Testicular tissue cryopreservation: 8 years of experience from a coordinated network of academic centers.

STUDY QUESTION: Is it feasible to disseminate testicular tissue cryopreservation with a standardized protocol through a coordinated network of centers and provide centralized processing/freezing for centers that do not have those capabilities? SUMMARY ANSWER: Centralized processing and freezing of testicular tissue from multiple sites is feasible and accelerates recruitment, providing the statistical power to make inferences that may inform fertility preservation practice. WHAT IS KNOWN ALREADY: Several centers in the USA and abroad are preserving testicular biopsies for patients who cannot preserve sperm in anticipation that cell- or tissue-based therapies can be used in the future to generate sperm and offspring. STUDY DESIGN, SIZE, DURATION: Testicular tissue samples from 189 patients were cryopreserved between January 2011 and November 2018. Medical diagnosis, previous chemotherapy exposure, tissue weight, and presence of germ cells were recorded. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human testicular tissue samples were obtained from patients undergoing treatments likely to cause infertility. Twenty five percent of the patient's tissue was donated to research and 75% was stored for patient's future use. The tissue was weighed, and research tissue was fixed for histological analysis with Periodic acid-Schiff hematoxylin staining and/or immunofluorescence staining for DEAD-box helicase 4, and/or undifferentiated embryonic cell transcription factor 1. MAIN RESULTS AND THE ROLE OF CHANCE: The average age of fertility preservation patients was 7.9 (SD = 5) years and ranged from 5 months to 34 years. The average amount of tissue collected was 411.3 (SD = 837.3) mg and ranged from 14.4 mg-6880.2 mg. Malignancies (n = 118) were the most common indication for testicular tissue freezing, followed by blood disorders (n = 45) and other conditions (n = 26). Thirty nine percent (n = 74) of patients had initiated their chemotherapy prior to undergoing testicular biopsy. Of the 189 patients recruited to date, 137 have been analyzed for the presence of germ cells and germ cells were confirmed in 132. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive study of testicular tissues obtained from patients who were at risk of infertility. The function of spermatogonia in those biopsies could not be tested by transplantation due limited sample size. WIDER IMPLICATIONS OF THE FINDINGS: Patients and/or guardians are willing to pursue an experimental fertility preservation procedure when no alternatives are available. Our coordinated network of centers found that many patients request fertility preservation after initiating gonadotoxic therapies. This study demonstrates that undifferentiated stem and progenitor spermatogonia may be recovered from the testicular tissues of patients who are in the early stages of their treatment and have not yet received an ablative dose of therapy. The function of those spermatogonia was not tested. STUDY FUNDING/COMPETING INTEREST(S): Support for the research was from the Eunice Kennedy Shriver National Institute for Child Health and Human Development grants HD061289 and HD092084, the Scaife Foundation, the Richard King Mellon Foundation, the Departments of Ob/Gyn & Reproductive Sciences and Urology of the University of Pittsburgh Medical Center, United States-Israel Binational Science Foundation (BSF), and the Kahn Foundation. The authors declare that they do not have competing financial interests.

[Three horizontal muscle surgery for infantile esotropia and decompensated microtropia]. / Horizontale Dreimuskelchirurgie bei frühkindlichem Innenschielen und dekompensiertem Mikrostrabismus.

BACKGROUND: Surgical management of large-angle infantile esotropia and decompensated microtropia has been controversially debated. There seems to be a relative bias against surgery on more than two horizontal muscles to avoid overcorrections. In our study we report on the sensory and motor outcomes after three horizontal muscle surgery. Furthermore we aim to suggest a table to guide surgery amounts. PATIENTS AND METHODS: This study was a retrospective interventional cohort of 27 patients (16 female, 11 male; mean age 7.9 [years], ranging from 1 to 27 [years]) with infantile esotropia and decompensated microtropia who underwent three horizontal muscle surgery (bilateral medial rectus muscle recession and lateral rectus muscle plication) between 2005 and 2013. RESULTS: Preoperative deviation in primary position measured 30.2 [°] (mean; range 21.9 to 48.0). Postoperatively esotropia was significantly reduced to 2.6 [°] (mean; range -16.7 to 16.7). After six months deviation measured 5.5 [°] (mean, range -14.1 to 21.9). An effect of 1.63°/mm was calculated for the combined three muscle surgery. CONCLUSIONS: For the correction of large-angle infantile esotropia and decompensated microtropia three horizontal muscle surgery is associated with a high success rate. Only a very low rate of consecutive exotropia does occur.

