A Nordic-Baltic perspective on indications for proton therapy with strategies for identification of proper patients.

The beneficial effects of protons are primarily based on reduction of low to intermediate radiation dose bath to normal tissue surrounding the radiotherapy target volume. Despite promise for reduced long-term toxicity, the percentage of cancer patients treated with proton therapy remains low. This is probably caused by technical improvements in planning and delivery of photon therapy, and by high cost, low availability and lack of high-level evidence on proton therapy. A number of proton treatment facilities are under construction or have recently opened; there are now two operational Scandinavian proton centres and two more are under construction, thereby eliminating the availability hurdle. Even with the advantageous physical properties of protons, there is still substantial ambiguity and no established criteria related to which patients should receive proton therapy. This topic was discussed in a session at the Nordic Collaborative Workshop on Particle Therapy, held in Uppsala 14-15 November 2019. This paper resumes the Nordic-Baltic perspective on proton therapy indications and discusses strategies to identify patients for proton therapy. As for indications, neoplastic entities, target volume localisation, size, internal motion, age, second cancer predisposition, dose escalation and treatment plan comparison based on the as low as reasonably achievable (ALARA) principle or normal tissue complication probability (NTCP) models were discussed. Importantly, the patient selection process should be integrated into the radiotherapy community and emphasis on collaboration across medical specialties, involvement of key decision makers and knowledge dissemination in general are important factors. An active Nordic-Baltic proton therapy organisation would also serve this purpose.

Development of an interlaced-crossfiring geometry for proton grid therapy.

BACKGROUND: Grid therapy has in the past normally been performed with single field photon-beam grids. In this work, we evaluated a method to deliver grid therapy based on interlacing and crossfiring grids of mm-wide proton beamlets over a target volume, by Monte Carlo simulations. MATERIAL AND METHODS: Dose profiles for single mm-wide proton beamlets (1, 2 and 3 mm FWHM) in water were simulated with the Monte Carlo code TOPAS. Thereafter, grids of proton beamlets were directed toward a cubic target volume, located at the center of a water tank. The aim was to deliver a nearly homogeneous dose to the target, while creating high dose heterogeneity in the normal tissue, i.e., high gradients between valley and peak doses in the grids, down to the close vicinity of the target. RESULTS: The relative increase of the beam width with depth was largest for the smallest beams (+6.9 mm for 1 mm wide and 150 MeV proton beamlets). Satisfying dose coverage of the cubic target volume (σ < ±5%) was obtained with the interlaced-crossfiring setup, while keeping the grid pattern of the dose distribution down to the target (valley-to-peak dose ratio <0.5 less than 1 cm before the target). Center-to-center distances around 7-8 mm between the beams were found to give the best compromise between target dose homogeneity and low peak doses outside of the target. CONCLUSIONS: A nearly homogeneous dose distribution can be obtained in a target volume by crossfiring grids of mm-wide proton-beamlets, while maintaining the grid pattern of the dose distribution at large depths in the normal tissue, close to the target volume. We expect that the use of this method will increase the tumor control probability and improve the normal tissue sparing in grid therapy.

Validation of automated post-adjustments of HDR prostate brachytherapy treatment plans by quantitative measures and oncologist observer study.

PURPOSE: The aim was to evaluate a postprocessing optimization algorithm's ability to improve the spatial properties of a clinical treatment plan while preserving the target coverage and the dose to the organs at risk. The goal was to obtain a more homogenous treatment plan, minimizing the need for manual adjustments after inverse treatment planning. MATERIALS AND METHODS: The study included 25 previously treated prostate cancer patients. The treatment plans were evaluated on dose-volume histogram parameters established clinical and quantitative measures of the high dose volumes. The volumes of the four largest hot spots were compared and complemented with a human observer study with visual grading by eight oncologists. Statistical analysis was done using ordinal logistic regression. Weighted kappa and Fleiss' kappa were used to evaluate intra- and interobserver reliability. RESULTS: The quantitative analysis showed that there was no change in planning target volume (PTV) coverage and dose to the rectum. There were significant improvements for the adjusted treatment plan in: V150% and V200% for PTV, dose to urethra, conformal index, and dose nonhomogeneity ratio. The three largest hot spots for the adjusted treatment plan were significantly smaller compared to the clinical treatment plan. The observers preferred the adjusted treatment plan in 132 cases and the clinical in 83 cases. The observers preferred the adjusted treatment plan on homogeneity and organs at risk but preferred the clinical plan on PTV coverage. CONCLUSIONS: Quantitative analysis showed that the postadjustment optimization tool could improve the spatial properties of the treatment plans while maintaining the target coverage.

