High resolution ArrayCGH and expression profiling identifies PTPRD and PCDH17/PCH68 as tumor suppressor gene candidates in laryngeal squamous cell carcinoma

Many classical tumor suppressor genes (TSG) were identified by delineation of bi-allelic losses called homozygous deletions. To identify systematically homozygous deletions in laryngeal squamous cell carcinoma (LSCC) and to unravel novel putative tumor suppressor genes, we screened 10 LSCC cell lines using high resolution array comparative genomic hybridization (arrayCGH) and array based expression analysis. ArrayCGH identified altogether 113 regions harboring protein coding genes that showed strong reduction in copy number indicating a potential homozygous deletion. Out of the 113 candidate regions, 22 novel homozygous deletions that affected the coding sequences of 15 genes were confirmed by multiplexPCR. Three genes were homozygously lost in two cell lines: PCDH17/PCH68, PRR20, and PTPRD. For the 15 homozygously deleted genes, four showed statistically significant downregulation of expression in LSCC cell lines as compared with normal human laryngeal controls. These were ATG7 (1/10 cell line), ZMYND11 (BS69) (1/10 cell line), PCDH17/PCH68 (9/10 cell lines), and PTPRD (7/10 cell lines). Quantitative real-time PCR was used to confirm the downregulation of the candidate genes in 10 expression array-studied cell lines and an additional cohort of cell lines; statistical significant downregulation of PCDH17/PCH68 and PTPRD was observed. In line with this also Western blot analyses demonstrated a complete absence of the PCDH17 and PTPRD proteins. Thus, expression profiling confirmed recurrent alterations of two genes identified primarily by delineation of homozygous deletions. These were PCDH17/PCH68, the protocadherin gene, and the STAT3 inhibiting receptor protein tyrosine phosphatase gene PTPRD. These genes are good candidates for novel TSG in LSCC.     

HCR, a candidate gene for psoriasis, is expressed differently in psoriasis and other hyperproliferative skin disorders and is downregulated by interferon-gamma in keratinocytes

We have previously shown that HCR is a good candidate gene for psoriasis based on its location in the PSORS1 locus, predicted secondary structure change of the associated allele, and expression pattern. To understand better the function of HCR, we studied how HCR expression is altered in hyperproliferative skin diseases other than psoriasis and in cancers. We examined also its regulation by different cytokines, growth factors, and antipsoriatic agents using quantitative RT-PCR (TaqMan) analysis and its location by immunostaining of keratinocyte cultures. Compared to psoriasis, HCR protein had a different distribution in chronic dermatitis, pityriasis rubra pilaris, mycosis fungoides, and chronic skin ulcers. In three of six grade III squamous cell carcinomas of the skin, four of four adenocarcinomas of the lung, and two of two ductal breast adenocarcinomas, positive cytoplasmic staining in cancer cells was detected. As in psoriasis, Ki67 did not colocalize with HCR. In cell cultures, HCR staining was detected perinuclearly in the cytoplasm and in the nuclei, suggesting that the protein may have a role in both compartments. A 2-fold downregulation of HCR mRNA expression was observed on stimulation with interferon-gamma. Based on the observations that HCR is detected in cancers of epithelial origin in Ki67-negative areas and that interferon-gamma downregulates its expression, we suggest it to have an antiproliferative function.     

Predictive value of procalcitonin decrease in patients with severe sepsis: a prospective observational study

Introduction  

This prospective study investigated the predictive value of procalcitonin (PCT) for survival in 242 adult patients with severe sepsis and septic shock treated in intensive care.     

Endoscopic laser surgery for laryngeal cancer

Endoscopic laser surgery is a novel treatment modality for laryngeal cancer. CO₂ laser combined with an operating microscope is the most frequently used instrumentation. In Finland we started large-scale laser surgery in 1994 in all five university hospitals, covering a population of about five million people. By 1998 we had operated on 140 patients, of whom 11 were females. Eighty-three per cent of the lesions were glottic. Because of the low number of stage III–IV patients, the recurrence and survival analyses included 132 patients with in situ, stage I or stage II tumours, numbering 8, 96 and 28 respectively. The mean follow-up time was 38 months. The 2-year recurrence frequencies were 5% for stage I, 31% for stage II, and 11% altogether. No patients developed recurrences after 2 years. Seven patients underwent a salvage laryngectomy and the adjusted cumulative survival rate was 95%. After laser surgery the quality of voice was good or excellent in 70% and only three patients suffered from severe aphonia. This study showed that the results of endoscopic laser surgery are comparable with those of radiation therapy, but this type of treatment is more convenient for the patients and much cheaper for society. Received: 20 June 2000 / Accepted: 10 April 2001     

Radiosensitivity of head and neck cancer cells in vitro. A 96-well plate clonogenic cell assay for squamous cell carcinoma

Radiation sensitivity was determined for nine University of Michigan squamous cell carcinoma (UM-SCC) cell lines, MCF-7 and HeLa, using a 96-well plate clonogenic assay. Plating efficiencies (PE) of the UM-SCC cell lines were between 0.16 and 0.36. Higher PE values obtained were with MCF-7 (0.4) and HeLa (0.5). The UM-SCC cell lines were used at low passages (passage 13 to passage 20) to minimize artifacts attributable to long-term culture. Cells were irradiated in suspension using a cobalt 60 gamma source at a dose rate of 0.94 Gy/min (94 rad/min). Survival data were fitted well by either a linear quadratic function F = e-(alpha D + beta D2) or by a monoexponential function F = Ae-alpha D. Mean inactivation dose, equivalent to the area under the survival curve (AUC), was used as a measure of radiation sensitivity. The UM-SCC-1, 9, 11A, 11B, MCF-7, and HeLa were the most radiation resistant lines we tested (AUC greater than 2.1), while UM-SCC-14A was the most sensitive (AUC = 1.591). The assay was highly reproducible, and the difference in radiation sensitivity between cell lines were statistically significant.     

