Glass fibre reinforced versus multistranded bonded orthodontic retainers: a 2 year prospective multi-centre study.

The objective of this study was to compare glass fibre reinforced (GFR) with multistranded bonded orthodontic retainers in terms of success rate and periodontal implications. A 2 year parallel study was conducted of 184 patients scheduled to receive bonded retainers in the upper and lower anterior segments. In three centres, the patients (mean age 14 years; 90 males and 94 females) were sequentially assigned to receive GFR retainers containing 500 unidirectional glass fibres (GFR500), 1000 unidirectional glass fibres (GFR1000), or multistranded retainers (gold standard). Retainer failures and periodontal conditions were monitored every 6 months. In a control group of 90 subjects without retainers, periodontal conditions were examined (negative control). Of the 274 recruited patients, 15 dropped out during the 2 year study period. Kaplan-Meier plots were drawn to assess survival of the different retainers. The Mantel-Cox log-rank test was used to identify significant differences in survival functions among the groups. Repeated measures analysis of variance and appropriate post hoc tests were adopted to evaluate periodontal conditions over time. GFR retainers showed unacceptably high failure rates in comparison with multistranded retainers (51 versus 12 per cent). The most significant periodontal conditions were found in patients with GFR retainers with no significant differences between the GFR500 and the GFR1000 group for any parameter at any time point. Subjects without retainers showed significantly lower levels of gingival inflammation and plaque accumulation when compared with patients in any retainer group. Multistranded retainers should remain the gold standard for orthodontic retention, although periodontal complications are common. The use of GFR retainers should be discouraged in daily practice.

A European network for virtual microscopy--design, implementation and evaluation of performance.

Web-based virtual microscopy has enabled new applications within pathology. Here, we introduce and evaluate a network of academic servers, designed to maximize image accessibility to users from all regions of Europe. Whole-slide imaging was utilized to digitize the entire slide set (n = 154) for the slide seminars of the 21st European Congress of Pathology. The virtual slides were mirrored to five academic servers across Europe using a novel propagation method. Functionality was implemented that automatically selects the fastest server connection in order to optimize the slide-viewing speed ( http://www.webmicroscope.net/ECP2007). Results show that during 6 months of monitoring the uptime of the network was 100%. The average viewing speed with the network was 3.1 Mbit/s, as compared to 1.9 Mbit/s using single servers. A good viewing speed (>2Mbit/s) was observed in 32 of 37 countries (86%), compared to 25 of 37 (68%) using single servers. Our study shows that implementing a virtual microscopy network spanning a large geographical area is technically feasible. By utilizing existing academic networks and cost-minimizing image compression, it is also economically feasible.

Evaluation of urinary biomarkers of exposure to benzene: correlation with blood benzene and influence of confounding factors.

PURPOSE: trans,trans-Muconic acid (t,t-MA) is generally considered as a useful biomarker of exposure to benzene. However, because of its lack of specificity, concerns about its value at low level of exposure have recently been raised. The aim of this study was (a) to compare t,t-MA, S-phenylmercapturic acid (SPMA) and benzene (B-U) as urinary biomarkers of exposure to low levels of benzene in petrochemical workers and, (b) to evaluate the influence of sorbic acid (SA) and genetic polymorphisms of biotransformation enzymes on the excretion of these biomarkers. METHOD: A total of 110 workers (including 24 smokers; 2-10 cigarettes/day) accepted to take part in the study. To assess external exposure to benzene, air samples were collected during the whole working period by a passive sampling device attached close to the breathing zone of 98 workers. Benzene was measured in blood (B-B) samples taken at the end of the shift, and was considered as the reference marker of internal dose. Urine was collected at the end of the shift for the determination of B-U, SPMA, t,t-MA, SA and creatinine (cr). B-U and B-B were determined by head-space/GC-MS, SPMA and SA by LC-MS, t,t-MA by HPLC-UV. RESULTS: Most (89%) personal measurements of airborne benzene were below the limit of detection (0.1 ppm); B-B ranged from <0.10 to 13.58 mug/l (median 0.405 microg/l). The median (range) concentrations of the urinary biomarkers were as follows: B-U 0.27 microg/l (<0.10-5.35), t,t-MA 0.060 mg/l (<0.02-0.92), SPMA 1.40 microg/l (0.20-14.70). Urinary SA concentrations ranged between <3 and 2,211 microg/l (median 28.00). Benzene concentration in blood and in urine as well as SPMA, but not t,t-MA, were significantly higher in smokers than in non-smokers. The best correlation between B-B and urinary biomarkers of exposure were obtained with benzene in urine (microg/l r = 0.514, P < 0.001; microg/g cr r = 0.478, P < 0.001) and SPMA (microg/l r = 0.495, P < 0.001; microg/g cr r = 0.426, P < 0.001) followed by t,t-MA (mg/l r = 0.363, P < 0.001; mg/g cr r = 0.300, P = 0.002). SA and t,t-MA were highly correlated (r = 0.618, P < 0.001; corrected for cr r = 0.637). Multiple linear regression showed that the variation of t,t-MA was mostly explained by SA concentration in urine (30% of the explained variance) and by B-B (12%). Variations of SPMA and B-U were explained for 18 and 29%, respectively, by B-B. About 30% of the variance of B-U and SPMA were explained by B-B and smoking status. Genetic polymorphisms for biotransformation enzymes (CYP2E1, EPHX1, GSTM1, GSTT1, GSTP1) did not significantly influence the urinary concentration of any of the three urinary biomarkers at this low level of exposure. CONCLUSION: At low levels of benzene exposure (<0.1 ppm), (1) t,t-MA is definitely not a reliable biomarker of benzene exposure because of the clear influence of SA originating from food, (2) SPMA and B-U reflect the internal dose with almost similar accuracies, (3) genetically based inter-individual variability in urinary excretion of biomarkers seems negligible. It remains to assess which biomarker is the best predictor of health effects.

