Exploration of clinical preferences in treatment planning of radiotherapy for prostate cancer using Pareto fronts and clinical grading analysis.

INTRODUCTION: Radiotherapy treatment planning is a multi-criteria problem. Any optimization of the process produces a set of mathematically optimal solutions. These optimal plans are considered mathematically equal, but they differ in terms of the trade-offs involved. Since the various objectives are conflicting, the choice of the best plan for treatment is dependent on the preferences of the radiation oncologists or the medical physicists (decision makers).We defined a clinically relevant area on a prostate Pareto front which better represented clinical preferences and determined if there were differences among radiation oncologists and medical physicists. METHODS AND MATERIALS: Pareto fronts of five localized prostate cancer patients were used to analyze and visualize the trade-off between the rectum sparing and the PTV under-dosage. Clinical preferences were evaluated with Clinical Grading Analysis by asking nine radiation oncologists and ten medical physicists to rate pairs of plans presented side by side. A choice of the optimal plan on the Pareto front was made by all decision makers. RESULTS: The plans in the central region of the Pareto front (1-4% PTV under-dosage) received the best evaluations. Radiation oncologists preferred the organ at risk (OAR) sparing region (2.5-4% PTV under-dosage) while medical physicists preferred better PTV coverage (1-2.5% PTV under-dosage). When the Pareto fronts were additionally presented to the decisions makers they systematically chose the plan in the trade-off region (0.5-1% PTV under-dosage). CONCLUSION: We determined a specific region on the Pareto front preferred by the radiation oncologists and medical physicists and found a difference between them.

Does hormone therapy, tibolone or raloxifene modify VEGF expression in cervical epithelial cells?

AIM: Vascular endothelial growth factor (VEGF) seems to be a critical molecule in cervical carcinogenesis. We aimed to investigate the possible associations between hormonal factors and VEGF expression in cervical epithelial cells from postmenopausal women. METHOD: A total of 105 healthy postmenopausal women (aged 45-68 years old) attending a university menopause clinic were enrolled in this cross-sectional study. Pap smears were derived from current users of 17ß-estradiol 1 mg + norethisterone acetate 0.5 mg (n = 28), tibolone 2.5 mg (n = 23), raloxifene HCl 60 mg (n = 21) and women not receiving treatment (n = 33). VEGF immunostaining was evaluated in squamous, glandular and metaplastic cells, using a semiquantitative method (rating scale: 0-3). RESULTS: Concerning endogenous hormones, higher Δ4-androstenedione levels were associated with more intense VEGF immunostaining in glandular (p = 0.041) and metaplastic cells (p = 0.004). Hormone therapy and raloxifene did not induce any changes in VEGF immunoreactivity in the examined cells. In contrast, tibolone administration was accompanied by diminished VEGF presence in metaplastic cells (p = 0.016 vs. controls). CONCLUSION: Our findings may in part reflect the molecular processes contributing to the safe profile of hormone therapy, tibolone and raloxifene in cervical carcinogenesis.

Odontogenic keratocyst expresses vascular endothelial growth factor: an immunohistochemical study.

BACKGROUND: Vascular endothelial growth factor (VEGF) expression may act as a sensitive measure of the angiogenic potential of a lesion. Furthermore, VEGF has been implicated in the pathogenesis of cystic tumors and inflammatory odontogenic cysts. Thus, we studied the expression of VEGF in the epithelium of odontogenic keratocyst (OK) in association with cell proliferation and apoptosis. METHODS: Forty-two cases of OK, 26 cases of dentigerous cyst (DC), and 15 cases of residual cyst (RC) were retrospectively examined by immunohistochemistry for VEGF, Ki67/Mib-1 and anti-caspase-3. For VEGF and caspase-3, the intensity of immunostaining was qualitatively assessed, while for the evaluation of Ki67 the average number of positively stained nuclei in 10 high-power microscopic fields (x 400) was calculated. RESULTS: The VEGF expression was stronger in OK when compared with DC (P < 0.007). The rate of nuclear Ki67 expression in OK was significantly higher than that in DC (P < 0.001) and RC (P < 0.001). Cytoplasmic caspase-3 expression was statistically more intense in RC than in OK (P = 0.001) or DC (P < 0.001). A statistically significant correlation was seen in OK for Ki67 (P < 0.001) and VEGF (P = 0.023), but not for caspase-3. Multiple regression analysis revealed a linear relationship between VEGF and Ki67. CONCLUSIONS: The VEGF was expressed in the epithelium of OK, DC, and RC with a variable intensity, and in OK VEGF expression was related to Ki67. It is suggested that VEGF expression by the odontogenic epithelium is not induced solely by inflammation.

