Increasing incidence of paediatric inflammatory bowel disease in northern Stockholm County, 2002-2007.

OBJECTIVES: A sharp increase in paediatric (younger than 16 years) inflammatory bowel disease (IBD) incidence was observed in northern Stockholm County, Sweden, in 1990-2001. The increasing incidence was primarily explained by a rising incidence of Crohn disease (CD). Here, we present an update on the trends in incidence of paediatric IBD, 2002-2007. METHOD: Medical records of all children diagnosed as having suspected IBD in northern Stockholm County, 2002-2007, were scrutinised using defined diagnostic criteria. Disease extension, localisation, and behaviour at diagnosis were classified within the framework of the Paris classification. RESULT: A total of 133 children were diagnosed as having IBD 2002-2007 corresponding to a sex- and age-standardised incidence (per 10 person-years) for paediatric IBD of 12.8 (95% CI 10.8-15.2). The standardised incidence was 9.2 (95% CI 7.5-11.2) for CD and 2.8 (95% CI 1.9-4.0) for ulcerative colitis (UC). A significant increasing incidence of UC (P < 0.05) was observed during the study period. No temporal trend was observed for the incidence of CD. CONCLUSIONS: The incidence rate of paediatric IBD in northern Stockholm was significantly higher in 2002-2007 than that observed in our earlier study covering 1990-2001. The former sharp increase in incidence of paediatric CD seems, however, to have levelled out, although at a higher rate than reported from most other regions in the world. Although CD was still predominant, the observed increase in incidence of UC during the study period is notable.

Harmful and potentially harmful constituents (HPHCs) in two novel nicotine pouch products in comparison with regular smokeless tobacco products and pharmaceutical nicotine replacement therapy products (NRTs).

BACKGROUND: Tobacco-free nicotine pouches is a novel category of oral nicotine-delivery products. Among current tobacco users such pouches may serve as a low-risk alternative to cigarettes or conventional, tobacco-based oral products e.g., snus and moist snuff. In the United States (U.S.), the market leading nicotine-pouch brand is ZYN®. However, no data on the chemical characteristics of ZYN have been published. METHODS: We screened for 43 compounds potentially present in tobacco products in seven oral nicotine-delivery products: ZYN (dry and moist), snus (General®), moist snuff (CRP2.1 and Grizzly Pouches Wintergreen), and two pharmaceutical, nicotine replacement therapy products (NRTs, Nicorette® lozenge and Nicotinell® gum). Thirty-six of the tested compounds are classified as harmful and potentially harmful constituents (HPHCs) by the Center for Tobacco Products at the U.S. Food and Drug Administration (FDA-CTP). Five additional compounds were included to cover the GOTHIATEK® product standard for Swedish snus and the last two compounds were chosen to include the four primary tobacco specific nitrosamines (TSNAs). RESULTS: The tested products contained nicotine at varying levels. The two ZYN products contained no nitrosamines or polycyclic aromatic hydrocarbons (PAHs) but low levels of ammonia, chromium, formaldehyde, and nickel. In the NRT products we quantified low levels of acetaldehyde, ammonia, cadmium, chromium, lead, nickel, uranium-235, and uranium-238. The largest number (27) and generally the highest levels of HPHCs were quantified in the moist snuff products. For example, they contained six out of seven tested PAHs, and seven out of ten nitrosamines (including NNN and NNK). A total of 19 compounds, none of which were PAHs, were quantified at low levels in the snus product. NNN and NNK levels were five to 12-fold lower in snus compared to the moist snuff products. CONCLUSIONS: No nitrosamines or PAHs were quantified in the ZYN and NRT products. Overall, the number of quantified HPHCs were similar between ZYN and NRT products and found at low levels.

Survival in patients with human papillomavirus positive tonsillar cancer in relation to treatment.