No detectable hypoxia in malignant salivary gland tumors: preliminary results.

PURPOSE: Hypoxia is detected in most solid tumors and is associated with malignant progression and adverse treatment outcomes. However, the oxygenation status of malignant salivary gland tumors has not been previously studied. The aim of this study was to investigate the potential clinical relevance of hypoxia in this tumor type. METHODS AND MATERIALS: Twelve patients scheduled for surgical resection of a salivary gland tumor were preoperatively injected with the hypoxia marker pimonidazole and the proliferation marker iododeoxyuridine. Tissue samples of the dissected tumor were immunohistochemically stained for blood vessels, pimonidazole, carbonic anhydrase-IX, glucose transporters-1 and -3 (Glut-1, Glut-3), hypoxia-inducible factor-1alpha, iododeoxyuridine, and epidermal growth factor receptor. The tissue sections were quantitatively assessed by computerized image analysis. RESULTS: The tissue material from 8 patients was of sufficient quality for quantitative analysis. All tumors were negative for pimonidazole binding, as well as for carbonic anhydrase-IX, Glut-1, Glut-3, and hypoxia-inducible factor-1alpha. The vascular density was high, with a median value of 285 mm(-2) (range, 209-546). The iododeoxyuridine-labeling index varied from <0.1% to 12.2% (median, 2.2%). Epidermal growth factor receptor expression levels were mostly moderate to high. In one-half of the cases, nuclear expression of epidermal growth factor receptor was observed. CONCLUSION: The absence of detectable pimonidazole binding, as well as the lack of expression of hypoxia-associated proteins in all tumors, indicates that malignant salivary gland tumors are generally well oxygenated. It is unlikely that hypoxia is a relevant factor for their clinical behavior and treatment responsiveness.

Expression of p14ARF, p16INK4a and p53 in relation to HPV in (pre-)malignant squamous skin tumours.

Studies in cervical dysplasia have reported overexpression of the tumour suppressors p14 and p16 - and absence of p53 - in high-risk human papilloma virus (HPV)- associated lesions. In skin carcinogenesis, the relation between these tumour suppressors and HPV remain unclear. We evaluated the expression of the tumour suppressors p14, p16 and p53 in pre-malignant and malignant squamous skin tumours, and its relation with risk factors for skin carcinogenesis (HPV, immune status and sun exposure). We performed immunohistochemical stainings for p14, p16 and p53 on paraffin embedded material of 71 pre-malignant squamous skin lesions and 34 squamous cell carcinomas, from 52 renal transplant recipients (RTRs) and 53 immunocompetent individuals. PCR-based assays were used for detection and genotyping of beta-papilloma virus (beta-PV) types and mucosal HPV types. P14 expression was independent of the expression of p16 and p53, irrespective of immune status and skin site. In 49 of 105 specimens (46.6%), one or more beta-PV types were detected. We found no significant association between p14, p16 or p53 protein expression and overall presence of beta-PV, irrespective of immune status. There was a significant association between presence of beta-PV and lesions from sun-exposed skin sites in the RTRs (P = 0.002). We conclude that in skin carcinogenesis, relations between the herein studied tumour suppressors and HPV are different from what one would expect based on findings in cervical neoplasia. P14, p16 and p53 expressions are independent of immune status. Our data indicate that in immunosuppressed patients, beta-PV together with ultraviolet radiation act synergetic in promoting carcinogenesis.