Preoperative short-course radiation therapy with PROtons compared to photons in high-risk RECTal cancer (PRORECT): Initial dosimetric experience.

Background and purpose: Neoadjuvant short-course radiotherapy (SCRT) followed by full-dose systemic chemotherapy is an established treatment modality in locally advanced rectal cancer (LARC). Until recently, SCRT has been exclusively delivered with photons. Proton beam therapy (PBT) may minimize acute toxicity, which in turn likely impacts favorably on the tolerability to subsequent chemotherapy. The aim of this study is a dosimetric comparison between SCRT with photons and protons in the randomized phase II trial PRORECT (NCT04525989). Materials and methods: From June 2021 to June 2022, twenty consecutive patients with LARC have been treated according to study protocol. For each patient, both a VMAT and a PBT treatment plans have been generated and compared pairwise. Results: Dose-volume histogram (DVH) analysis revealed that SCRT with protons significantly reduced radiation dose to pelvic organs at risk including bladder, bones, and bowel in comparison to SCRT with photons. Photon and proton treatment plans had equivalent conformity and homogeneity indexes. Conclusion: Preoperative SCRT with protons offers a significant reduction of radiation dose to normal tissues compared with current photon-based radiotherapy technique. Demonstrated dosimetric advantages may translate into measurable clinical benefits in patients with LARC. Clinical implications of the dosimetric superiority of SCRT with protons will be presented in the coming reports from the PRORECT trial.

Biochemical Recurrence and Risk of Mortality Following Radiotherapy or Radical Prostatectomy.

Importance: Stratifying patients with biochemical recurrence (BCR) after primary treatment for prostate cancer based on the risk of prostate cancer-specific mortality (PCSM) is essential for determining the need for further testing and treatments. Objective: To evaluate the association of BCR after radical prostatectomy or radiotherapy and its current risk stratification with PCSM. Design, Setting, and Participants: This population-based cohort study included a total of 16 311 male patients with 10 364 (64%) undergoing radical prostatectomy and 5947 (36%) undergoing radiotherapy with curative intent (cT1-3, cM0) and PSA follow-up in Stockholm, Sweden, between 2003 and 2019. Follow-up for all patients was until death, emigration, or end of the study (ie, December 31, 2018). Data were analyzed between September 2022 and March 2023. Main Outcomes and Measures: Primary outcomes of the study were the cumulative incidence of BCR and PCSM. Patients with BCR were stratified in low- and high-risk according to European Association of Urology (EAU) criteria. Exposures: Radical prostatectomy or radiotherapy. Results: A total of 16 311 patients were included. Median (IQR) age was 64 (59-68) years in the radical prostatectomy cohort (10 364 patients) and 69 (64-73) years in the radiotherapy cohort (5947 patients). Median (IQR) follow-up for survivors was 88 (55-138) months and 89 (53-134) months, respectively. Following radical prostatectomy, the 15-year cumulative incidences of BCR were 16% (95% CI, 15%-18%) for the 4024 patients in the low D'Amico risk group, 30% (95% CI, 27%-32%) for the 5239 patients in the intermediate D'Amico risk group, and 46% (95% CI, 42%-51%) for 1101 patients in the high D'Amico risk group. Following radiotherapy, the 15-year cumulative incidences of BCR were 18% (95% CI, 15%-21%) for the 1230 patients in the low-risk group, 24% (95% CI, 21%-26%) for the 2355 patients in the intermediate-risk group, and 36% (95% CI, 33%-39%) for the 2362 patients in the high-risk group. The 10-year cumulative incidences of PCSM after radical prostatectomy were 4% (95% CI, 2%-6%) for the 1101 patients who developed low-risk EAU-BCR and 9% (95% CI, 5%-13%) for 649 patients who developed high-risk EAU-BCR. After radiotherapy, the 10-year PCSM cumulative incidences were 24% (95% CI, 19%-29%) for the 591 patients in the low-risk EAU-BCR category and 46% (95% CI, 40%-51%) for the 600 patients in the high-risk EAU-BCR category. Conclusions and Relevance: These findings suggest the validity of EAU-BCR stratification system. However, while the risk of dying from prostate cancer in low-risk EAU-BCR after radical prostatectomy was very low, patients who developed low-risk EAU-BCR after radiotherapy had a nonnegligible risk of prostate cancer mortality. Improving risk stratification of patients with BCR is pivotal to guide salvage treatment decisions, reduce overtreatment, and limit the number of staging tests in the event of PSA elevations after primary treatment.