Evaluation of combined h-TERT, bcl-2, and caspases 3 and 8 expression in cutaneous malignant melanoma based on tissue microarrays and computerized image analysis.

PURPOSE: Deregulation of apoptotic pathways in cutaneous malignant melanoma appears to be correlated with chemoresistance and poor prognosis. Furthermore, telomerase (especially h-TERT) expression induces proliferation and also represents a potential target for vaccination regarding some types of malignancies. MATERIALS AND METHODS: Using tissue microarrays (TMA) technology, 25 paraffin-embedded tissue samples of histologically confirmed malignant melanomas were cored at a diameter of 2 mm and re-embedded into one recipient block (final TMA density 24/25-96%). Immunohistochemistry (IHC) was performed by the use of anti-bcl-2, anti-caspase 3, anti-caspase 8 and anti- h-TERT antibodies. Protein expression levels were evaluated using a computerized image analysis system (CIA). SPSS (chi square test and inter-rater kappa) was used for statistical analysis. RESULTS: Strong protein expression was observed in 1/24 (4.1%), 1/24 (4.1%), 2/24 (8.2%), and 4/24 (16.4%) cases regarding h-TERT, caspase 3, caspase 8 and bcl-2, respectively. Moderate was observed in 7/24 (29.1%), 8/24 (32.2%), 5/24 (20.2%), and 8/24(32.2%) cases, whereas reduced or absent expression demonstrated 16/24 (65%), 15/24 (60.2%), 17/24 (68.5%), and 12/24 (50 %) cases. Statistical significance was assessed correlating age to caspase 3 (p=0.05), Breslow's thickness to telomerase (p=0.013) and to bcl-2 (p=0.053), Clark's level to telomerase (p=0.008) and to bcl-2 (p=0.022), and finally ulceration to telomerase expression (p=0.007). CONCLUSION: bcl-2 and telomerase expression are correlated to critical parameters of malignant melanoma, affecting its biological behavior. Furthermore, downregulation of proteins such as caspases 3/8, which normally induce apoptosis, is perhaps associated with resistance of the applied chemotherapeutic strategies in this type of malignancy.

Temperature and serum proinflammatory cytokine changes in patients with NSCLC after BAL.

We examined the effects of bronchoalveolar lavage (BAL) and BAL fluid characteristics on the systemic proinflammatory cytokine expression and their relation to clinical and laboratory findings. Thirty patients suspected to have lung cancer were subjected to fiber-optic bronchoscopy (FOB) and BAL. Clinical and laboratory findings were determined at baseline, 4 h, and 24 h, including lung auscultation, temperature, chest X-ray, WBC, neutrophils, and serum IL-1beta, IL-6, and TNF-alpha. BAL fluid characteristics were determined including cytokine levels. Fifteen volunteers served as controls to determine serum variation of the same cytokines. Significant temperature elevation was defined as 1 degrees C increase compared to baseline. BAL was associated with temperature and serum TNF-alpha and IL-6 but not IL-1beta increase at 4 h. Four patients (13.3%) developed temperature over 38 degrees C. In controls there were no significant changes between baseline and 24 h measurements for the same cytokines. Eleven patients (36.6%) developed a significant temperature elevation 4 h after BAL. These patients had a statistically significant ( p < 0.05) increase in serum IL-6 at 4 h and in TNF-alpha at both 4 and 24 h after BAL compared with the nonsignificant temperature increase group. BAL characteristics were not different between the two groups. On the other hand, BAL fluid IL-6 and TNF-alpha levels were significantly higher ( p < 0.05) in the nonfever group. Significant temperature increase was observed in 36.6% of the patients undergoing BAL and associated with significant serum TNF-alpha and IL-6 increase at 4 h. Lung cytokines levels, alveolar macrophages, and BAL fluid characteristics are not related to temperature and serum proinflammatory cytokine increase. The hypothesis of alveolar macrophages derive from cytokine production and shift to the systemic circulation cannot be supported by our data.