A panel of p16(INK4A), MIB1 and p53 proteins can distinguish between the 2 pathways leading to vulvar squamous cell carcinoma.

Two pathways leading to vulvar squamous cell carcinoma (SCC) exist. The expression of proliferation- and cell-cycle-related biomarkers and the presence of high-risk (hr) HPV might be helpful to distinguish the premalignancies in both pathways. Seventy-five differentiated vulvar intra-epithelial neoplasia (VIN)-lesions with adjacent SCC and 45 usual VIN-lesions (32 solitary and 13 with adjacent SCC) were selected, and tested for hr-HPV DNA, using a broad-spectrum HPV detection/genotyping assay (SPF(10)-LiPA), and the immunohistochemical expression of MIB1, p16(INK4A) and p53. All differentiated VIN-lesions were hr-HPV- and p16-negative and in 96% MIB1-expression was confined to the parabasal layers. Eighty-four percent exhibited high p53 labeling indices, sometimes with parabasal extension. Eighty percent of all usual VIN-lesions were hr-HPV-positive, p16-positive, MIB1-positive and p53-negative. Five (of seven) HPV-negative usual VIN lesions, had an expression pattern like the other HPV-positive usual VIN lesions. In conclusion, both pathways leading to vulvar SCC have their own immunohistochemical profile, which can be used to distinguish the 2 types of VIN, but cannot explain differences in malignant potential.

Recurrent melanotic neuroectodermal tumor of infancy: a proposal for treatment protocol with surgery and adjuvant chemotherapy.

The case of a 4-month-old male infant treated with combined surgery and chemotherapy for an aggressive recurrent melanotic neuroectodermal tumor of infancy (MNTI) on the top of the alveolar process of the mandible with a long-term follow-up is presented. Initial treatment comprised conservative local excision and curettage of the mandible. After several local recurrences and because radical surgical excision would give gross functional and aesthetic mutilation, finally complete, long-lasting remission was achieved with adjuvant chemotherapy, according to a neuroblastoma protocol (10-year follow-up). The reason for this protocol was because molecular genetic studies of this tumor showed loss of heterozygosity of chromosome 1p and gain of chromosome 7q analogue to neuroblastomas. A combination of surgery and chemotherapy should be the preferred treatment in case of a recurrence MNTI because optimal functional and aesthetic outcome.

Improved identification of malignant cells in serous effusions using a small, robust panel of antibodies on paraffin-embedded cell suspensions.

OBJECTIVE: To evaluate the expression of these markers individually and to find out which markers would be the most effective in a diagnostic panel to reliably discriminate these lesions. STUDY DESIGN: Sections from cell blocks of these fluids were stained with antibodies against calretinin, EMA, HMFG-2, BerEp4, B72.3 and CEA. A preliminary diagnosis was formulated based on cytomorphologic criteria. Subsequently, results of all 6 immunocytochemical stainings were evaluated, the most effective diagnostic pane of antibodies was proposed and staining results using this panel were compared to results obtained by solely cytomorphologic evaluation and to the ultimate diagnosis. RESULTS: Additional immunocytochemical staining with the proposed panel of calretinin, EMA, HMFG-2 and CEA improved sensitivity for malignancy in general from 78% to 96% and specificity from 73% to 91%. Sensitivity for malignant mesothelioma increased from 45% to 91%, with an increase in specificity from 87% to 96%. Sensitivity for adenocarcinoma decreased slightly from 100% to 92%, but specificity increased from 86% to 100%. Overall, diagnostic accuracy increased from 76% to 94%. CONCLUSION: Immunocytochemical staining of standardized cell block reparations of serous fluid cells with a small panel of 4 antibodies significantly improves diagnostic results compared to cytomorphologic evaluation alone.