Early onset of acquired comitant non-accommodative esotropia in childhood.

BACKGROUND: The aim of this study was to describe the clinical characteristics and surgical outcome of acquired comitant non-accommodative esotropia without obvious cause in children with an early onset ≤ three years of age. PATIENTS AND METHODS: 24 consecutive patients (13 females) with acquired comitant non-accommodative esotropia underwent strabismus surgery. Outcome measures included amount of deviation and level of binocularity at last follow-up. RESULTS: An acute onset of strabismus was observed in 83 % of patients. Accompanying features such as diplopia, covering one eye or stumbling were observed in 40 % of the children. Cycloplegic retinoscopy revealed a mild to moderate hypermetropia in all children (mean hypermetropia + 2.2 diopters). 90.5 % of children were aligned within 8 PD of orthotropia. 81 % regained normal stereovision (Lang I/II). CONCLUSIONS: All children exhibited the typical features of acute acquired comitant esotropia (AACE) type II (Burian-Franceschetti). Collective characteristics of this form of strabismus are a comitant and relatively large deviation, mild hypermetropia, a potential of normal binocular cooperation and absence of an accommodative component and neurological pathology. Time and suddenness of strabismus onset may be less helpful in defining this entity.

Who is prone to high levels of distress after prophylactic mastectomy and/or salpingo-ovariectomy?

BACKGROUND: The present study aimed to assess predictors of distress after 'prophylactic mastectomy (PM) and salpingo-ovariectomy (PSO), in order to enable the early identification of patients who could benefit from psychological support. PATIENTS AND METHODS: General distress and cancer-related distress were assessed in 82 women at increased risk of hereditary breast and/or ovarian cancer undergoing PM and/or PSO, before and 6 and 12 months after prophylactic surgery. Neurotic lability and coping were assessed before surgery. RESULTS: Cancer-related distress and general distress at both follow-up moments were best explained by the level of cancer-related and general distress at baseline. Being a mutation carrier was predictive of increased cancer-related distress at 6-month follow-up (but not at 12 months), and of lower general distress 12 months after prophylactic surgery. Also, coping by having comforting thoughts was predictive of less cancer-related distress at 6-month follow-up. CONCLUSIONS: Genetically predisposed women who are at risk of post-surgical distress can be identified using one or more of the predictors found in this study. Exploration of and/or attention to cancer-related distress and coping style before prophylactic surgery may help physicians and psychosocial workers to identify women who might benefit from additional post-surgical support.

Effectiveness of breast cancer surveillance in BRCA1/2 gene mutation carriers and women with high familial risk.

PURPOSE: Women with a high breast cancer risk due to a familial predisposition may choose between preventive surgery and regular surveillance. The effectiveness of surveillance in high-risk women and especially BRCA1/2 mutation carriers is unknown. We present first results from a single large family cancer clinic. PATIENTS AND METHODS: Women with breast cancer risk over 15% were examined by physical examination every 6 months and mammography every year. Detection rates and screening parameters were calculated for the total group and separately for different age and genetic risk groups. RESULTS: At least one examination was performed in 1,198 women: 449 moderate and 621 high-risk women and 128 BRCA1/2 mutation carriers. Within a median follow-up of 3 years, 35 breast cancers were detected (four ductal carcinoma-in-situ; 31 invasive tumors); the average detection rate was 9.7 per 1,000. Detection rates (95% confidence interval) for moderate and high-risk women and BRCA1/2 carriers were 3.3 (1.1 to 8.6), 8.4 (5.4 to 13.2), and 33 (17 to 63) per 1,000 person-years, respectively. The ratio of observed cases versus breast cancers expected in an average-risk population of comparable age was 2.7, 7.0 and 23.7 respectively. Overall, node negativity was 65%; 34% of primary tumors were less than 10 mm; sensitivity was 74%. Results with respect to tumor stage and sensitivity were less favorable in BRCA1/2 carriers and in women under the age of 40. CONCLUSION: It is possible to identify young women at high risk for breast cancer. The number of cancers detected was significantly greater than expected in an age-matched average-risk population and related to the risk category. Overall, screening parameters were comparable to population screening data, with less favorable results in the youngest age group (< 40) and BRCA1/2 carriers.