The incidence of tonsillar cancer and the proportion of human papillomavirus (HPV) positive tonsillar cancer cases have increased in the last decades. In parallel, treatment for tonsillar cancer has been intensified e.g., by accelerated radiotherapy, and chemotherapy, resulting in more side effects. Patients with HPV-positive tonsillar cancer have better prognosis than those with HPV-negative tumors, and the former group could hypothetically benefit from reduced, less-toxic treatment without compromising survival. Here, we therefore evaluated possible differences in overall and disease-specific survival after different oncological treatments in 153 patients with HPV DNA- and P16-positive tonsillar cancer who were diagnosed and treated with intent to cure between 2000 and 2007, in Stockholm, Sweden. Of these patients, 86 were treated with conventional radiotherapy, 40 were treated with accelerated radiotherapy and 27 were treated with chemoradiotherapy. There were no significant differences in overall or disease-free survival between the groups. However, there was a trend, implying a beneficial effect of the intensified treatment, with chemoradiotherapy being better than radiotherapy despite that more patients had stage IV disease in the former group; and accelerated radiotherapy being better than conventional radiotherapy. This needs to be followed further in larger more homogenous groups of patients. In conclusion, patients with HPV-positive tonsillar cancer treated with conventional- or accelerated radiotherapy or chemoradiotherapy disclosed similar survival rates. The trend for better survival and less metastasis after intensified treatment underlines the need for large prospective studies comparing less intense to more intense treatment (chemoradiotherapy).

Nitric oxide pathway-related gene alterations in inflammatory bowel disease.

OBJECTIVE: To reveal specific gene activation in nitric oxide (NO)-related inflammation we studied differential gene expression in inflammatory bowel disease (IBD). METHODS: Total RNA was isolated from 20 biopsies of inflamed mucosa from Crohn's disease (CD) and ulcerative colitis (UC) patients each as well as from six controls, labeled with (32)P-dCTP and hybridized to a human NO gene array. Significant genes were analyzed for functional gene interactions and heatmaps generated by hierarchical clustering. A selection of differentially expressed genes was further evaluated with immunohistochemical staining. RESULTS: Significant gene expression differences were found for 19 genes in CD and 23 genes in UC compared to controls, both diseases with high expression of ICAM1 and IL-8. Correlation between microarray expression and corresponding protein expression was significant (r = 0.47, p = 0.002). Clustering analysis together with functional gene interaction analysis revealed clusters of coregulation and coexpression in CD and UC: transcripts involved in angiogenesis, inflammatory response mediated by the transcription factor hypoxia-inducible factor 1, and tissue fibrosis. Also, a fourth cluster with transcripts regulated by the transcription factor Sp1 was found in UC. CONCLUSIONS: Expression analysis in CD and UC revealed disease-specific regulation of NO-related genes, which might be involved in perpetuating inflammatory disease activity in IBD.

Human papillomavirus and survival in patients with base of tongue cancer.

The incidence of base of tongue cancer is increasing in Sweden and the proportion of human papillomavirus (HPV) positive cancer has increased in Stockholm, Sweden. Between 2006 and 2007, 84% of base of tongue cancer cases in Stockholm were HPV-positive. The objective of this study was to assess the impact of HPV status on prognosis for base of tongue cancer patients. One-hundred and nine patients were diagnosed with base of tongue cancer between 1998 and 2007 in Stockholm County and 95 paraffin-embedded diagnostic tumor biopsies were obtained and tested for HPV by PCR. Eighty-seven patients had available biopsies, were treated with intention to cure and could be included in the survival analysis. Age, sex, TNM-stage, stage, treatment and survival were recorded from patient charts. Kaplan-Meier curves were used to present survival data. In multivariable analyses, a Cox proportional hazards model was used to adjust for covariates. In total 68 (78%) tumor biopsies from the 87 included patients were HPV DNA positive. Kaplan-Meier estimates showed that the overall survival for patients with HPV-positive cancer was significantly better (p = 0.0004), (log-rank test) than that of patients with HPV-negative cancer. Patients with HPV-positive tumors also had significantly better disease-free survival (p = 0.0008), (log-rank test) than those with HPV-negative tumors. These results further strengthen the option to consider HPV-status when planning prospective studies on treatment for base of tongue cancer.

The role of human papillomavirus in the increased incidence of base of tongue cancer.