Objective assessment of cancer biomarkers using semi-rare event detection.

Objective and reproducible assessment of cancer biomarkers may be performed using rare event detection systems. Because many biomarkers are not true 'rare events', in this study a semi-rare event detection system was developed. The system is capable of assigning a discriminant score to detected positive cells, expressing the extent and intensity of the immunocytochemical staining. A gallery image is constructed showing the diagnostically most interesting cells as well as quantitative data expressing the biomarker staining pattern. To increase scanning speed, an adaptive scanning strategy is studied in which scanning is aborted when a sufficient number of positive cells has been identified. System performance was evaluated using liquid based cervical smears, stained with an antibody directed against p16(INK4a) tumor suppressor protein. Overexpression of p16(INK4a) in cervix is related to high-risk HPV infection, which is associated with carcinogenesis. Reproducibility of the system was tested on specimens containing limited positivity. Quantitative analysis was evaluated using 10 cases within normal limits and 10 high grade lesions. The system was highly reproducible in detecting positive cells and in calculating discriminant scores (average CV 0.7%). Quantitative features were significantly increased in high grade lesions (p<0.001). Adaptive scanning decreased scanning time with only minor impact on scanning results. The system is capable of automated, objective and reproducible assessment of biomarker expression and may be useful for a variety of applications.

Two-piece implants with turned versus microtextured collars.

Implant companies have been promoting two-piece implants with microtextured collars in the interest of hard tissue preservation and/or soft tissue integration. However, this rationale may not be justified. Based on comparative studies currently available, it is unclear whether microroughened implant necks reduce crestal bone loss. A possible effect may be overruled by the establishment of a biologic width or by other factors influencing crestal bone remodeling. In addition, the orientation and attachment of the collagen fibers in the peri-implant mucosa are a little different because the surface roughness varies at the level of the implant neck. The clinician should be cautious when using these modified implants because the impact of microtextured collars on the initiation and progression of peri-implant pathology is currently unknown.

Multiple complications due to osteoradionecrosis in a patient with thromboangiitis obliterans.

A case is presented of a patient with thromboangiitis obliterans (TAO) who developed severe necrosis of the intraoral soft tissues and maxillary and mandibular bone after radiotherapy for a cT2N0M0 squamous cell carcinoma of the soft palate. Multiple surgical procedures including partial resection of the mandible and maxilla and reconstruction of intraoral and extraoral defects with a pectoralis major myocutaneous flap and anterolateral thigh flap were performed with partial success. Although a causal relationship between TAO and the described complications cannot be verified, close monitoring of patients with TAO who are being treated with radiotherapy is advised.

MIB1 expression in basal cell layer: a diagnostic tool to identify premalignancies of the vulva.

Lichen sclerosus, high-grade usual vulvar intraepithelial neoplasia (VIN) and differentiated VIN have a different malignant potential. The objective of this study was to quantify the proliferative activity in the basal region of the epithelium of vulvar premalignancies. Furthermore, we investigated whether MIB1 expression in the basal region of vulvar epithelium can be helpful in diagnosing differentiated VIN, which may be hard to discern from normal epithelium. MIB1 was used to immunohistochemically visualise proliferating cells within formalin-fixed, paraffin-embedded, archival tissue sections of different vulvar premalignancies (N=48) and normal vulvar epithelium (N=16). Automatic digital image analysis software was developed to quantify the proliferating fraction in different parts of the epithelium (MIB1 positivity index). MIB1 expression differed among the various vulvar premalignancies; a MIB1-negative basal cell layer was a distinct feature of normal vulvar epithelium. No MIB1-negative basal cell layer was noted in differentiated VIN or other vulvar premalignancies. Owing to this negative cell layer, the MIB1 proliferation index in normal vulvar epithelium was significantly lower than in vulvar premalignancies. In conclusion, MIB1 expression can be a helpful tool in diagnosing a premalignancy and has additional value especially to distinguish differentiated VIN neoplasia from normal vulvar epithelium, but cannot explain the differences in malignant potential.

The prognostic value of endogenous hypoxia-related markers for head and neck squamous cell carcinomas treated with ARCON.