Autosegmentation based on different-sized training datasets of consistently-curated volumes and impact on rectal contours in prostate cancer radiation therapy.

Background and purpose: Autosegmentation techniques are emerging as time-saving means for radiation therapy (RT) contouring, but the understanding of their performance on different datasets is limited. The aim of this study was to determine agreement between rectal volumes by an existing autosegmentation algorithm and manually-delineated rectal volumes in prostate cancer RT. We also investigated contour quality by different-sized training datasets and consistently-curated volumes for retrained versions of this same algorithm. Materials and methods: Single-institutional data from 624 prostate cancer patients treated to 50-70 Gy were used. Manually-delineated clinical rectal volumes (clinical) and consistently-curated volumes recontoured to one anatomical guideline (reference) were compared to autocontoured volumes by a commercial autosegmentation tool based on deep-learning (v1; n = 891, multiple-institutional data) and retrained versions using subsets of the curated volumes (v32/64/128/256; n = 32/64/128/256). Evaluations included dose-volume histogram metrics, Dice similarity coefficients, and Hausdorff distances; differences between groups were quantified using parametric or non-parametric hypothesis testing. Results: Volumes by v1-256 (76-78 cm3) were larger than reference (75 cm3) and clinical (76 cm3). Mean doses by v1-256 (24.2-25.2 Gy) were closer to reference (24.2 Gy) than to clinical (23.8 Gy). Maximum doses were similar for all volumes (65.7-66.0 Gy). Dice for v1-256 and reference (0.87-0.89) were higher than for v1-256 and clinical (0.86-0.87) with corresponding Hausdorff comparisons including reference smaller than comparisons including clinical (5-6 mm vs. 7-8 mm). Conclusion: Using small single-institutional RT datasets with consistently-defined rectal volumes when training autosegmentation algorithms created contours of similar quality as the same algorithm trained on large multi-institutional datasets.

Development of a consensus-based delineation guideline for locally recurrent rectal cancer.

BACKGROUND AND PURPOSE: Neoadjuvant chemoradiotherapy (nCRT) is used in locally recurrent rectal cancer (LRRC) to increase chances of a radical surgical resection. Delineation in LRRC is hampered by complex disease presentation and limited clinical exposure. Within the PelvEx II trial, evaluating the benefit of chemotherapy preceding nCRT for LRRC, a delineation guideline was developed by an expert LRRC team. MATERIALS AND METHODS: Eight radiation oncologists, from Dutch and Swedish expert centres, participated in two meetings, delineating GTV and CTV in six cases. Regions at-risk for re-recurrence or irradical resection were identified by eleven expert surgeons and one expert radiologist. Target volumes were evaluated multidisciplinary. Inter-observer variation was analysed. RESULTS: Inter-observer variation in delineation of LRRC appeared large. Multidisciplinary evaluation per case is beneficial in determining target volumes. The following consensus regarding target volumes was reached. GTV should encompass all tumour, including extension into OAR if applicable. If the tumour is in fibrosis, GTV should encompass the entire fibrotic area. Only if tumour can clearly be distinguished from fibrosis, GTV may be reduced, as long as the entire fibrotic area is covered by the CTV. CTV is GTV with a 1 cm margin and should encompass all at-risk regions for irradical resection or re-recurrence. CTV should not be adjusted towards other organs. Multifocal recurrences should be encompassed in one CTV. Elective nodal delineation is only advised in radiotherapy-naïve patients. CONCLUSION: This study provides a first consensus-based delineation guideline for LRRC. Analyses of re-recurrences is needed to understand disease behaviour and to optimize delineation guidelines accordingly.

Beta-tubulin III expression in prostate cancer.