Numerous reports have shown that the incidence for oropharyngeal cancer is increasing and that human papillomavirus (HPV) is a risk factor. However, few studies have investigated the specific subsites of the oropharynx. Following our previous research on tonsillar cancer, we assessed the increase in the incidence of base of tongue cancer and the prevalence of HPV in this disease. Between 1998 and 2007, 109 patients were diagnosed for base of tongue cancer in Stockholm county. Ninety-five paraffin-embedded diagnostic tumor biopsies from patients were obtained and tested for HPV, both by general HPV PCR and HPV-16/HPV-33 type-specific PCR. Expression of HPV-16 RNA was analyzed to confirm E6 and/or E7 expression. Incidence data were obtained from the Swedish Cancer Registry. An overall increase in the incidence of base of tongue cancer from 0.15/100,000 person-years during 1970-1974 to 0.47/100,000 person-years during 2005-2007 was found in Sweden. The prevalence of HPV in base of tongue cancer in Stockholm county increased from 58% during 1998-2001 to 84% during 2004-2007 (p < 0.05). In the HPV-positive tumors, HPV-16 dominated (86%) but interestingly, HPV33 was detected in as many as 10%. E6 and/or E7 RNA were found in 85% of the samples tested. The incidence of base of tongue cancer, as well as the proportion of HPV-positive tumors, has increased in Sweden during the study period, suggesting that HPV may contribute to this increase.

Incidence of human papillomavirus (HPV) positive tonsillar carcinoma in Stockholm, Sweden: an epidemic of viral-induced carcinoma?

In the county of Stockholm, between 1970 and 2002, we have previously reported a 3-fold parallel increase in the incidence of tonsillar squamous cell carcinoma (SCC) and the proportion of human papillomavirus (HPV) positive tonsillar SCC. Here, we have followed the above parameters in all patients (n = 120) diagnosed with tonsillar SCC during 2003-2007 in the same area, and also in correlation to our previous data. Ninety-eight pretreatment biopsies were available and presence of HPV DNA and HPV-16 E6 and E7 RNA were tested by polymerase chain reaction (PCR) and RT-PCR. Incidence data were obtained from the Swedish Cancer Registry. Data reported from 1970 to 2002 were also obtained for comparison. HPV DNA was present in 83 of 98 (85%) of the tonsillar SCC biopsies from 2003 to 2007 and 77 of these were HPV-16 positive. HPV-16 E6 and E7 RNA were found in 98% of 52 analyzed HPV-16 positive cases. The proportion of HPV-positive cancers had significantly increased both from 1970 to 2007 (p < 0.0001) as well from 2000 to 2007 (p < 0.01), with 68% (95% confidence interval (CI), 53-81) 2000-2002; 77% (95% CI, 63-87) 2003-2005; and 93% (95% CI, 82-99) 2006-2007. The incidence rate of HPV-positive tumors almost doubled each decade between 1970 and 2007, in parallel with a decline of HPV-negative tumors. In conclusion, the incidence of HPV-positive cancers is still increasing in the County of Stockholm, suggesting an epidemic of a virus-induced carcinoma, with soon practically all tonsillar SCC being HPV positive, as in cervical cancer.

The importance of rectal cancer MRI protocols on interpretation accuracy.

BACKGROUND: Magnetic resonance imaging (MRI) is used for preoperative local staging in patients with rectal cancer. Our aim was to retrospectively study the effects of the imaging protocol on the staging accuracy. PATIENTS AND METHODS: MR-examinations of 37 patients with locally advanced disease were divided into two groups; compliant and noncompliant, based on the imaging protocol, without knowledge of the histopathological results. A compliant rectal cancer imaging protocol was defined as including T2-weighted imaging in the sagittal and axial planes with supplementary coronal in low rectal tumors, alongside a high-resolution plane perpendicular to the rectum at the level of the primary tumor. Protocols not complying with these criteria were defined as noncompliant. Histopathological results were used as gold standard. RESULTS: Compliant rectal imaging protocols showed significantly better correlation with histopathological results regarding assessment of anterior organ involvement (sensitivity and specificity rates in compliant group were 86% and 94%, respectively vs. 50% and 33% in the noncompliant group). Compliant imaging protocols also used statistically significantly smaller voxel sizes and fewer number of MR sequences than the noncompliant protocols CONCLUSION: Appropriate MR imaging protocols enable more accurate local staging of locally advanced rectal tumors with less number of sequences and without intravenous gadolinium contrast agents.