Clonogenic cell assay for anchorage-dependent squamous carcinoma cell lines using limiting dilution

Clonogenic assays under either anchorage-dependent or -independent conditions are very useful for testing the sensitivity of tumor cells to cytotoxic drugs and radiation. These assays have not been widely used with squamous-cell carcinomas (SCC) because of poor tumor-cell viability and poor cloning efficiency, especially in semi-solid media. To find a clonogenic assay suitable for use with human squamous cancers we tested SCC lines, derived in our laboratory from patients with head and neck cancer, for the capacity to form colonies in soft agar and in 96-well plates. Of 13 UM-SCC lines tested for colony formation in agarose, only UM-SCC-11A was capable of growth in conventional semi-solid media. One other line, UM-SCC-14C, produced colonies in agarose only in the presence of epidermal growth factor. In contrast, all 17 of the SCC lines tested exhibited colony formation in adherent cell culture using limiting dilution in 96-well plates. The plating efficiencies of the SCC lines in the 96-well plate assay ranged from 0.02 to 0.52 colonies (wells)/cell whereas the PE values in soft agar were lower, ranging from 0.0055 to 0.0086 colonies/cell. The 96-well plate assay is not affected by cell migration, a problem encountered with some cell lines when clonogenic assays are performed in Petri dishes. UM-SCC-11A was tested for radiation sensitivity both in soft agar and in the 96-well plate assay. Comparable results were obtained. In summary, the majority of SCC cell lines did not form viable colonies in soft agar but the 96-well plate assay was applicable to a broad spectrum of anchorage-dependent human SCC cell lines and provides an efficient method for evaluating clonogenic cell survival.     

Buprenorphine versus methadone maintenance therapy: a randomized double-blind trial with 405 opioid-dependent patients

Aims To assess the efficacy of buprenorphine compared with methadone maintenance therapy for opioid dependence in a large sample using a flexible dosing regime and the marketed buprenorphine tablet. Design Patients were randomized to receive buprenorphine or methadone over a 13‐week treatment period in a double‐blind, double‐dummy trial. Setting Three methadone clinics in Australia. Participants Four hundred and five opioid‐dependent patients seeking treatment. Intervention Patients received buprenorphine or methadone as indicated clinically using a flexible dosage regime. During weeks 1–6, patients were dosed daily. From weeks 7–13, buprenorphine patients received double their week 6 dose on alternate days. Measurements Retention in treatment, and illicit opioid use as determined by urinalysis. Self‐reported drug use, psychological functioning, HIV‐risk behaviour, general health and subjective ratings were secondary outcomes. Findings Intention‐to‐treat analyses revealed no significant difference in completion rates at 13 weeks. Methadone was superior to buprenorphine in time to termination over the 13‐week period (Wald χ² = 4.371, df = 1, P = 0.037), but not separately for the single‐day or alternate‐day dosing phases. There were no significant between‐group differences in morphine‐positive urines, or in self‐reported heroin or other illicit drug use. The majority (85%) of the buprenorphine patients transferred to alternate‐day dosing were maintained in alternate‐day dosing. Conclusions Buprenorphine did not differ from methadone in its ability to suppress heroin use, but retained approximately 10% fewer patients. This poorer retention was due possibly to too‐slow induction onto buprenorphine. For the majority of patients, buprenorphine can be administered on alternate days.     

Multiprofessional collaboration promoting home care clients' personal resources: perspectives of older clients

Home care can be decisive in supporting older people in the home environment. However, one professional in home care cannot take the whole responsibility for promotion alone; on the contrary multiprofessional collaboration is needed. The aim of the study is to describe the experiences of multiprofessional collaboration in promoting personal resources among older home care clients (75+ years) in Finland. The data were collected by unstructured interviews with 21 older home care clients. Their mean age was 83.5 years, ranging from 75 to 91, with 17 female and four male participants. Inductive content analysis was used to analyze the data. The interviewees described the work of professionals from four perspectives: expertise, communication, decision-making and responsibility. Multiprofessional collaboration promoted the personal resources of interviewees with physical, psychological and social support. This study showed that the professionals worked as being expert-oriented: in the multiprofessional collaboration, each expert took care of his/her own part of the client's situation. This included the risk,, that the client's overall situations remained uncharted. However, the client's overall situation is a very important aspect when professionals suppport older people living in their own homes as long as possible. This study revealed the need for developing collaboration skills between social and health care professionals so that the staffs serve the needs of aged clients better together.