Igor Sirák

High-dose rate brachytherapy in the treatment of penile carcinoma--first experience.

PURPOSE Interstitial low-dose rate brachytherapy (BRT) allows a conservative treatment of T1-T2 penile carcinoma. High-dose rate (HDR) BRT is often considered as a dangerous method for interstitial implants because of higher risk of complications. However, numerous reports suggest that results of HDR-BRT may be comparable to low-dose rate BRT. There are no data available in the literature regarding HDR interstitial BRT for carcinoma of the penis. METHODS AND MATERIALS Ten patients with early penile carcinoma were treated by interstitial hyperfractionated HDR-BRT at the dose of 18 times 3Gy twice daily between years 2002 and 2009. Breast interstitial BRT template was used for fixation and precise geometry reconstruction of stainless hollow needles. RESULTS Median followup was 20 months. Our BRT technique and fractionation schedule was well tolerated by all patients. Acute reaction consisted predominantly of penis edema and Grade 2 radiation mucositis that dissolved during 8 weeks after the treatment. We neither observed any postradiation necrosis nor urethral stenosis. The worst late side effects recorded were mild telanagiectasias in the treatment region. At the last followup, all patients were alive without evidence of the tumor and with fully functional organ. CONCLUSIONS Hyperfractionated interstitial HDR-BRT with 18 times 3 Gy per fraction twice daily is a promising method in selected patients of penile carcinoma and deserves further evaluation in a larger prospective study.

Epidermal Growth Factor Receptor as a Predictor of Tumor Response to Preoperative Chemoradiation in Locally Advanced Gastric Carcinoma

Purpose:The purpose of our study was a retrospective evaluation whether the intensity of epidermal growth factor receptor (EGFR) expression predicts tumor response to preoperative chemoradiotherapy in patients with locally advanced gastric carcinoma.Patients and Methods:Thirty-six patients with gastric adenocarcinoma (cT2–4 or N+) were studied. Preoperative treatment consisted of 30–45 Gy of gastric irradiation with continuous 5-fluorouracil and weekly cisplatin. Surgical resection was performed 4–6 weeks later. EGFR expression in pretreatment tumor biopsies was assessed by immunohistochemistry. Level of EGFR expression was determined from the intensity and extent of staining. Tumor response was defined as a reduction of at least one T-stage level and/or finding of intense tumor regression in histopathologic examination.Results:Seventeen patients responded to preoperative chemoradiation – 8 patients (22%) had pathologic complete response, 9 patients (25%) were downstaged. Positive EGFR expression was found in 8 tumors (22%), and represented a significant predictive marker of poor tumor response in multivariate logistic regression analysis (p = 0.015). Response to chemoradiotherapy was found in 60% (16/28) of EGFR negative patients and in 13% (1/8) of EGFR positive patients (p = 0.044). None of the eight EGFR positive patients achieved pathologic complete response in comparison with 8/28 (29%) of patients with EGFR negative staining (p = 0.16).Conclusion:EGFR may represent a molecular marker predictive for poor response to preoperative chemoradiotherapy in locally advanced gastric carcinoma.Ziel:Ziel dieser Studie war eine retrospektive Evaluierung, ob die Expressionsintensität des epidermalen Wachstumsfaktor-Rezeptors (EGFR) die Tumorantwort auf präoperative Chemoradiotherapie vorhersagt bei Patienten mit lokal fortgeschrittenem Magenkrebs.Patienten und Methodik:36 Patienten mit Adenokarzinom des Magens (cT2–4 oder N+) wurden untersucht. Die präoperative Behandlung bestand aus einer Bestrahlung des Magens mit 30–45 Gy mit fortlaufendem 5-Fluorouracil und wöchentlichem Cisplatin. Die operative Resektion wurde 4–6 Wochen später durchgeführt. Die Expression des EGFR in den vorbehandelten Biopsiepräparaten des Tumors wurde mittels Immunhistochemie gemessen. Die Höhe der EGFR-Expression wurde festgelegt nach der Intensität und dem Ausmaß der Färbung. Das Downstaging wurde definiert als eine Reduktion von mindestens einem T-Stadium und/oder einem Befund von Tumorregression in der histopathologischen Untersuchung.Ergebnisse:17 Patienten haben auf die präoperative Chemoradiotherapie angesprochen – 8 Patienten (22%) hatten eine pathologisch komplette Reaktion, 9 Patienten (25%) wurden „downgestaged“. Eine positive EGFR-Expression wurde in 8 Tumoren gefunden (22%), und stellte einen signifikanten Vorhersagefaktor dar für eine geringe Tumorantwort in multivariater logistischer Regressionsanalyse (p = 0,015). Eine Reaktion auf die Chemoradiotherapie wurde bei 60% (16/28) der EGFR-negativen Patienten festgestellt und bei 13% (1/8) der EGFR-positiven Patienten (p = 0,044). Keiner der 8 EGFR-positiven Patienten erreichte eine pathologisch komplette Reaktion im Vergleich zu 8/28 (29%) der Patienten mit EGFR-negativer Färbung (p = 0,16).Schlussfolgerung:Der EGFR stellt einen Vorhersagefaktor für eine geringe Reaktion auf die präoperative Chemoradiotherapie beim lokal fortgeschrittenen Magenkarzinom dar.