Fecal calprotectin: a quantitative marker of colonic inflammation in children with inflammatory bowel disease.

OBJECTIVES: The protein calprotectin (S100 A8/A9) is present in neutrophils, monocytes, and macrophages. Colorectal inflammation can be detected by increased excretion of fecal calprotectin (FC). The aim of this study was to evaluate FC as a quantitative marker of inflammatory activity in children with inflammatory bowel disease (IBD). PATIENTS AND METHODS: Thirty-nine children with IBD delivered a fecal spot sample and underwent colonoscopy. The samples were examined with an enzyme-linked immunosorbent assay for FC (Calprest, Eurospital, Trieste, Italy). The concentrations were correlated to macroscopic and microscopic assessments of extent and severity of inflammation in 8 colonic segments for each patient. RESULTS: FC correlated significantly to the macroscopic extent (Spearman rho = 0.61) and the severity (Spearman rho = 0.52) of colonic inflammation and to a macroscopic, combined extent and severity score (Spearman rho = 0.65). Significant correlations also were found to the microscopic extent (Spearman rho = 0.71) and severity (Spearman rho = 0.72) of colonic inflammation and to a microscopic, combined extent and severity score (Spearman rho = 0.75). The median FC was 392 mug/g (95% confidence interval [CI], 278-440) in children with clinical IBD symptoms (n = 23) and 32.9 mug/g (95% CI, 9.4-237) in asymptomatic IBD patients (n = 16). Of the asymptomatic children, 56% had a complete microscopic mucosal healing, and their median FC was 9.9 mug/g (95% CI, 5.9-41.9). CONCLUSIONS: FC can be used as a surrogate marker for estimation of colonic inflammation in pediatric IBD. Normalized FC concentration seems to indicate complete mucosal healing. FC is simple to obtain and analyze; this should facilitate objective assessment and monitoring of IBD activity.

Brush cytology is superior to biopsies obtained by a new device in bile duct strictures.

BACKGROUND/AIMS: Brush cytology during ERCP has been reported to have a low sensitivity. A new device, Howell biliary system (Wilson-Cook), enables targeted biopsies for histopathologic assessment. The aim was to compare histopathology with brush cytology. METHODOLOGY: Brush cytology followed by biopsies obtained by the Howell device was taken consecutively from bile duct strictures. Coded slides were scored by 3 pathologists and 2 cytologists in a 3-graded scale; 2: benign; 3: suspicious of malignancy; 4: malignant. The clinical outcome including autopsy served as the gold standard for the definite diagnoses. RESULTS: Twenty-one malignant and 6 benign strictures were evaluated. The histopathology revealed 11 out of the 21 malignant as certain or suspected malignant (score > or = 3) (sensitivity: 0.52). The cytology scored 17 out of 21 > or = 3 (sensitivity: 0.80). The in pair kappa values for the 3 pathologists were: (0.37; 0.26; 0.41) vs. 0.56 for the 2 cytologists. Among the evaluable strictures the pathologists scorings were; (median: 3.0, SD: 0.72) for the malignant and (median: 2.3, SD: 0.98) for the benign (p = 0.27) and the cytology scorings were; (median: 3.5, SD: 0.73) for the malignant and (median: 2.7, SD: 0.65) for the benign (p = 0.09). CONCLUSIONS: Brush cytology has a higher accuracy than the targeted biopsies and should be used in combination with other methods to reach the correct diagnosis.

Integrin alpha2beta1 regulates neutrophil recruitment and inflammatory activity in experimental colitis in mice.