Renal tolerance to nonhomogenous irradiation: comparison of observed effects to predictions of normal tissue complication probability from different biophysical models.

PURPOSE: A patient series was analyzed retrospectively as an example of whole organ kidney irradiation with an inhomogenous dose distribution to test the validity of biophysical models predicting normal tissue tolerance to radiotherapy. METHODS AND MATERIAL: From 1969 to 1984, 142 patients with seminoma were irradiated to the paraaortic region using predominantly rotational techniques which led to variable but partly substantial exposure of the kidneys. Median follow up was 8.2 (2.1-21) years and actuarial 10-year survival (Kaplan-Meier estimate) 82.8%. For all patients 3-dimensional dose distributions were reconstructed and normal tissue complication probabilities for the kidneys were generated from the individual dose volume histograms. To this respect different published biophysical algorithms had been introduced in a 3-dimensional-treatment planning system. RESULTS: In seven patients clinical manifest renal impairment was observed (interval 10-84 months). An excellent agreement between predicted and observed effects was seen for two volume-oriented models, whereas complications were overestimated by an algorithm based on critical element assumptions. CONCLUSIONS: Should these observations be confirmed and extended to different types of organs corresponding algorithms could easily be integrated into 3-dimensional-treatment planning programs and be used for comparing and judging different plans on a more biologically oriented basis.

Photogrammetric accuracy measurements of head holder systems used for fractionated radiotherapy.

PURPOSE: We describe how stereo photogrammetry can be used to determine immobilization and repositioning accuracies of head holder systems used for fractionated radiotherapy of intracranial lesions. METHODS AND MATERIALS: The apparatus consists of two video cameras controlled by a personal computer and a bite block based landmark system. Position and spatial orientation of the landmarks are monitored by the cameras and processed for the real-time calculation of a target point's actual position relative to its initializing position. The target's position is assumed to be invariant with respect to the landmark system. We performed two series of 30 correlated head motion measurements on two test persons. One of the series was done with a thermoplastic device, the other one with a cast device developed for stereotactic treatment at the German Cancer Research Center. Immobilization and repositioning accuracies were determined with respect to a target point situated near the base of the skull. The repositioning accuracies were described in terms of the distributions of the mean displacements of the single motion measurements. RESULTS: Movements of the target in the order of 0.05 mm caused by breathing could be detected with a maximum resolution in time of 12 ms. The data derived from the investigation of the two test persons indicated similar immobilization accuracies for the two devices, but the repositioning errors were larger for the thermoplastic device than for the cast device. Apart from this, we found that for the thermoplastic mask the lateral repositioning error depended on the order in which the mask was closed. CONCLUSION: The photogrammetric apparatus is a versatile tool for accuracy measurements of head holder devices used for fractionated radiotherapy.

Estimation of pneumonitis risk in three-dimensional treatment planning using dose-volume histogram analysis.

PURPOSE: Investigations to study correlations between the estimations of biophysical models in three dimensional (3D) treatment planning and clinical observations are scarce. The development of clinically symptomatic pneumonitis in the radiotherapy of thoracic malignomas was chosen to test the predictive power of Lyman's normal tissue complication probability (NTCP) model for the assessment of side effects for nonuniform irradiation. METHODS AND MATERIALS: In a retrospective analysis individual computed-tomography-based 3D dose distributions of a random sample of 46/20 patients with lung/esophageal cancer were reconstructed. All patients received tumor doses between 50 and 60 Gy in a conventional treatment schedule. Biological isoeffective dose-volume histograms (DVHs) were used for the calculation of complication probabilities after applying Lyman's and Kutcher's DVH-reduction algorithm. Lung dose statistics were performed for single lung (involved ipsilateral and contralateral) and for the lung as a paired organ. RESULTS: In the lung cancer group, about 20% of the patients (9 out of 46) developed pneumonitis 3-12 (median 7.5) weeks after completion of radiotherapy. For the majority of these lung cancer patients, the involved ipsilateral lung received a much higher dose than the contralateral lung, and the pneumonitis patients had on average a higher lung exposure with a doubling of the predicted complication risk (38% vs. 20%). The lower lung exposure for the esophagus patients resulted in a mean lung dose of 13.2 Gy (lung cancer: 20.5 Gy) averaged over all patients in correlation with an almost zero complication risk and only one observed case of pneumonitis (1 out of 20). To compare the pneumonitis risk estimations with observed complication rates, the patients were ranked into bins of mean ipsilateral lung dose. Particularly, in the bins with the highest patient numbers, a good correlation was achieved. Agreement was not reached for the lung functioning as a paired organ. CONCLUSIONS: Realistic assessments for the prediction of radiation-induced pneumonitis seem to be possible. In this respect, the implementation of DVH-analysis in 3D planning could be a helpful tool for the evaluation of treatment plans.