BACKGROUND AND PURPOSE: Hypoxic radioresistance is an important cause for treatment failure in a number of tumor types including head and neck cancers. Recent studies suggest that outcome can be improved by oxygenation modifying treatments such as ARCON. A robust endogenous marker of hypoxia might be a valuable aid to select patients for such treatments. The aim of this investigation was to study associations between the putative endogenous hypoxia markers CA-IX, Glut-1 and Glut-3 and clinical tumor and patient characteristics and to evaluate the prognostic value of these markers. PATIENTS AND METHODS: Tumor biopsies from 58 patients treated with ARCON in a phase II trial were included. Tumor sections were immunohistochemically stained for CA-IX, Glut-1 and Glut-3. Sections were scored for relative tumor area stained by the markers (CA-IX and Glut-3) and for intensity of staining (Glut-1 and Glut-3). Further, sections were stained for CD34, an endothelial marker to assess microvascular density. RESULTS: Staining of CA-IX and Glut-3 was observed at some distance from vessels and adjacent to necrosis. Glut-1 staining was generally very diffuse. The distribution of clinical characteristics was equal between tumors with high and low marker expression. Significant differences were found for locoregional control (P = 0.04) and for freedom of distant metastases (P = 0.02) in favour of patients with high CA-IX positivity (>25% of tumor area). High Glut-3 expression was associated with a better locoregional control (P = 0.04). Higher Glut-1 intensity was associated with an increased rate of distant metastases (P = 0.0005) and a worse overall survival (P = 0.001). CONCLUSIONS: The inconsistent associations with outcome of CA-IX and the glucose transporters indicate that different factors play a role in up-regulation of these markers. Compared to studies with conventional treatment, the correlation between CA-IX expression and Glut-3 expression and outcome was reversed after treatment with ARCON. This does not support the potential of any of these proteins as very specific and robust hypoxia markers.

Treatment results of patients with a squamous cell carcinoma of the buccal mucosa.

The purpose of this study was to evaluate outcome and value of follow-up of squamous cell carcinoma (SCC) of the buccal mucosa in patients treated at the Radboud University Nijmegen Medical Centre, The Netherlands. A longitudinal cohort study was performed involving 32 patients treated with curative intent (surgery on indication followed by radiotherapy) for SCC of the buccal mucosa from 1987 to 2002, with a minimum follow-up of three years. The prognosis of SCC of the buccal mucosa is comparable to other sites of the oral cavity. The success rate of therapy for a local and/or regional recurrence is very limited. Patients with a second primary tumour (SPT) can be cured if the tumour is detected in an early stage. Routine follow-up used to detect local recurrence or SPT has almost no value after five years and seems of limited value after three years.

p14ARF and p16INK4A, two products of the same gene, are differently expressed in cervical intraepithelial neoplasia.

OBJECTIVE: To study the expression patterns of two different tumor suppressor proteins p16INK4A and p14ARF in cervical lesions. Both proteins are encoded by the same INK4A/ARF gene on chromosome 9p21. The expression patterns of these two proteins, both playing a central role in the cell cycle, were analyzed in detail in CIN, carcinomas, and normal epithelium to test the hypothesis that p16INK4A positive cells also demonstrate p14ARF expression. METHODS: Serial tissue sections of 9 CIN1 lesions, 10 CIN2 lesions, 12 CIN3 lesions, and 7 carcinomas were stained with monoclonal antibodies against p16INK4A and p14ARF. The short fragment polymerase chain reaction hybridization line probe assay was used to detect HPV. RESULTS: Normal epithelium was negative for both proteins. Marked immunoreactivity (++) for p16INK4A and p14ARF was observed in 5/7 carcinomas, 10/12 CIN3, and 1/10 CIN2 lesions and 0/9 CIN1 lesions. Simultaneous expression (+ or ++) was found in 19/22 CIN2/3 and not in CIN1 lesions. The fraction of p16INK4A-stained cells increased with CIN-grade. Overexpression of p14ARF was observed in a subpopulation of p16INK4A positive cells, and exclusively found in lesions infected with high-risk HPV. In two CIN3 lesions with early stromal invasion, p14ARF positivity was mainly found in the invasive cells. In carcinomas, all cells showed p16INK4A expression, whereas p14ARF was limited to the peripheral cells of the invasive tumor nests and individual migrating tumor cells. CONCLUSIONS: Overexpression of p14ARF is limited to a fraction of the p16INK4A-expressing cells and therefore it is likely that p14ARF- and p16INK4A expression are not induced by the same mechanisms. Before expression of p14ARF can be linked to invasion or invasive phenotype, larger series of (micro-) invasive squamous lesions need to be studied.