OBJECTIVE: Beta-tubulin isotype III is a microtubule component associated with resistance to chemotherapy and poor outcome in various cancers. This study aimed to investigate its expression in prostate cancer and its role as a prognostic factor in this setting. MATERIAL AND METHODS: A tissue microarray was constructed of 289 prostate cancers from radical prostatectomy specimens with a median follow-up of 48.9 months. Slides were immunostained for ß-tubulin III. The intensity and extent of immunoreactivity and their product [immunoreactivity product (IRP)] were evaluated. RESULTS: Tubulin III was expressed in the cytoplasm of prostate cancer cells but not in benign glands. Only 11.6% of cancers were positive for tubulin III. Among low-grade (Gleason score 5-6) and high-grade (Gleason score 7-10) cancers, 6.0% and 16.6% were positive, respectively (p = 0.006). ß-Tubulin III expression was more often seen in high-stage disease and more often in metastases (62.5%) than in primary lesions (11.6%) (p < 0.001). The intensity, extent and IRP of tubulin III all predicted biochemical recurrence in univariate Cox analysis (p = 0.02, p = 0.048 and p = 0.012, respectively). IRP was an independent predictor of prognosis when adjusted for serum prostate-specific antigen in a multivariate Cox analysis (p = 0.005), but not when the Gleason score was added to the model (p = 0.17). CONCLUSION: ß-Tubulin III predicts biochemical recurrence after radical prostatectomy in a subset of patients. Its practical utility is limited by the low number of cases positive for this biomarker.

Initial experience with introducing national guidelines for CT- and MRI-based delineation of organs at risk in radiotherapy.

A fundamental problem in radiotherapy is the variation of organ at risk (OAR) volumes. Here we present our initial experience in engaging a large Radiation Oncology (RO) community to agree on national guidelines for OAR delineations. Our project builds on associated standardization initiatives and invites professionals from all radiotherapy departments nationwide. Presently, one guideline (rectum) has successfully been agreed on by a majority vote. Reaching out to all relevant parties in a timely manner and motivating funding agencies to support the work represented early challenges. Population-based data and a scalable methodological approach are major strengths of the proposed strategy.

Expression of PDX-1 in prostate cancer, prostatic intraepithelial neoplasia and benign prostatic tissue.

Pancreatic duodenal homeobox 1 (PDX-1), a Hox type transcription factor, is necessary for differentiation of exocrine and endocrine pancreas, and regulates insulin gene transcription. PDX-1 expression was studied by immunohistochemistry on a tissue microarray (TMA) of 289 primary prostate cancers (PCa) from radical prostatectomy (RP) specimens with median follow-up of 48.9 months. We separately arrayed benign prostatic tissue, atrophy, high-grade prostatic intraepithelial neoplasia (HGPIN) and PCa from 40 men and also 17 lymph node metastases. Intensity and extent of immunoreactivity and their product (IRp) were evaluated by two independent observers. PDX-1 was overexpressed in cancer vs benign tissue (p<0.001), but also in atrophy and HGPIN vs cancer (p<0.001 and p=0.022, respectively). PDX-1 expression did not correlate with biochemical recurrence, but decreased with higher Gleason pattern (p<0.001) and in metastases vs primary PCa (p<0.001). Weighted kappa for interobserver agreement of intensity, extent and IRp was 0.65, 0.13 and 0.54, respectively. Presence of PDX-1 protein in benign and malignant prostatic tissue was confirmed by Western blot. In view of recent attention to the role of insulin systems in men with PCa, this protein is of interest in the pathogenesis of PCa.

Quantitative evaluation of potential irradiation geometries for carbon-ion beam grid therapy.