Stereotactically guided fractionated radiotherapy: technical aspects.

A system for high precision radiotherapy in the head and neck region has been developed. The components of the system are a head mask connected to a stereotactic frame, a localization unit that can be used during CT- and MR-imaging and a stereotactic target positioner. Conformal precision radiotherapy is planned with a new treatment planning system (Voxelplan-Heidelberg). Three different multi-leaf collimator systems are used. An evaluation of the precision and accuracy of the head fixation system, which was performed with a photogrammetry system, is presented.

Influence of the positioning error on 3D conformal dose distributions during fractionated radiotherapy.

The influence of patient immobilization error on 3D planned conformal radiation therapy in tumors of the thorax and pelvis was studied. The mean positioning error in 43 patients with carcinomas of the thorax and pelvis undergoing 3D conformal radiotherapy (laser supported alignment, no immobilization device) was measured. A total of 194 portal films were superposed with the corresponding simulator radiographs according to anatomic landmarks and using a subtrascope. x-, y- and z-axis deviation was determined within a coordinate system. Using specialized software including Fourier transformation the mean positioning error was employed to recalculate the dose distributions of all cases under the influence of random (Gaussian) immobilization uncertainty. The mean two-dimensional positioning error using the data from all patients was 5.5 (+/- 3.7) mm. The distribution was Gaussian. Dose volume histograms (DVHs) of each patient with and without consideration of positioning uncertainty were compared on the base of tumor control probability estimations (TCP) using published DVH reduction and TCP algorithms. Inclusion of the positioning error resulted in a mean decrease in TCP (given as the difference between the TCP assuming no positioning error and the TCP modified by the positioning error) of 2% in a series of esophagus carcinomas and of 5% in the prostate carcinomas when looking at gross tumor volume (GTV), only. Planning target volume (PTV) exhibited a relative decrease in TCP of 5% and 11%, respectively.

Early mycosis fungoides: molecular analysis for its diagnosis and the absence of p53 gene mutations in cases with progression.

The histological diagnosis of initial mycosis fungoides (MF) and the molecular mechanisms that are responsible for its progression and transformation to the more highly malignant variants of MF remain largely unknown. Because of the rare occurrence of these tumours, the need for snap frozen skin biopsy specimens and the difficulty to obtain suitable material for karyotypic and genotypic analysis, specific cytogenetic and molecular lesions have not yet been identified. In particular the role of known oncogenes and tumour suppressor genes, including the p53 gene, in the pathogenesis and clinical progression of MF has not been extensively investigated. The present study was carried out using the polymerase chain reaction (PCR) technique combined with temperature gradient gel electrophoresis (TGGE) to detect mutations of the p53 gene in 58 patients with MF. TGGE analysis was also used in combination with clonality analysis by means of T-cell receptor gamma (TCRG) gene rearrangement studies to distinguish parapsoriasis en plaque and initial MF from patch/plaque stage MF. More than 83% of the diagnoses of initial MF could be confirmed using PCR-TGGE analysis. However, although the sensitive TGGE analysis was used for all exons, p53 gene polymorphisms were found in 4 and p53 gene mutation in only 1 of 58 biopsy specimens. It appears unlikely that p53 gene mutations play a role in either the pathogenesis of parapsoriasis and initial MF or their progression to advanced stages of MF. However, TCRG gene rearrangement studies by means of TCR-TGGE analysis may be useful for distinguishing histologically discordant cases of initial MF.