Effectiveness of routine follow-up of patients treated for T1-2N0 oral squamous cell carcinomas of the floor of mouth and tongue.

BACKGROUND: The duration of follow-up after treatment for head and neck cancer, the depth of the routine visits, and the diagnostic tools used are determined on the basis of common acceptance rather than evidence-based practice. Patients with early-stage tumors are more likely to benefit from follow-up programs, because they have the best chance for a second curative treatment after recurrence. The purpose of this study was to determine the benefit of our 10-year follow-up program in patients with stage I and II squamous cell carcinoma (SCC) of the floor of mouth and tongue. METHODS: In a longitudinal cohort study involving 102 patients who were treated with curative intent for a pT1-2N0M0 SCC of the floor of mouth and tongue from 1989-1998 with a minimum follow-up of 5 years, we evaluated the effect of routine follow-up. RESULTS: During the follow-up (mean, 61 months; SD, 4 months), 10 patients had a recurrence, and 20 patients had a second primary tumor. No regional lymph node recurrences in the neck were detected. Location, T classification of the primary tumor, choice of therapy, or measure of tumor-free margins in the resection did not significantly affect the occurrence of a secondary event (p >or= .1). The secondary event was discovered during a patient-initiated visit for complaints in 14 patients and was found during routine follow-up visits in 16 patients. Only seven second primary tumors were detected after 60 months, four on routine follow-up and three on a self-initiated visit. The mean disease-free survival time after treatment of the secondary event was 72 months (SD, 17 months) in the "own initiative" group and 65 months (SD, 13 months) in the routine follow-up group; this difference was not statistically significant (p=.3). CONCLUSIONS: The effectiveness of a 10-year routine follow-up, even in patients with early-stage oral SCC, is very limited. These visits on routine basis can be stopped after 5 years.

Interleukin-12 has no effect on vascular density, perfusion, hypoxia and proliferation of an implanted human squamous cell carcinoma xenograft tumour despite up-regulation of ICAM-1.

BACKGROUND: Interleukin-12 is an anti-angiogenic and antitumor agent in many transplanted murine tumour models. In a previous clinical study in head and neck squamous cell carcinoma patients treated with rhIL-12 the tumour turned pale, after an initial reddening. The aim of this study was to investigate the effects of rmIL-12 on the vasculature, blood perfusion, hypoxia and proliferation of tumour cells in an implanted human head and neck squamous cell carcinoma xenograft tumour, with a relatively large diameter, in Balb/c nu/nu mice over time. MATERIALS AND METHODS: Established human squamous cell carcinoma xenograft tumours were intratumorally injected for 3 days with either 200 ng rmIL-12 or PBA. Mice were sacrificed at 4 different time points (between 8 hours and 8 days after the last injection), after administration of Pimonidazole, BrdUrd and Hoechst 33342. The tumour sections were quantitatively analysed with a semi-automatic method based on a computerised digital image analysis system, after immunohistochemical staining. RESULTS: Despite a faster and higher up-regulation of anti-mouse ICAM-1 in the IL-12-treated tumours, no significant differences in vascular density, perfusion fraction, hypoxic fraction and BrdUrd labelling index were detected between IL-12-treated tumour and control tumours. CONCLUSION: We suggest that the main reason why the observation made in humans could not be confirmed in this mice study is the combination of a lack of an intact immune system in the Balb/c nu/nu mice and a relatively large tumour with probably a lot of mature vessels.

Intratumoral recombinant human interleukin-12 administration in head and neck squamous cell carcinoma patients modifies locoregional lymph node architecture and induces natural killer cell infiltration in the primary tumor.

