In 471 breast cancer patients the influence of a positive history of oral contraceptive (OC) use on survival was investigated. 297 (63%) patients used OCs during any period of their life and 92 (20%) used them still at the time of diagnosis. Sixty months after diagnosis OC users had a significantly increased overall survival (p = 0.037). Survival rates amounted to 79.5% and 70.3% for OC users and non-users, respectively. The effect persisted after adjustment for other prognostic factors and was mainly attributed to women who had taken OCs four years or longer (p = 0.025). Comparing the survival after a 56 months median follow-up dependent on duration of OC use (never, 1-48 months,or = 49 months) in subgroups of prognostic factors, the most significant influence on survival was observed among long-term users with tumors more than 2 cm in diameter (p = 0.005), with axillary node-positive tumors (1-3 nodes, p = 0.055/or = 4 nodes, p = 0.019), and with tumors of low estrogen receptor (p = 0.015) or progesterone receptor content (p = 0.04). The difference in survival between OC users and non-users cannot be explained by the distribution of prognostic factors investigated (histological type, histological grade, tumor size, lymph node involvement, hormonal receptor content). OC users had an even higher percentage of poorly differentiated tumors (p = 0.003). These results suggest an effect of OC use on tumor biology during the preclinical phase of the disease.

KP-1 (CD68) is a recently described monoclonal antibody to a cytoplasmic epitope present on tissue histiocytes and macrophages. To determine the specificity and sensitivity of this marker in the evaluation of cases of malignant fibrous histiocytoma (MFH), this reagent and a panel of commercially antibodies were used to stain formalin-fixed paraffin sections from 25 cases of MFH and 25 other tumors, including a variety of soft-tissue sarcomas. Eighteen of 25 cases of MFH stained for KP-1 (72%), whereas all other tumors were negative, including 12 cases of pleomorphic soft-tissue sarcoma other than MFH. The percentage of tumor cells staining for KP-1 varied. In 11 cases KP-1 was only focally present, but staining was of a high intensity and associated with minimal nonspecific or background staining. Pleomorphic histiocytic cells and spindle cells from storiform tumors were strongly decorated with antibodies to KP-1 in most cases, and antigen also was present on tumor giant cells. Although alpha-1-antitrypsin and alpha-1-chymotrypsin stained a higher percentage of cases of MFH (92%), immunoreactivity for these markers also was noted in other tumors. Because of its specificity as a histiocyte marker, KP-1 is a useful component in a panel of antibodies for the characterization of soft-tissue sarcomas and the diagnosis of MFH.

Keratin profiles of exfoliated mesothelial and adenocarcinoma cells were determined using antisera to different molecular weight keratins (45, 46, 55, 63 kdaltons) and the immunoperoxidase technic. Most metastatic adenocarcinomas in effusions stained for low (45, 46 kdaltons) and intermediate (55 kdaltons) molecular weight keratins but were negative for 63 kdalton keratin. In contrast, most reactive and malignant mesothelial cells in effusions stained strongly for 63 kdalton keratin and keratins of lower molecular weight. This is the first report of high molecular weight (greater than 60 kdaltons) keratin in exfoliated cells of nonepidermal origin. Differences in staining for 63 kdalton keratin between mesothelial and adenocarcinoma cells may help to distinguish these cells in effusions.

Lymph nodes from homosexual men with persistent generalized adenopathy were evaluated for distributions of T-cell phenotypic subsets and surface immunoglobulin (SIg)-bearing lymphocytes. Electron microscopy revealed tubulovesicular structures within lymphocytes but no multivesicular rosettes. Eight to 33 per cent of the lymphocytes within germinal centers were supressor T cells, compared with germinal centers from control lymph nodes, in which these cells were rare ( P =0.002). Significantly greater percentages of suppressor/cytotoxic T lymphocytes were also present in the paracortex and follicular mantles of lymph nodes from the homosexual group ( P =0.002 and 0.007, respectively). Percentages of helper T lymphocytes were significantly decreased in germinal centers ( P =0.008) and paracortical regions ( P =0.002). Florid follicular hyperplasia with aberrations in follicular architecture was the most common histologic pattern, but one node with diffuse hyperplasia and subtotal effacement of architecture revealed depletion of SIg-bearing lymphocytes and increased numbers of suppressor T cells. Reversed helperto-suppressor T-cell ratios in lymph nodes from homosexuals with generalized adenopathy may be related to viral infection and contribute to immune deficiency in this group.