BACKGROUND: Human inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), is associated with leukocyte accumulation in the inflamed intestinal tissue. Recent studies strongly suggest a role of beta1 integrin receptors in regulating tissue damage and disease symptoms related to inflammatory bowel disease. The aim of this study was to investigate the role of the collagen-binding alpha2beta1 integrin (CD49b/CD29) in dextran sodium sulfate-induced colitis in mice. METHODS: Colitis was induced in mice through oral administration of 2% dextran sodium sulfate in drinking water. Rectal administration of anti-alpha2-monoclonal antibody (mAb) in 1 group was compared with oral treatment with betamethasone in another group and rectal administration of a control antibody in a third group. Clinical and histological signs of colitis, neutrophil infiltration into the colon mucosa, and gene expression of metalloproteinases were assessed. RESULTS: Rectal administration of anti-alpha2-mAb was found to significantly reduce weight loss from 13.5% +/- 6.5% to 2.2% +/- 0.2% (P = 0.013 versus control mAb) and mucosal neutrophil infiltration from 47.2 +/- 10.0 to 6.6 +/- 8.0 neutrophils per counted area (P < 0.05 versus control mAb). Metalloproteinase gene expression was suppressed through anti-alpha2-mAb treatment. The protective effect against colitis seen after anti-alpha2beta1 integrin treatment was found to be favorable to the effect seen after high-dose oral betamethasone. CONCLUSIONS: We demonstrate an alleviating action of the collagen-binding alpha2beta1 integrin in experimental colitis in mice and suggest that this effect is mediated by inhibition of neutrophil migration and activation. Local administration of function-blocking antibodies against integrin alpha2beta1 may provide novel avenues to treat inflammatory bowel disease.

Reliability of the reported size of removed colorectal polyps.

BACKGROUND: The size of colorectal polyps is important in the clinical management of these lesions. AIM: To audit the accuracy in calculating the size of "polyps" by various specialists. MATERIALS AND METHODS: Eighteen pathologists and four surgeons measured, with a conventional millimetre ruler, the largest diameter of 12 polyp phantoms. The results of two independent measurements (two weeks apart) were compared with the gold standard-size assessed at The Royal Institute of Technology, Sweden. RESULTS: Thirty-one percent (83/264-trial 1) and 33% (88/264-trial 2) of the measurements underestimated or overestimated the gold standard size by >1 mm. Of the 22 experienced participants, 95% (21/22-trial 1) and 91% (20/22-trial 2) misjudged by >1 mm the size of one or more polyps. Values given by 13 participants (4.9%) in trial 1 and by 15 participants (5.7%) in trial 2, differed by > or = +/-4 mm from the gold standard size. In addition, a big difference between the highest and the lowest values was recorded in some polyps (up to 11.4 mm). Those disparate values were regarded as a human error in reading the scale on the ruler. CONCLUSION: Using a conventional ruler (the tool of pathologists worldwide) unacceptably high intra-observer and inter-observer variations in assessing the size of polyp-phantoms was found. The volume and the shape of devices, as well as human error in reading the scale of the ruler were confounding factors in size assessment. In praxis, the size is crucial in the management of colorectal polyps. Considering the clinical implications of the results obtained, the possibility of developing a method that will allow assessment of the true size of removed clinical polyps is being explored.

Improving the Prediction of Prostate Cancer Overall Survival by Supplementing Readily Available Clinical Data with Gene Expression Levels of IGFBP3 and F3 in Formalin-Fixed Paraffin Embedded Core Needle Biopsy Material.

BACKGROUND: A previously reported expression signature of three genes (IGFBP3, F3 and VGLL3) was shown to have potential prognostic value in estimating overall and cancer-specific survivals at diagnosis of prostate cancer in a pilot cohort study using freshly frozen Fine Needle Aspiration (FNA) samples. METHODS: We carried out a new cohort study with 241 prostate cancer patients diagnosed from 2004-2007 with a follow-up exceeding 6 years in order to verify the prognostic value of gene expression signature in formalin fixed paraffin embedded (FFPE) prostate core needle biopsy tissue samples. The cohort consisted of four patient groups with different survival times and death causes. A four multiplex one-step RT-qPCR test kit, designed and optimized for measuring the expression signature in FFPE core needle biopsy samples, was used. In archive FFPE biopsy samples the expression differences of two genes (IGFBP3 and F3) were measured. The survival time predictions using the current clinical parameters only, such as age at diagnosis, Gleason score, PSA value and tumor stage, and clinical parameters supplemented with the expression levels of IGFBP3 and F3, were compared. RESULTS: When combined with currently used clinical parameters, the gene expression levels of IGFBP3 and F3 are improving the prediction of survival time as compared to using clinical parameters alone. CONCLUSION: The assessment of IGFBP3 and F3 gene expression levels in FFPE prostate cancer tissue would provide an improved survival prediction for prostate cancer patients at the time of diagnosis.