The objective of this study was to evaluate the histologic and immunohistopathologic effects of intratumorally given recombinant human interleukin-12 on the immune cells in the primary tumors and regional lymph nodes. Ten previously untreated patients with head and neck squamous cell carcinoma (HNSCC) were injected in the primary tumor twice to thrice, once weekly, at two dose levels of 100 or 300 ng/kg, before surgery. These patients were compared with 20 non-IL-12-treated control HNSCC patients. In the primary tumor, the number of CD56+ natural killer (NK) cells was increased in IL-12-treated patients compared with control patients. In some IL-12-treated patients, an impressive peritumoral invasion of CD20+ B cells was noticed. No differences were seen in the CD8+ or CD4+ T lymphocytes. Interestingly, major differences were apparent in the architecture of the enlarged lymph nodes of IL-12-treated patients; in particular, the distribution of B cells differed and fewer primary and secondary follicles with smaller germinal centers were observed. In addition, a decrease of dendritic cell lysosyme-associated membrane glycoprotein-positive cells in the paracortex was noted, resulting in a reduction of paracortical hyperplasia. In the lymph nodes, especially the CD56+ NK cells but also the CD8+ and CD4+ T lymphocytes, produced a high amount of IFN-gamma. Patients, irrespectively of IL-12 treatment, with a high number of CD56+ cells in the primary tumor had a better overall survival than those with a low number. In conclusion, after i.t. IL-12 treatment in HNSCC patients, the largest effect was seen on the NK cells, with a higher number in the primary tumor and a high IFN-gamma mRNA expression in the lymph nodes. Significant effects were noted on B cells, with altered lymph node architecture in every IL-12-treated patient and excessive peritumoral infiltration in some patients.

INK4-ARF and p53 mutations in metastatic cutaneous squamous cell carcinoma: case report and archival study on the use of Ink4a-ARF and p53 mutation analysis in identification of the corresponding primary tumor.

So far, histopathologic, immunohistochemical and molecular properties of metastatic cutaneous squamous cell carcinomas (CSCCs) are relatively unexplored. In patients with multiple CSCCs, as for instance renal transplant recipients (RTRs), it might prove difficult to identify the primary tumor responsible for metastasis. We report a case of an RTR with multiple CSCCs, one of which metastasized. By using p53 and INK4a-ARF mutation analysis, we identified the responsible primary tumor due to an identical mutation in exon 2 of the INK4a-ARF locus. Archival study yielded 14 cases of metastatic CSCC (present case included). In only 8 of 14 metastases, DNA quality was sufficient to perform PCR reactions. In 7 of 8 metastases, either an INK4a-ARF (6 of 8 cases) and/or p53 (3 of 8 cases) mutation was present. In 6 of 7 cases, the corresponding primary could be identified by an identical mutation in p53 and/or INK4a-ARF. In conclusion, molecular analysis using a combination of p53 and INK4a-ARF mutation analysis can identify the corresponding primary skin tumor in case of CSCC metastases in the majority of cases. This is facilitated by the high frequency of these mutations in metastatic CSCC when compared with frequency spectra reported in the literature in primary CSCCs. The major limitation was formed by insufficient DNA quality in archival tissue.

Quantitation of Ki-67 expression in the differential diagnosis of reserve cell hyperplasia vs. small cell lung carcinoma.

OBJECTIVE: To investigate whether the detection of proliferation-associated Ki-67 antigen may be of value in differentiating between reserve cell hyperplasia (RCH) and small cell lung cancer (SCLC). STUDY DESIGN: Retrospectively, 20 Papanicolaou-stained bronchial brushes or washings from 20 patients were selected. Ten were diagnosed as RCH (and had no SCLC in follow-up) and the other 10 as SCLC (histologically confirmed). All 20 Papanicolaou-stained slides were restained with the monoclonal antibody MIB1, directed against Ki-67 antigen; that simple and reliable procedure was described recently. In each specimen 5 coherent cell groups were identified, corresponding to RCH or SCLC, respectively; photographed; and studied for Ki-67 antigen expression after MIB1 staining of the slides. At least 3 cell groups remained in each specimen. The Ki-67 labeling index (LI) of the specimens was determined as the number of MIB1-positive cells divided by the total number of cells in the remaining cell groups. RESULTS: All cases of SCLC showed a mean Ki-67 LI of at least .415 (mean .684, SD .151), whereas in the cases with RCH the mean Ki-67 LI never was more than .158 (mean .048, SD .049). The difference was highly significant (P<.001, Student's t test). Linear discriminant analysis resulted in a classifier with which we were able to discriminate correctly between SCLC and RCH in 100% of the 20 bronchial brushings and washings. CONCLUSION: The results clearly demonstrate that measuring proliferative activity in Papanicolaou-stained bronchial brushings and washings by MIB1 restaining of the slides may be of great practical value in accurately discriminating RCH from SCLC. The method is simple and can be performed in any laboratory that is able to carry out immunocytochemical staining. However, an additional (prospective) study with a series of difficult cases is necessary to confirm these findings.