HER-2/neu overexpression in breast cancer: an immunohistochemical study including correlations with clinicopathologic parameters, p53 oncoprotein and cathepsin-D.

PURPOSE: To evaluate the relationship between HER-2/neu overexpression and standard as well as investigational prognostic factors in Greek females with invasive breast carcinoma. MATERIALS AND METHODS: Paraffin-embedded tumor sections from 128 consecutive primary breast cancer patients were screened for HER-2/neu oncoprotein (p185) overexpression by immunohistochemistry using the polyclonal antibody A 0485. The relationship between HER-2/neu staining and other established markers of prognosis were examined using both a univariate and a multivariate analysis. RESULTS: HER-2/neu overexpression was detected in 46.1% of tumors. No statistical correlation was found between HER-2/neu and age, tumor diameter, histologic type, nodal status, tumor grade, stage, estrogen and progesterone receptor status or cathepsin-D. A significant correlation was noted between HER-2/neu and p53 overexpression in the total sample (p=0.014) as well as in node-positive patients (p=0.026). In those groups, HER-2/neu and p53 co-expression was found in 19.5% and 21.6% of cases, respectively. In node-negative patients, HER-2/neu overexpression did not correlate with any other marker. CONCLUSION: HER-2/neu overexpression seemed to correlate only with p53 oncoprotein.

Additional characterization of a hexanucleotide polymorphic site in the first intron of human H-ras gene: comparative study of its alterations in non-small cell lung carcinomas and sporadic invasive breast carcinomas.

Intron 1 of the human H-ras gene possesses a polymorphism consisting of repetitions of the GGGCCT consensus. Three alleles have been reported at this locus. We confirmed that two, P1 and P2, display four and two repeats, respectively, with their internal sequence structure similar to that previously described. The third, P3, previously assigned as a three-unit repetition allele according to its electrophoretic mobility and with no other information regarding its internal structure, was also found. Sequence analysis of the P3 allele revealed that it consists of three perfect repeats of the GGGCCT consensus. This polymorphism is present only in human c-H-ras gene, although single hexanucleotide repeats are found scattered within intron 1 of this gene in rodents. Analysis of this locus in matched tumor/distant normal samples from: (i) 38 patients with non-small-cell lung carcinoma (NSCLC), and (ii) 35 patients with sporadic invasive breast carcinoma, revealed: (1) 6.6% and 19% loss of heterozygosity (LOH) respectively, and (2) 10.5% and 2.9% hexanucleotide instability (HI) respectively, detected by the presence of shifted in length alleles. Shifted alleles exhibited altered internal sequence structure in comparison to normal ones, suggesting complex mutational events. The same pattern of alterations was also detected in tissues adjacent to lung adenocarcinomas and dysplasias adjacent to squamous cell carcinomas (7.7% LOH, 5.9% HI), implying that abnormalities at this locus may be early events in lung carcinogenesis. The frequency of alterations (LOH vs. HI) was significantly different among NSCLC and breast cancer (P=.005), probably due to the different tumor biology of each system. Finally, altered mRNA expression of H-ras gene was detected in all cases with HI, but this finding was also observed in samples without HI. In view of reports showing that elements in intron 1 of H-ras gene potentially influence its transcriptional regulation, from our results we cannot exclude that the hexanucleotide locus could be an element with possible involvement in expressional regulation of this gene.

"High risk" HPV types are frequently detected in potentially malignant and malignant oral lesions, but not in normal oral mucosa.