Carrier Molecules and Extraction of Circulating Tumor DNA for Next Generation Sequencing in Colorectal Cancer

The aims of the study were: i) to compare circulating tumor DNA (ctDNA) yields obtained by different manual extraction procedures, ii) to evaluate the addition of various carrier molecules into the plasma to improve ctDNA extraction recovery, and iii) to use next generation sequencing (NGS) technology to analyze KRAS, BRAF, and NRAS somatic mutations in ctDNA from patients with metastatic colorectal cancer. Venous blood was obtained from patients who suffered from metastatic colorectal carcinoma. For plasma ctDNA extraction, the following carriers were tested: carrier RNA, polyadenylic acid, glycogen, linear acrylamide, yeast tRNA, salmon sperm DNA, and herring sperm DNA. Each extract was characterized by quantitative real-time PCR and next generation sequencing. The addition of polyadenylic acid had a significant positive effect on the amount of ctDNA eluted. The sequencing data revealed five cases of ctDNA mutated in KRAS and one patient with a BRAF mutation. An agreement of 86% was found between tumor tissues and ctDNA. Testing somatic mutations in ctDNA seems to be a promising tool to monitor dynamically changing genotypes of tumor cells circulating in the body. The optimized process of ctDNA extraction should help to obtain more reliable sequencing data in patients with metastatic colorectal cancer.

High-dose-rate brachytherapy in early oral cancer with close or positive margins

PURPOSE Retrospective evaluation of high-dose-rate brachytherapy (HDR BT) in early oral cancer and factors influencing tumor control. METHODS AND MATERIALS A total of 30 patients with T1-T3N0 tongue and floor of mouth cancer were treated with tumor excision±elective neck dissection and HDR BT 18×3 Gy b.i.d. The Kaplan-Meier model was used for survival analyses, and the log-rank test and Cox regression analyses were used to evaluate the influence of T-stage, histologic grade, resection margin, depth of invasion, and vascular endothelial growth factor (VEGF) intensity on local control (LC), nodal control (NC), disease-free survival (DFS), and overall survival (OS). Median followup was 40 months (6-145). RESULTS Actuarial 3-year LC, NC, DFS, DFS after salvage treatment, and OS were 85.4%, 69.2%, 65.4%, 75.6%, and 73.0%, respectively. The log-rank test and univariate Cox regression analysis revealed the following correlations, namely tumor grade correlated with LC, DFS, and OS; T-stage with NC and DFS; depth of invasion and VEGF intensity with NC, DFS, and OS. Associations detected on the multivariate analysis were as follows: tumor grade with LC, depth of invasion with NC, depth of invasion and tumor grade with DFS, and VEGF intensity with DFS after salvage treatment. Only one case of osteoradionecrosis and two cases of soft tissue necrosis occurred. CONSLUSION The HDR BT 18×3 Gy b.i.d. is a safe treatment of early oral cancer with a good LC. The T-stage, tumor grade, depth of invasion, and intensity of VEGF were significant predictors of locoregional control.

Daily prostate volume anD position monitoring using implanteD golD markers anD on-boarD imaging During raDiotherapy

PURPOSE This study aimed to evaluate prostate volume changes and prostate motions during radiotherapy. METHODS In 2010, twenty-five patients were treated for prostate cancer by external beam radiotherapy with implanted fiducial markers. Coordinates of three gold markers on kilovoltage images were calculated daily. Volume changes in target structure were observed through changes in intermarker distances. Differences in patient position between laser-tattoo alignment and gold marker localization were evaluated. Intrafraction motion was assessed by measuring marker displacement on kilovoltage images acquired before and after fraction delivery. RESULTS Prostate shrinkage was observed in 60% of patients. The average shrinkage was 7% of the prostates initial volume. Corrections after laser-tattoo alignment remained mostly below 1 cm. The difference between marker centroid position on the actual images and the planning images was 2 +/- 1 mm on average. The extension of intrafraction movements was 7.6 +/- 0.2 mm on average. CONCLUSIONS In our retrospective study, the possibility for prostate volume changes during radiotherapy was revealed. Intrafraction movements turned out to be the limiting factor in safety margin reduction.