Investigation of the adsorption behaviour of a chiral model compound on a tartardiamide-based network-polymeric chiral stationary phase.

The adsorption behaviour of the enantiomers of 2-phenylbutyric acid on the chiral stationary phase (CSP) Kromasil CHI-TBB was studied using hexane/MTBE (90/10) as eluent. Adsorption isotherms were acquired at 40 different enantiomer concentrations in the interval between 7.6 microM and 305 mM, an approximately 40,000-fold dynamic range. The adsorption data fitted well to the bi-Langmuir model, indicating a heterogeneous surface with two different types of adsorption sites having different equilibrium constants and capacities; namely one chiral site and one non-chiral site. A comparison with earlier adsorption studies on modern CSPs revealed that the capacity value of the "true" chiral site of Kromasil CHI-TBB is the largest reported so far. The elution profiles simulated with these parameters show excellent agreement with the corresponding experimental profiles. Guidelines for comparisons of loading capacities of CSPs are presented.

Presentation and progression of childhood-onset inflammatory bowel disease in Northern Stockholm County.

BACKGROUND: Some studies have suggested that childhood-onset inflammatory bowel disease (IBD) is characterized by extensive intestinal involvement and rapid progression to complications. Here, we report the presentation and progression of patients diagnosed with IBD during childhood in a population-based cohort from northern Stockholm County. METHODS: Medical records for all 280 patients diagnosed in the period 1990-2007 with childhood-onset IBD in northern Stockholm County were followed until 2011 (median follow-up time, 8.8 yr). Disease phenotypes were classified according to the Paris pediatric IBD classification. RESULTS: Among the 74 patients with ulcerative colitis, 72% presented with pancolitis. Among the 200 patients with Crohn's disease (CD), 75% presented with colitis. Complicated disease behavior was observed in 18% of patients with CD by end of follow-up. Extension of the disease territory was observed in 22% of patients with ulcerative colitis and 15% of patients with CD. The cumulative risk of intra-abdominal surgery after 10 years was 8% (95% confidence interval, 4%-20%) for ulcerative colitis and 22% (95% confidence interval, 15%-28%) for patients with CD. Nonmucosal healing at 1 year was associated with a complicated disease course in patients with CD (hazard ratio = 14.56; 95% confidence interval, 1.79-118.68; P = 0.01). CONCLUSIONS: Patients with childhood-onset IBD were characterized by extensive colitis that was relatively stable over time and associated with a relatively low risk of complications and abdominal surgery. Our findings confirm the more extensive disease location in pediatric IBD but did not identify the proposed dynamic and aggressive nature of the childhood-onset phenotype. The association of nonmucosal healing with a complicated disease course suggests that endoscopy should guide treatment intensity in childhood-onset CD.

Theoretical and experimental study of binary perturbation peaks with focus on peculiar retention behaviour and vanishing peaks in chiral liquid chromatography.

The perturbation peak theory was recently developed for acquiring binary isotherm data using the perturbation method (PM) and it was applied for some chiral systems. However, the binary plateaus of these systems were only weakly to moderately nonlinear. In this article the perturbation theory for LC, is developed for both retention times and peak areas and is verified by systematic experiments over the whole range of non-linearity. Attention is focused on non-linear effects that complicate the proper use of the PM method under moderately to strongly non-linear conditions. A serious complication was that the second perturbation peak vanished already at moderate plateau concentrations. A solution to this problem based on a firm theoretical basis and verified experimentally is presented. We also investigated a peculiar retention dependence on the binary plateau concentration, as the retentions of the two perturbation peaks of the binary plateau was compared with the single plateau peak of the more retained enantiomer.

Use of liquid chromatography-diode-array detection and mass spectrometry for rapid product identification in biotechnological synthesis of a hydroxyprogesterone.