Involucrin is a precursor of the cross-linked envelope protein of human stratum corneum, and its appearance in the upper layers of the epidermis is a function of the normal differentiation of the keratinocyte. Cases of basal cell and squamous cell carcinoma were evaluated for the presence of involucrin using immunoperoxidase techniques on paraffin sections. Basal cell carcinomas were negative for involucrin with staining restricted to squamous horn cysts, while squamous cell carcinomas stained strongly, particularly in large keratinized cells. Cases of squamous cell carcinoma in situ (Bowen's disease) revealed increased staining for involucrin with staining of dyskeratotic cells at all levels in the epithelium. Abnormal patterns of staining were also noted in non-neoplastic epidermis adjacent to carcinomas. Immunohistochemical staining for involucrin identifying abnormal or premature keratinization is a sensitive marker for dyskeratosis in squamous epithelia and may have applications in the histopathologic evaluation of skin specimens.

The authors report at adult age 7 patients (6 men, one woman) with the syndrome of "acquired aphasia-epilepsy", 6 of which had been previously studied as children. The results of the language, neuropsychological and socio-educational evaluation detailed many years after the onset of the aphasia are the subject of this report. One man has recovered completely, one has a normal oral language but is severely dyslexic, one has recovered normal comprehension but has severe expressive language problems. Four have absent language comprehension and lack of expressive speech, and only one of them has learned and is using sign language with some efficiency. None has developed functional written language. Attempts to offer a substitutive language to children with prolonged inability to understand and use oral language appears important but is fraught with problems. Although there are no conclusive data about the role of the continuous paroxysmal EEG discharges and the effect of their suppression with drug treatment on the prognosis of the aphasia, the definite fluctuations of the aphasia in some cases, the isolated recent case reports of definitive improvement with drug treatment justify further trials in this potentially severe and chronic condition.

Sixty-four lung tumors were evaluated for the presence of immunoreactive neuron-specific enolase (NSE), bombesin (Bn), and chromogranin (Cg) to assess their value as markers for neuroendocrine cells in the histologic diagnosis of pulmonary neoplasms. Staining was correlated with the presence and density of neurosecretory granules (number of neurosecretory granules per unit cytoplasmic cross-sectional area) as determined by planimetry on electron micrographs. The cytoplasmic density of neurosecretory granules was significantly greater in the carcinoid tumors than in the small cell carcinomas (P less than 0.001). Neuron-specific enolase was localized in all of the neuroendocrine granule-bearing tumors but was also present in 57 per cent of the nonneuroendocrine carcinomas. Bombesin was present in 68 per cent of the neuroendocrine tumors and in less than 1 per cent of the nonneuroendocrine tumors. Staining for Cg appeared to correlate with the density of neuroendocrine granules, with staining in carcinoid tumors but no staining in small cell anaplastic carcinomas. A panel of antibodies may be required for the reliable identification of neuroendocrine lung tumors by immunohistochemical techniques.

The absence of keratin staining in tumor cells from localized fibrous mesotheliomas in both paraffin-embedded and frozen sections with sensitive peroxidase-antiperoxidase and avidin-biotin techniques is described. In addition ot the absence of staining for whole-stratum corneum keratin proteins, sections were negative for keratins of different molecular weights (45, 55, and 63 kilodaltons) that are characteristically present in mesothelial cells. Ultrastructurally, the cells most closely resembled mesenchymal cells of the fibroblastic type. These findings are in accordance with recent theories that relate the derivation of localized fibrous mesotheliomas to nonmesothelial cells, including subpleural connective tissue. Based on differences in immunohistochemical staining, the tumors appear to be unrelated to diffuse malignant mesotheliomas.

Immunoreactivity for epithelial membrane antigen (EMA) was evaluated in exfoliated benign and malignant cells using immunoperoxidase and immunogold techniques. In addition, protein A-colloidal gold was used for ultrastructural localization of EMA. Our results suggest that EMA is useful in distinguishing adenocarcinoma cells (strongly positive) from reactive mesothelial cells (negative or weakly positive), lymphoid cells (negative), and a variety of nonepithelial neoplasms (negative) with which they may be confused. Exfoliated cells from two mesotheliomas were also strongly positive for EMA. Ultrastructurally, EMA was distributed in a dense, even, linear pattern along the cell membrane and microvillous surface processes of adenocarcinoma cells. A similar but sparse distribution pattern was observed in reactive mesothelial cells. These observations reflect the increased sensitivity and higher resolution of the immunogold technique. Diagn Cytopathol 1987;3:41–49.