Preoperative neoadjuvant chemoradiation for locally advanced gastric adenocarcinoma

Abstract Aims and Background To evaluate toxicity and the radical resection rate in gastric adenocarcinoma treated with preoperative neoadjuvant chemoradiation. Materials & Methods 32 patients, 22 males and 10 females with gastric adenocarcinoma, were treated with chemoradiation and hyperthermia. Results The neoadjuvant regimen was completed as planned in 19/32 (59 %) patients; in the remaining patients the intensity of chemotherapy had to be reduced because of haematological and gastrointestinal toxicity. Surgical stage was as follows: 2 patients pathologically complete response, 3 patients AJCC stage I.A, 5 patients stage I.B, 7 patients stage II, 7 patients stage III.A, 1 patient stage III.B, 7 patients stage IV. R0 resection was achieved in 19/32 (59%) patients, R1 in 2/32 (6%) patients and R2 in 11 (34%) patients. Downstaging after neoadjuvant chemoradiotherapy was achieved in 17/32 (53%) patients. At the date of evaluation (31 March 2009), 4 patients were still alive 58, 81, 86 and 98 months from the date of diagnosis. Median survival was 18 months (95% confidence interval: 13–38 months). One-year survival was 69% (95% confidence interval: 53%–85%). Four-year survival was 19% (95% C.I.: 5%–34%). Conclusions Preoperative neoadjuvant chemoradiotherapy has acceptable toxicity, and can lead to a high rate of R0 resections.

SMARCB1/INI1-deficient sinonasal carcinoma shows methylation of RASSF1 gene: A clinicopathological, immunohistochemical and molecular genetic study of a recently described entity.

The aim of the study was detailed clinicopathological investigation of SMARCB1/INI1-deficient sinonasal carcinomas, including molecular genetic analysis of mutational status and DNA methylation of selected protooncogenes and tumor suppressor genes by means of next generation sequencing (NGS) and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). A total of 4/56 (7%) cases of SMARCB1/INI1-deficient carcinomas were detected among 56 sinonasal carcinomas diagnosed over a 19year period using immunohistochemical screening. The series comprised 3 males and 1 female, aged 27-76 years (median 64 years). All tumors arose in the nasal cavity. Three neoplasms were diagnosed in advanced stage pT4. During the follow-up period (range 14-111 months (median 72 months)), three tumors recurred locally, but none of the patients developed regional or distant metastases. Ultimately, two patients died due to the tumor. Microscopically, all tumors consisted of infiltrating nests of polygonal basaloid cells with a variable component of rhabdoid cells with eosinophilic cytoplasm. Immunohistochemically, there was almost diffuse expression of cytokeratins (CK), p16, p40 and p63 in all cases, while expression of CK5/6, CK7 and vimentin was only focal or absent. The detection of NUT gave negative results. In three cases, the absence of SMARCB1/INI1 expression was due to deletion of SMARCB1/INI1 gene. Methylation of SMARCB1/INI1 gene was not found. One tumor harbored HPV18 E6/E7 mRNA. All 12 genes (BRAF, BRCA1, BRCA2, KIT, EGFR, KRAS, NRAS, PDGFRA, PIK3CA, PTEN, RET, and ROS1) tested for mutations using NGS were wild-type. Regarding DNA methylation, all four SMARCB1/INI1-deficient tumors showed methylation of RASSF1 gene by means of MS-MLPA. There was a statistically significant difference in RASSF1 gene methylation between SMARCB1/INI1-deficient and SMARCB1/INI1-positive tumors (p=0.0095). All other examined genes (ATM, BRCA1, BRCA2, CADM1, CASP8, CD44, CDKN1B, CDKN2A, CDKN2B, CHFR, DAPK1, ESR1, FHIT, GSTP1, HIC1, KLLN, MLH1a, MLH1b, RARB, and VLH) were unmethylated. In summary, we described four cases of SMARCB1/INI1-deficient sinonasal carcinoma with detailed clinicopathological data indicating that these tumors can be regarded as a distinct entity with aggressive behaviour. For the first time, we performed analysis of DNA methylation in SMARCB1/INI1-deficient sinonasal carcinomas, reporting on significantly higher methylation of RASSF1 gene in this neoplasm.