Studies on the involvement of the human papillomavirus (HPV) in initiation and progression of oral neoplasia have generated conflicting results. The observed discrepancy is attributable mainly to the varying sensitivity of the applied methodologies and to epidemiologic factors of the examined patient groups. To evaluate the role of HPV in oral carcinogenesis, we analyzed 53 potentially neoplastic and neoplastic oral lesions consisting of 29 cases of hyperplasia, 5 cases of dysplasia, and 19 cases of squamous cell carcinomas, as well as 16 oral specimens derived from healthy individuals. A highly sensitive nested polymerase chain reaction (PCR) assay was used, along with type-specific PCR, restriction fragment length polymorphism analysis, dot blotting, and nonisotopic in situ hybridization. Nested PCR revealed the presence of HPV DNA in 48 of the 53 (91%) pathologic samples analyzed, whereas none (0%) of the normal specimens was found to be infected. Positivity for HPV was independent of histology and the smoking habits of the analyzed group of patients. At least one "high risk" type, such as HPV 16, 18, and 33, was detected by type-specific PCR in 47 (98%) infected specimens, whereas only 1 (2%) squamous cell carcinoma was solely infected by a "low risk" type (HPV 6). HPV 16 was the prevailing viral type, being present in 71% of infected cases. Single HPV 16 and HPV 18 infections were confirmed by restriction fragment length polymorphism. HPV 58 was detected by dot blotting in three hyperplastic lesions. HPV positivity and genotyping were further confirmed, and the physical status of this virus was evaluated by nonisotopic in situ hybridization. Diffuse and punctate signals, indicative of the episomal and integrative pattern of HPV infection, were observed for low- and high-risk types, respectively. Our findings are suggestive of an early involvement of high-risk HPV types in oral carcinogenesis.

Double partial nephrectomy in a patient with two adenocarcinomas in a solitary kidney.

We present a 67 year-old female patient with two synchronous adenocarcinomas in a solitary kidney. She underwent thorough clinical and radiographic evaluation. Selective angiography proved to be the most reliable examination in preoperative diagnosis. Double partial nephrectomy was performed and the patient has normal renal function and no evidence of recurrence 27 months postoperatively. Partial nephrectomy in patients with one tumor in a solitary kidney is well documented, though we believe that partial nephrectomy can also be performed in selected cases with multifocal tumors.

Human papilloma virus (HPV) is possibly involved in laryngeal but not in lung carcinogenesis.

Data on human papilloma virus (HPV) involvement in preneoplastic and neoplastic lesions of the larynx and lung are limited and conflicting. The presence of HPV was investigated in a series of laryngeal specimens and non-small cell lung carcinomas (NSCLCs). The laryngeal samples (154) comprised 14 cases with hyperplasia without dysplasia, 49 with dysplasia, and 91 squamous cell carcinomas (SqCCs). The NSCLCs included 31 SqCCs, 32 adenocarcinomas, and 5 undifferentiated large cell carcinomas. Furthermore, we examined, for HPV DNA sequences, 14 bronchial metaplastic squamous lesions located next to cancerous areas. We used a sensitive nested polymerase chain reaction assay (NPCR), dot blotting, and in situ hybridization. The findings were correlated with clinicopathologic features of the patients. In the laryngeal specimens, NPCR analysis showed HPV DNA in 20 (13%) of the 154 specimens. Notably, 19 of 20 HPV-positive cases were carcinomas and only one was a mild dysplastic lesion. Typing of the carcinomas showed single HPV 6, 16, 18, and 33 infection in 1 (1.1%), 12 (13.2%), 2 (2.2%), and 1 (1.1%) samples, respectively, and HPV 6/33, 16/33, and 6/18 coinfection in three carcinomas. In situ hybridization findings were in agreement with PCR results, with the exception of two cases in which HPV 18 DNA was detected only by PCR. HPV was more frequently observed in heavy smokers than in patients with low daily cigarette consumption and nonsmokers (P = .03). There was no correlation between virus infection and gender, grade, and lymph node status of the carcinomas. None of the NSCLCs or adjacent metaplastic squamous epithelium contained HPV DNA sequences. The presented data suggest a contributory role of HPV in late stages of laryngeal carcinogenesis, because all premalignant lesions were negative but one. This study does not support a potential role of HPV in the development of NSCLCs.

Alterations of the p16-pRb pathway and the chromosome locus 9p21-22 in non-small-cell lung carcinomas: relationship with p53 and MDM2 protein expression.