PURPOSE: Radiotherapy using grids containing cm-wide beam elements has been carried out sporadically for more than a century. During the past two decades, preclinical research on radiotherapy with grids containing small beam elements, 25 µm-0.7 mm wide, has been performed. Grid therapy with larger beam elements is technically easier to implement, but the normal tissue tolerance to the treatment is decreasing. In this work, a new approach in grid therapy, based on irradiations with grids containing narrow carbon-ion beam elements was evaluated dosimetrically. The aim formulated for the suggested treatment was to obtain a uniform target dose combined with well-defined grids in the irradiated normal tissue. The gain, obtained by crossfiring the carbon-ion beam grids over a simulated target volume, was quantitatively evaluated. METHODS: The dose distributions produced by narrow rectangular carbon-ion beams in a water phantom were simulated with the PHITS Monte Carlo code. The beam-element height was set to 2.0 cm in the simulations, while the widths varied from 0.5 to 10.0 mm. A spread-out Bragg peak (SOBP) was then created for each beam element in the grid, to cover the target volume with dose in the depth direction. The dose distributions produced by the beam-grid irradiations were thereafter constructed by adding the dose profiles simulated for single beam elements. The variation of the valley-to-peak dose ratio (VPDR) with depth in water was thereafter evaluated. The separation of the beam elements inside the grids were determined for different irradiation geometries with a selection criterion. RESULTS: The simulated carbon-ion beams remained narrow down to the depths of the Bragg peaks. With the formulated selection criterion, a beam-element separation which was close to the beam-element width was found optimal for grids containing 3.0-mm-wide beam elements, while a separation which was considerably larger than the beam-element width was found advantageous for grids containing 0.5-mm-wide beam elements. With the single-grid irradiation setup, the VPDRs were close to 1.0 already at a distance of several cm from the target. The valley doses given to the normal tissue at 0.5 cm distance from the target volume could be limited to less than 10% of the mean target dose if a crossfiring setup with four interlaced grids was used. CONCLUSIONS: The dose distributions produced by grids containing 0.5- and 3.0-mm wide beam elements had characteristics which could be useful for grid therapy. Grids containing mm-wide carbon-ion beam elements could be advantageous due to the technical ease with which these beams can be produced and delivered, despite the reduced threshold doses observed for early and late responding normal tissue for beams of millimeter width, compared to submillimetric beams. The treatment simulations showed that nearly homogeneous dose distributions could be created inside the target volumes, combined with low valley doses in the normal tissue located close to the target volume, if the carbon-ion beam grids were crossfired in an interlaced manner with optimally selected beam-element separations. The formulated selection criterion was found useful for the quantitative evaluation of the dose distributions produced by the different irradiation setups.

Proton Grid Therapy: A Proof-of-Concept Study.

In this work, we studied the possibility of merging proton therapy with grid therapy. We hypothesized that patients with larger targets containing solid tumor growth could benefit from being treated with this method, proton grid therapy. We performed treatment planning for 2 patients with abdominal cancer with the suggested proton grid therapy technique. The proton beam arrays were cross-fired over the target volume. Circular or rectangular beam element shapes (building up the beam grids) were evaluated in the planning. An optimization was performed to calculate the fluence from each beam grid element. The optimization objectives were set to create a homogeneous dose inside the target volume with the constraint of maintaining the grid structure of the dose distribution in the surrounding tissue. The proton beam elements constituting the grid remained narrow and parallel down to large depths in the tissue. The calculation results showed that it is possible to produce target doses ranging between 100% and 130% of the prescribed dose by cross-firing beam grids, incident from 4 directions. A sensitivity test showed that a small rotation or translation of one of the used grids, due to setup errors, had only a limited influence on the dose distribution produced in the target, if 4 beam arrays were used for the irradiation. Proton grid therapy is technically feasible at proton therapy centers equipped with spot scanning systems using existing tools. By cross-firing the proton beam grids, a low tissue dose in between the paths of the elemental beams can be maintained down to the vicinity of a deep-seated target. With proton grid therapy, it is possible to produce a dose distribution inside the target volume of similar uniformity as can be created with current clinical methods.

Microbeam radiation therapy - grid therapy and beyond: a clinical perspective.

Microbeam irradiation is spatially fractionated radiation on a micrometer scale. Microbeam irradiation with therapeutic intent has become known as microbeam radiation therapy (MRT). The basic concept of MRT was developed in the 1980s, but it has not yet been tested in any human clinical trial, even though there is now a large number of animal studies demonstrating its marked therapeutic potential with an exceptional normal tissue sparing effect. Furthermore, MRT is conceptually similar to macroscopic grid based radiation therapy which has been used in clinical practice for decades. In this review, the potential clinical applications of MRT are analysed for both malignant and non-malignant diseases.

Tissue shrinkage after fixation with formalin injection of prostatectomy specimens.