ATM and TGFB1 genes polymorphisms in prediction of late complications of chemoradiotherapy in patients with locally advanced cervical cancer.

The purpose of our study was to evaluate a possible correlation between genetic polymorphisms in ATM and TGFB1 genes and late toxicity of chemoradiotherapy for locally advanced cervical cancer. Fifty five patients with FIGO stage IIB and higher without a disease recurrence with a mean follow up of 6 years were included. Late toxicity was assessed by EORTC/RTOG late toxicity criteria. Univariate and multivariate logistic regression model was used for statistical analysis. Degree of association between polymorphisms and late toxicity of chemotherapy was assessed on the basis of phi-coefficient (φ) as well. We did not find any association between 5557G>A polymorphism in the ATM gene or single TGFB1 polymorphisms and late toxicity. TGFB1 compound homozygosity (-1552delAGG, -509C>T, L10P) was a significant predictive factor of grade III-IV and any grade of complications in both univariate and multivariate logistic regression analyses and statistical significance of association between polymorphisms and late toxicity of chemoradiotherapy was confirmed also by the evaluation of phi-coefficient (φ). We conclude that haplotypes instead of single nucleotide polymorphic sites in the genes may better characterize the individual radiosensitivity. Keywords: cervical cancer, radiotherapy, ATM, TGFB1, late toxicity.

Analysis of D1853N ATM polymorphism in radiosensitive patients with cervical carcinoma.

UNLABELLED Clinical oncologists have been focusing their efforts on attempting to define risk groups of patients with unusual biological reactions to the recommended therapy regimens using molecular biology techniques. THE AIMS OF OUR STUDY WERE (i) to find a design and validate a method for fast and reliable analysis of the D1853N (5557G>A) genetic polymorphism in the ATM (ataxia-telangiectasia mutated) gene; (ii) to use side-directed mutagenesis to generate ATM 5557A-positive DNA (reference ATM5557A DNA); and (iii) to analyze a group of patients suffering from cervical carcinoma with adverse responses to radiotherapy. The 5557A variant was found in three of twenty women (15%). Our data show that the prevalence of the 5557A allelic variant in cervical cancer subjects with adverse responses after irradiation probably does not differ from the prevalence common in Caucasians. A larger population study should confirm these preliminary results.

Analysis of DNA methylation and microRNA expression in NUT (nuclear protein in testis) midline carcinoma of the sinonasal tract: a clinicopathological, immunohistochemical and molecular genetic study

The aim of this study was a detailed clinicopathological investigation of sinonasal NUT midline carcinoma (NMC), including analysis of DNA methylation and microRNA (miRNA) expression. Three (5%) cases of NMC were detected among 56 sinonasal carcinomas using immunohistochemical screening and confirmed by fluorescence in situ hybridization. The series comprised 2 males and 1 female, aged 46, 60, and 65 years. Two tumors arose in the nasal cavity and one in the maxillary sinus. The neoplasms were staged pT1, pT3, and pT4a (all cN0M0). All patients were treated by radical resection with adjuvant radiotherapy. Two patients died 3 and 8 months after operation, but one patient (pT1 stage; R0 resection) experienced no evidence of disease at 108 months. Microscopically, all tumors consisted of infiltrating nests of polygonal cells with vesicular nuclei, prominent nucleoli and basophilic cytoplasm. Abrupt keratinization was present in only one case. Immunohistochemically, there was a diffuse expression of cytokeratin (CK) cocktail, CK7, p40, p63, and SMARCB1/INI1. All NMCs tested negative for EBV and HPV infection. Two NMCs showed methylation of RASSF1 gene. All other genes (APC, ATM, BRCA1, BRCA2, CADM1, CASP8, CD44, CDH13, CDKN1B, CDKN2A, CDKN2B, CHFR, DAPK1, ESR1, FHIT, GSTP1, HIC1, KLLN, MLH1a, MLH1b, RARB, TIMP3, and VHL) were unmethylated. All NMCs showed upregulation of miR-9 and downregulation of miR-99a and miR-145 and two cases featured also upregulation of miR-21, miR-143, and miR-484. In summary, we described three cases of sinonasal NMCs with novel findings on DNA methylation and miRNA expression, which might be important for new therapeutic strategies in the future.