The p16-pRb and p53-MDM2 pathways represent vital cell cycle checkpoints. Recent studies provide evidence that these pathways are directly linked via MDM2-pRb interaction and p53 suppression of the RB1 gene. In the present study we investigated the alterations of this G1 phase protein network using immunohistochemical and molecular methods in a series of 68 non-small-cell lung carcinomas (NSCLCs) and correlated the findings with clinicopathological features and prognosis of the patients. Aberrant expression (Ab) of p16 and pRb was observed in 33 (49%) and 27 (40%) of the carcinomas, respectively. Analysis of the region that encodes for p16 by deletion mapping, a polymerase chain reaction (PCR)-based methylation assay and PCR single-strand conformation polymorphism (SSCP) analysis revealed that deletions and transcriptional silencing by methylation might represent the main mechanisms of CDKN2/p16ink4a inactivation in NSCLCs. The results of deletion mapping also suggest that other tumor suppressor genes may reside at the 9p21-22 region, which encodes for CDKN2/MTS1/p16ink4a, p14ARF, and MTS2/p15ink4b. In addition, microsatellite instability was observed with a frequency of 16% in the 9p21-22 chromosome area. Overexpression (P) of p53 and MDM2 proteins was found in 39 (58%) and 47 (70%) of the cases, respectively. A highly significant association was observed between p53 overexpression and p53 mutations (P = 0.006). Statistical analysis of the expression patterns of the biologically relevant molecules (p16/pRb, p53/MDM2, MDM2/pRb, and p53/pRb) showed coincident overexpression of p53 and MDM2 (P = 0.04) and that abnormal pRb was correlated with elevated levels of MDM2 (P = 0.013) and p53 (P = 0.01), respectively. We suggest that deregulated expression of these molecules may act synergistically. An important finding of the study was that multiple impairments (three and four molecules affected) of the p16/pRb/p53/MDM2 network occurred in a large proportion (43%) of the carcinomas. This finding in addition to the absence of correlation with clinical stage of the tumors suggests that multiple hits of this network may be a relatively early event in the development of a subset of NSCLCs. The relationship between the factors examined in the present study, clinicopathological features, and survival of the patients did not reveal any significant correlations with the exception of smoking, which was associated with microsatellite alterations (loss of heterozygosity and microsatellite instability) at the 9p21-22 locus (P = 0.04) and the immunophenotypes p53(P)/MDM2(P) (P = 0.04) and p16(Ab)/pRb(Ab)/p53(P)/MDM2(P) (P = 0.03), respectively. We suggest that in a subset of NSCLCs, simultaneous deregulation of the members of this network may represent one way of initiating the oncogenic procedure whereas in other NSCLC subgroups alternative pathways may play this role.

Angiomyolipoma of the left ureterovesical junction associated with ipsilateral renal agenesis.

To our knowledge we report the first case of retroperitoneal angiomyolipoma located at the left ureterovesical junction associated with ipsilateral renal agenesis. Radiologically, the mass had the characteristics of a cystic soft tissue tumor. Final diagnosis was determined by histopathological examination, which revealed the specific characteristics of angiomyolipoma. The tumor is theorized to be a result of developmental malformation of fetal mesenchymal elements along the ureteral bud during fetal development since it was associated with ipsilateral renal agenesis.

Clonal evolution of an immunoblastic type non-Hodgkin's lymphoma with der(6)t(1;6)(q11;p11) as its primary cytogenetic abnormality.

Recurrent pleural effusions from a 45-year-old man who was diagnosed as having non-Hodgkin's lymphoma of immunoblastic type were studied cytogenetically. The majority of the metaphases were tetraploid, but there were also lymphoma cells observed with pseudodiploid chromosome constitutions. Cytogenetic analysis by G-banding revealed the existence of at least two cell populations. The karyotype of the minor pseudodiploid clone, which exhibited partial trisomy of 1q11qter and monosomy of 6p11pter as sole abnormalities, was 46,XY,der(6)t(1;6)(q11;p11). The karyotype of the major clone was 92,XXYY,-1,der(6)t(1;6)(q11;p11)x2, +9. The ancestral diploid clone, carrier of the balanced translocation involving chromosomes 1 and 6, was not observed even in the first pleural effusion harvest. The high proportion of tetraploid cells in the recurrent effusions was an indication that these cells were favorably selected in the environment of the somatic cavity. Our cytogenetic findings suggest that partial trisomy of 1q may be a crucial secondary chromosomal abnormality in highly malignant non-Hodgkin's lymphoma. This genetic imbalance was predetermined from the primary abnormality and may be responsible for further tumor progression, as suggested from the clonal evolution in this particular case and, therefore, may be associated with the aggressive biologic behavior of malignant cells.