Prostate cancer volume correlates with stage, grade, and progression after prostatectomy. When tumor volume is measured planimetrically, results are multiplied by a correction factor to compensate for tissue shrinkage caused by processing. Injection of formalin into prostatectomy specimens was suggested for improved fixation. Our aim was to investigate how this affects the prostate volume. We studied 142 radical prostatectomy specimens. All prostates were immersed in 10% formalin. In 84 prostates (59%) we also injected 20 ml of formalin before routine fixation. The prostates were weighed unfixed after injection and after final fixation. The specimens were sliced and totally embedded. The transverse diameters of the prostates were measured on unfixed specimens and microscopic sections. The average weight loss after final fixation was 5.8 and 8.6% for formalin-injected specimens and standard-fixed specimens, respectively (p<0.001). However, when total shrinkage was estimated from the transverse diameters, there was no difference related to fixation technique (p=0.59). The average linear shrinkage was 4.5%, corresponding to a volume correction factor of 1.15. We conclude that formalin injection for fixation of prostate tissue does not influence tumor volume calculation compared to conventional fixation.

Cytological features of prostatic intraepithelial neoplasia.

Fine-needle aspiration cytology (FNAC) is an acknowledged method for diagnosing prostate cancer. False-positive results are uncommon, but concerns have been raised that prostatic intraepithelial neoplasia (PIN) could be misinterpreted as carcinoma. Therefore, we attempted to describe cytological features of PIN. Cells were scraped from macroscopically normal areas of 177 radical prostatectomy specimens, smeared and Giemsa-stained. Histological slides from these areas were reviewed, and 17 samples with high-grade PIN and with no invasive cancer were selected. Smears from 17 invasive cancers were used for comparison. Cancer showed high cellularity and dissociation, while PIN smears only contained a few clusters of atypical cells. Pronounced nuclear atypia, prominent or multiple nucleoli and mucin were more common in cancer, while cytoplasmic granules, crystalloids and cluster size did not distinguish between PIN and cancer. In conclusion, PIN should not be diagnosed by FNAC alone. However, a highly cellular smear with pronounced atypia seems to preclude PIN.

A national approach for automated collection of standardized and population-based radiation therapy data in Sweden.

PURPOSE: To develop an infrastructure for structured and automated collection of interoperable radiation therapy (RT) data into a national clinical quality registry. MATERIALS AND METHODS: The present study was initiated in 2012 with the participation of seven of the 15 hospital departments delivering RT in Sweden. A national RT nomenclature and a database for structured unified storage of RT data at each site (Medical Information Quality Archive, MIQA) have been developed. Aggregated data from the MIQA databases are sent to a national RT registry located on the same IT platform (INCA) as the national clinical cancer registries. RESULTS: The suggested naming convention has to date been integrated into the clinical workflow at 12 of 15 sites, and MIQA is installed at six of these. Involvement of the remaining 3/15 RT departments is ongoing, and they are expected to be part of the infrastructure by 2016. RT data collection from ARIA®, Mosaiq®, Eclipse™, and Oncentra® is supported. Manual curation of RT-structure information is needed for approximately 10% of target volumes, but rarely for normal tissue structures, demonstrating a good compliance to the RT nomenclature. Aggregated dose/volume descriptors are calculated based on the information in MIQA and sent to INCA using a dedicated service (MIQA2INCA). Correct linkage of data for each patient to the clinical cancer registries on the INCA platform is assured by the unique Swedish personal identity number. CONCLUSIONS: An infrastructure for structured and automated prospective collection of syntactically interoperable RT data into a national clinical quality registry for RT data is under implementation. Future developments include adapting MIQA to other treatment modalities (e.g. proton therapy and brachytherapy) and finding strategies to harmonize structure delineations. How the RT registry should comply with domain-specific ontologies such as the Radiation Oncology Ontology (ROO) is under discussion.

Mutation analysis of the BRG1 gene in prostate cancer clinical samples.

Previous studies in hereditary and sporadic prostate cancer have indicated the existence of a tumor suppressor gene in chromosomal region 19p13. The BRG1 gene in this region is one of the possible candidates, based on both the frequency of inactivating mutations in human cancer cell lines, including the prostate cancer cell line DU145, and its functional properties. To our knowledge, no studies have been done to evaluate possible involvement of the BRG1 gene in clinical prostate cancer. To accomplish this, we carried out a complete mutation analysis of all 35 BRG1 exons in tumor and constitutional DNA samples from 21 prostate cancer patients. We report the absence of somatic mutations in the panel of samples employed, but the existence of five germline single nucleotide polymorphisms (SNPs) in CpG islands of the BRG1 gene, among them, three novel ones. In conclusion, the study excludes the presence of common BRG1 mutations in prostate cancer.

Clinical significance of intratumoral CD8+ regulatory T cells in prostate carcinoma.

