OCT2, SSX and SAGE1 reveal the phenotypic heterogeneity of spermatocytic seminoma reflecting distinct subpopulations of spermatogonia.

Spermatocytic seminoma (SS) is a rare testicular neoplasm that occurs predominantly in older men. In this study, we aimed to shed light on the histogenesis of SS by investigating the developmental expression of protein markers that identify distinct subpopulations of human spermatogonia in the normal adult testis. We analysed the expression pattern of OCT2, SSX2-4, and SAGE1 in 36 SS cases and four intratubular SS (ISS) as well as a series of normal testis samples throughout development. We describe for the first time two different types of SS characterized by OCT2 or SSX2-4 immunoexpression. These findings are consistent with the mutually exclusive antigenic profile of these markers during different stages of testicular development and in the normal adult testis. OCT2 was expressed predominantly in A(dark) spermatogonia, SSX2-4 was present in A(pale) and B spermatogonia and leptotene spermatocytes, whilst SAGE1 was exclusively present in a subset of post-pubertal germ cells, most likely B spermatogonia. The presence of OCT2 and SSX2-4 in distinct subsets of germ cells implies that these markers represent germ cells at different maturation stages. Analysis of SAGE1 and SSX2-4 in ISS showed spatial differences suggesting ongoing maturation of germ cells during progression of SS tumourigenesis. We conclude that the expression pattern of OCT2, SSX2-4, and SAGE1 supports the origin of SS from spermatogonia and provides new evidence for heterogeneity of this tumour, potentially linked either to the cellular origin of SS or to partial differentiation during tumour progression, including a hitherto unknown OCT2-positive variant of the tumour likely derived from A(dark) spermatogonia.

Handling of radical prostatectomy specimens: total or partial embedding?

AIMS: Proper examination and accurate reporting of radical prostatectomy specimens (RPS) is essential in determining post-surgical treatment and predicting patient outcome. Surveys have demonstrated the absence of consensus on handling of RPS. The aim of this study was to determine whether significant information is lost when only half the horizontal tissue sections are examined. METHODS AND RESULTS: During a 1-year period, 238 RPS were sectioned into horizontal slices. Apex and basis was cut sagittally, and remaining slices were embedded in quadrants. Glass slides from every second horizontal slice were withheld. The remaining slides were evaluated microscopically, and essential pathological parameters were recorded. Subsequently, a full report was compiled, including the withheld slides. A median of 12 slides (30%) were withheld during initial assessment. In eight RPS (3.2%) the pTNM stage had to be changed; in six cases (2.6%) from pT2b to pT2c and in two cases (0.8%) from pT2c to pT3a. In one RPS (0.4%) the surgical margin status was changed. CONCLUSIONS: Only little information is lost with systematic partial embedding, overlooking features significant for the postoperative treatment in only 1.2%. Partial embedding as suggested, decreasing the laboratory workload by 30%, is concluded to be acceptable for valid histopathological assessment.

Activating mutations in FGFR3 and HRAS reveal a shared genetic origin for congenital disorders and testicular tumors.

Genes mutated in congenital malformation syndromes are frequently implicated in oncogenesis, but the causative germline and somatic mutations occur in separate cells at different times of an organism's life. Here we unify these processes to a single cellular event for mutations arising in male germ cells that show a paternal age effect. Screening of 30 spermatocytic seminomas for oncogenic mutations in 17 genes identified 2 mutations in FGFR3 (both 1948A>G, encoding K650E, which causes thanatophoric dysplasia in the germline) and 5 mutations in HRAS. Massively parallel sequencing of sperm DNA showed that levels of the FGFR3 mutation increase with paternal age and that the mutation spectrum at the Lys650 codon is similar to that observed in bladder cancer. Most spermatocytic seminomas show increased immunoreactivity for FGFR3 and/or HRAS. We propose that paternal age-effect mutations activate a common 'selfish' pathway supporting proliferation in the testis, leading to diverse phenotypes in the next generation including fetal lethality, congenital syndromes and cancer predisposition.