In exploratory scale biotechnological process development, the product must be rapidly identified although a reference compound may not always be available. LC-diode-array detection and MS were used for this purpose in a process producing 9alpha-hydroxyprogesterone from progesterone as substrate. The electrospray ionization mass spectrometer was combined with an ion trap mass spectrometer for the second generation MS. The preliminary identification, which could be carried out within the course of a day, confirmed that the product was a hydroxyprogesterone. The final identification step, which was much more material intensive and hence time consuming, involved a two-step preparative separation to yield quantities necessary for definitive product identification based on 1H- and 13C NMR.

Laminin-5 gamma 2: a marker to identify oral mucosal lesions at risk for tumor development?

Mucosal lesions of various types are frequently encountered in the oral cavity and a limited proportion of these will eventually progress to invasive cancer. Neither macroscopic nor microscopic evaluation can give reliable information concerning risk for cancer development which is why a marker indicating high risk for tumor progression would be of significant value. Laminins are a family of alpha beta gamma heterotrimeric proteins found in basement membranes and earlier studies have shown that laminin-5 gamma 2 expression is a useful marker of invasiveness in head and neck squamous cell carcinomas. Unique material comprising 36 different oral mucosal lesions from 15 patients, prior to the development of invasive carcinoma (12 cases) or cancer in situ (CIS) (3 cases) on the same site, was investigated. For controls we had mucosal lesions from 15 patients without malignant progression. These 15 lesions matched with the specific site in the oral cavity of the initial patients. The minimum follow-up time for the control group was at least 36 months. We performed immunohistochemical staining with a monoclonal antibody against the recombinant gamma 2 chain of laminin-5 in the archived biopsies of preneoplastic oral lesions. Nine out of 15 cases of preneoplastic lesions which became invasive/CIS were laminin-5-positive (60%) whereas only 3 out of 13 (2 were excluded due to sparse material for laminin-5 analysis) lesions which did not progress were laminin-5-positive (23%) (p = 0.05). The results indicate that oral mucosal lesions expressing laminin-5 show an increased risk for tumor progression.

The gamma2 chain of laminin-5 as an indicator of increased risk for recurrence in T1 stage tongue cancer.

BACKGROUND: Treatment of small, localized tongue cancer is an act of balance between sufficiently extensive surgery and/or radiotherapy for safe cure, and minimizing treatment-related morbidity. The biological aggressiveness of these cancers is variable and conventional histopathology does not give sufficient information concerning the risk for local or regional recurrence. Laminin-5 is an extracellular matrix protein noted to have special importance for epithelial cell invasion. PATIENTS AND METHODS: We investigated 43 diagnostic biopsies from patients with T1 tongue cancer treated with wedge excision only. Immunohistochemical analysis was performed using a polyclonal antibody against the gamma2 chain of laminin-5. RESULTS: Thirty-six specimens were evaluated and 16 patients had a recurrence. A staining pattern of 50% or more was seen in the majority of patients (75%) with tumor recurrence and in fewer patients (45%) without recurrence. CONCLUSION: In order to optimize treatment for the individual T1 tongue cancer patient, immunostaining against laminin-5 appears to be one possible useful tool.

Guidelines for analytical method development and validation of biotechnological synthesis of drugs. Production of a hydroxyprogesterone as model.

In connection with biotechnological synthesis of pharmaceutical drugs, validated methods for quantification of both product and substrate at different time intervals are essential for proper calculation of rate coefficients. In this field, there still exist no guidelines for analytical validation, unlike the situation in the bioanalytical field. Therefore, in this study the detailed guidelines by FDA for bioanalytical method validation were applied to a typical biotechnological process; the enzymatic synthesis of 9alpha-hydroxyprogesterone in E. coli using progesterone as substrate. The process liquid was extracted and analyzed using an HPLC-DAD system. The quality control (QC) samples of the product demonstrated excellent precision (C.V.<1.5%) and accuracy between 99.3 and 107%. The study showed that the recommendations and the validation terms for bioanalytical methods can be used also for biotechnological production, but with some important exceptions. The tolerances (C.V. values) of the validation terms should be much narrower; the internal standard (I.S.) must be present in the process liquid before the start of the process and must be much different in structure from the substrate (so as not to participate in the biotechnological process). In addition, the selectivity must be checked very frequently during the process due to the changes in the blank process liquid with time.