The retroperitoneal resection margin and vessel involvement are important factors determining survival after pancreaticoduodenectomy for ductal adenocarcinoma of the head of the pancreas.

The prognosis of ductal adenocarcinoma of the pancreas is still poor. We analysed the factors that have a major influence on the survival of patients. Surgical specimens from 51 patients with ductal adenocarcinoma of the head of the pancreas were examined for tumour size, histological type, grade and local extension. In 7 patients the retroperitoneal resection margin was involved either macroscopically or histologically. Their mean survival was 10.6 months (1-17 months), compared with 22.7 months for the 44 patients with curative R0 resection. In 10 patients large vessels (portal and/or mesenteric vein) had to be resected; they survived for only 2-11 months, with a mean of 5 months (P<0.05). Non-R0-resected patients and patients in whom tumour-invaded vessels had to be resected constitute a high-risk group with a significantly shorter mean survival of 8.8 months, compared with 24.3 months for R0 resected patients without vessel invasion (P<0.05). Lymph node metastases were seen in 35 of 51 patients. Survival analysis based on nodal status revealed a mean survival of 33 months for patients staged as N0, 21.4 for N1a patients and 14 month for N1b patients. The differences were not statistically significant, however. Our data suggest that tumour invasion of the retroperitoneal resection margin and large vessel involvement are the major factors determining survival in patients with pancreatic cancer.

Duct changes and K-ras mutations in the disease-free pancreas: analysis of type, age relation and spatial distribution.

Recent molecular studies have suggested that hyperplastic duct lesions of the pancreas are potential precursors of pancreatic ductal carcinoma. This study examines the type, distribution, age-related incidence and K-ras codon 12 mutation rate of duct lesions in the normal pancreas. Postmortem pancreases from 140 patients were screened for the presence of mucinous cell hypertrophy (MHT), ductal papillary hyperplasia (DPH), adenomatoid ductal hyperplasia (ADH), and squamous metaplasia (SQM). Microdissected cell samples were analyzed for K-ras codon 12 mutations by polymerase chain reaction amplification of exon 1 of the K-ras gene, combined with constant denaturing gel electrophoresis, and analyzed by sequencing. Of the 140 specimens 114 showed duct lesions. The lesions were evenly distributed throughout the pancreas. They were more common beyond the age of 40. MHT was present in 68%, DPH in 36%, ADH in 40%, and SQM in 36% of the cases. K-ras mutations were found in 19 samples from 15 out of 79 pancreases (18%), including all types of duct lesions and a variant of ADH with dense stroma. 67% of the K-ras-positive specimens showed the transition GGT to GAT (8) or GTT (5). Hyperplastic/metaplastic duct changes of the pancreas increase with age, but their distribution pattern in the pancreas differs from that of ductal carcinomas.

Detection of specific DNA lesions by a combination of comet assay and FISH in plants.

We have studied comet formation on Vicia faba nuclei embedded in agarose and treated with the endonucleases DNase I (to produce SSBs or DSBs at random sites), FokI (to produce DSBs preferentially within FokI repeats), or EcoRI (to produce DSBs at random sites but not within FokI elements). DNase I-induced SSBs were detected when enzyme treatment was followed by alkaline denaturation. DSBs efficiently mediated comet formation using neutral conditions. FISH with DNA probes, detecting specific chromosomal domains such as FokI element-containing heterochromatin, NORs, or telomeres, was done on comets. The distribution of FISH signals between the head and tail of comets indicated to which degree these domains were damaged and reflected the distribution of cleavage sites for the applied restriction endonucleases within these domains. The data confirmed the expectation that the observed comet formation was based on enzyme-specific DNA breakage.

Adaptation to alkylation damage in DNA measured by the comet assay.

The alkylating mutagens N-methyl-N-nitrosourea (MNU) and methyl methanesulfonate (MMS) were studied for their potential to induce DNA strand breaks and abasic (AP) sites in meristematic nuclei of Vicia faba root tips by the comet assay. The alkaline unwinding/neutral electrophoresis (A/N) and alkaline unwinding/alkaline electrophoresis (A/A) protocols were used for detection of DNA damage. With the A/N comet assay, less DNA damage was seen after conditioning pretreatment with a low dose prior to a high challenging dose of alkylating mutagens as compared to application of the high dose only, whereas a nearly additive effect was seen when the A/A comet assay was used. Adaptation was even more obvious when AP sites were revealed by the AP-endonuclease activity of exonuclease III. The adaptation observed with the A/N comet assay was abolished by pretreatment with the protein synthesis inhibitor cycloheximide. These data suggest that the comet assay is able to detect on molecular level a phenomenon resembling clastogenic adaptation.