OBJECTIVE: To explore Foxp3, a member of the forkhead box family of transcription factors, which is a major gene for regulatory T (Treg) cell development of CD4+CD25+ or CD8+CD25+ phenotype. STUDY DESIGN: We constructed tissue microarrays from 82 patients after radical prostatectomy (RP). Three cores of neoplastic tissue and 3 from normal/inflammatory tissue were taken. Sections were immunostained for Foxp3 and CD8. Numbers of Foxp3 and CD8 positive cells were counted. Serum prostate-specific antigen levels before and after RP, Gleason score (GS), surgical margin status and pathologic stage were available. RESULTS: Among 82 patients aged 55-76 years (mean 66.1, SD +/- 5.8), 64 (78%) were staged pT2 and 18 (22%) pT3. Twenty-nine patients (35%) had positive margins, 24 (29%) increasing prostate-specific antigen levels (biochemical relapse) 6 months to 2 years (SD +/- 24.8 months) after RP. GS was distributed as follows: 41 (50%) patients with GS 6 (3+3), 34 (41%) with GS 7 [19 (3+4) and 15 (4+3)], 7 (9%) with GS > or = 8. In tumor cores, Foxp3 and CD8 counts correlated (p = 0.012). Foxp3 counts also correlated with biochemical relapse (p = 0.04). CONCLUSION: Treg cells were more common in cancer than in benign prostatic tissue. There are links between Foxp3 and CD8+ cells and between Foxp3 positive cells and biochemical relapse. Patients with prostate cancer show an immunosuppressive regulatory profile, including nonresponsive Tregs. It would be important to find mechanisms to target these cells for successful immunotherapy.

Distribution of Foxp3-, CD4- and CD8-positive lymphocytic cells in benign and malignant prostate tissue.

Foxp3 is a transcription factor that inhibits antitumor immune response and is expressed in regulatory T cells (Tregs). High levels of Tregs have been reported in several human cancers. This study investigates the distribution of cells positive for Foxp3, CD4 and CD8 in benign prostatic tissues and prostatic carcinoma. Tissue microarrays were constructed from radical prostatectomy specimens of 36 patients. From each patient, six cores were taken: two cores from cancer, one from benign tissue of each of the peripheral (PZ), transition (TZ) and central zones (CZ) and one from atrophy. Foxp3-, CD4- and CD8-positive cells were more common in cancer than in non-atrophic benign tissue (p < 0.01) and more common in atrophy than in non-atrophic PZ, but did not differ significantly between cancer and atrophy. Cells positive for Foxp3 and CD4 were less prevalent in CZ than in PZ and TZ. Tregs infiltrate more in prostate cancer (PC) than in benign tissue. Their presence in atrophy may have relevance for the hypothesis on atrophy as a potential precursor lesion of PC. CZ has the lowest Treg levels, and a possible role for the low rate of cancer in this zone remains to be investigated.

Endothelin-1 expression in prostate cancer and high grade prostatic intraepithelial neoplasia.

OBJECTIVE: To investigate the prognostic value of endothelin-1 (ET-1), a vasoconstrictor involved in differentiation and growth of cancer, in prostate cancer. STUDY DESIGN: A tissue microarray was constructed of 287 prostate cancers from radical prostatectomy (RP) specimens with median follow-up of 48.9 months. Slides were immunostained for ET-1. Intensity and extent of immunoreactivity and their product (IRp) were evaluated. We separately arrayed benign prostatic tissue, atrophy, high grade prostatic intraepithelial neoplasia (HGPIN) and prostate cancer from 40 men. RESULTS: ET-1 expression was stronger in both HGPIN and cancer than in benign tissue (p < 0.001). The intensity and the IRp of ET-1 predicted biochemical recurrence (p < 0.001 and p = 0.044, respectively), while the staining extent showed no significant correlation with outcome (p = 0.68). Recurrence-free survival in patients with strong ET-1 staining was shorter than in those with weaker expression (hazard ratio [CI 95%] 2.44 [1.55-3.84], p < 0.001). In a multivariate analysis including ET-1 expression, preoperative serum prostate-specific antigen (PSA), extraprostatic extension, margin status, seminal vesicle invasion and Gleason score, only PSA, margins and Gleason score remained significant. CONCLUSION: ET-1 is overexpressed in localized prostate cancer and predicts outcome after RP in univariate analysis.