The prognostic influence of neuroendocrine differentiation in breast cancer: results of a long-term follow-up study.

Reports about neuroendocrine (NE) differentiation in breast carcinomas and its possible relation with prognosis are scarce. Furthermore the results of some studies have not been subjected to multivariate survival analysis and the follow-up periods were relatively short. Therefore, in the present long-term follow-up study, the prognostic influence of immunohistochemically defined NE cells, present in the tumours of 40 out of 317 (12.6%) curatively operated breast cancer patients, was studied. The mean follow-up period was 104 months. NE differentiation (NED) was determined by the immunohistochemical detection of chromogranin A and/or synaptophysin. This is concordant with other studies focussing on NED in breast cancer. In contrast to the literature in our series only in 9 out of 40 cases (23%) we were able to detect coexpression of chromogranin A and synaptophysin. This might be due to the characteristics of the antibodies we used. Although most tumours in our series were of the usual type, some tumours with NED were of a special type. Neither univariately, nor taking account of various known prognostic factors, does focal NED appear to carry a special prognostic significance. This finding is in line with results of previous studies.

The comet assay detects adaptation to MNU-induced DNA damage in barley.

We have established the comet assay for detection of DNA damage in barley. Immediately after treatment with the monofunctional alkylating agent MNU, a dose-dependent increase of DNA damage (mainly DNA breaks) was detected by the alkaline denaturation/neutral gel electrophoresis (A/N) variant of the comet assay in nuclei isolated from root tip meristems and from young leaves. A reduction of damage was observed within meristematic nuclei but not in differentiated leaf nuclei 48h after treatment. Adaptive pretreatment with a nontoxic dose of cadmium chloride prior to challenge treatment with MNU reduced the frequency of chromatid type aberrations, micronuclei and aneuploid cells as well as the amount of DNA in comet tails of meristematic nuclei.

DNA damage and repair in Arabidopsis thaliana as measured by the comet assay after treatment with different classes of genotoxins.

The three protocols of the comet assay A/N, A/A and N/N were for the first time applied to the plant species Arabidopsis thaliana. The purpose of the experiments was to establish conditions for genotoxic exposure causing DNA damage in Arabidopsis nuclei. This is required for comprehensive gene expression profiling with the intention to screen for genes involved in response of Arabidopsis cells to genotoxic stress. Five chemicals belonging to different classes of mutagens (the monofunctional alkylating agents N-methyl-N-nitrosourea and methyl methanesulfonate, the polyfunctional alkylating agent mitomycin C, the radiomimetic bleomycin and the herbicide maleic hydrazide) were tested. Except for maleic hydrazide, dose-dependent increases in DNA damage were found using the A/N comet assay protocol. While a rapid repair of bleomycin-mediated SSBs and DSBs was found, no significant reduction of DNA migration was observed up to 48h after treatment with the monofunctional alkylating agents.

Basement membrane proteins in synovial membrane: distribution in rheumatoid arthritis and synthesis by fibroblast-like cells.

Rheumatoid arthritis is a complex disease of unknown origin. In consequence of some immunological reactions, proliferative invading synovial tissue leads to destruction of normal joint architecture. The aim of this study was to investigate qualitative changes in extracellular matrix distribution of proliferating rheumatoid synovium and their cellular origin. Synovial tissues from 57 clinically indicated arthrotomies were investigated with immunofluorescence, using specific antibodies against extracellular matrix proteins in tissue slides and cultured cells, which were also studied for collagen biosynthesis. Results indicated that synovial fibroblast-like cells synthesize and secrete basement membrane proteins laminin and collagen type IV as e.g. endothelial cells or organogenic fibroblasts. Laminin and collagen type IV were specifically demonstrated pericellularly in the hyperplastic lining layer of active rheumatoid synovitis. These findings are discussed with respect to the possible implication of altered cell-matrix interactions in rheumatoid synovial proliferation.