A comparison of endoscopic ultrasound guided biopsy and positron emission tomography with integrated computed tomography in lung cancer staging.

BACKGROUND AND STUDY AIMS: Exact staging of patients with non-small-cell lung cancer (NSCLC) is important to improve selection of resectable and curable patients for surgery. Positron emission tomography with integrated computed tomography (PET/CT) and endoscopic ultrasound guided fine needle aspiration biopsy (EUS-FNA) are new and promising methods, but indications in lung cancer staging are controversial. Only few studies have compared the 2 methods. The aim of this study was to assess and compare the diagnostic values of PET/CT and EUS-FNA for diagnosing advanced lung cancer in patients, who had both procedures performed. PATIENTS AND METHODS: 27 patients considered to be potential candidates for resection of NSCLC underwent PET/CT and EUS-FNA. Diagnoses were confirmed either by open thoracotomy, mediastinoscopy or clinical follow-up. Advanced lung cancer was defined as tumour-stage ≥ IIIA(N2), corresponding to T4- and/or N2-N3- and/or M1 disease. Diagnostic values of PET/CT and EUS-FNA, with regard to the diagnosis of advanced lung cancer, were assessed and compared. RESULTS: The sensitivity of PET/CT and EUS-FNA were respectively 60% and 60% for T4 disease, 56% versus 100% for N2-N3 disease (p=0.12) and 100% versus 33% for M1 disease (p=0.50). For diagnosing advanced lung cancer PET/CT had a sensitivity of 79%, specificity of 61%, positive predictive value (PPV) of 69%, negative predictive value (NPV) of 73%, and an accuracy of 70%. EUS-FNA had a sensitivity of 79%, specificity of 100%, PPV of 100%, NPV of 81%, and an accuracy of 89% for advanced lung cancer. CONCLUSIONS: PET/CT and EUS-FNA had a comparable sensitivity and NPV for diagnosing advanced lung cancer, but EUS-FNA had superior specificity and PPV. The two methods seem to complement each other.

Tumor infiltrating lymphocytes in seminoma lesions comprise clonally expanded cytotoxic T cells.

Seminoma lesions are characterized by a brisk inflammatory infiltrate containing both CD4 and CD8 T cells, which is of prognostic significance. However, whether seminoma cells express the HLA molecules required for classical T-cell recognition remains controversial. In the present study, we conducted a molecular, phenotypical and functional characterization of tumor infiltrating lymphocytes (TILs) from seminoma lesions. T-cell receptor clonotype mapping demonstrated the presence of clonally expanded T cells in the majority of the lesions. The cytotoxic capacity of TILs was indicated by expression of CD107a, which is a recently described surrogate marker for cytolytic activity. Indeed, the frequency of CD107a positive cells was substantially higher in TILs when compared to peripheral blood mononuclear cells. Moreover, fluorescence activated cell sorting of CD107a positive TILs allowed comparison of the clonotypic T-cell receptor fingerprint and demonstrated the ability of expanded clones to express this cytotoxic marker, suggesting cytotoxic activity at the tumor site. The cytotoxicity was confirmed by in situ granzyme B expression. Furthermore, by staining with multimeric HLA-peptide complexes, we could demonstrate the presence of Mage-3 specific T cells among TILs. In summary, specific and functional T-cell responses are operative in seminoma, indicating that the inflammatory infiltrate is indeed involved in the immunological control of the tumor.

Endoscopic ultrasound guided biopsy performed routinely in lung cancer staging spares futile thoracotomies: preliminary results from a randomised clinical trial.

BACKGROUND: Up to 45% of operations with curative intent for non-small-cell lung cancer (NSCLC) can be regarded as futile, apparently because the stage of the disease is more advanced than expected preoperatively. During the past decade several studies have evaluated the usefulness of endoscopic ultrasound guided fine needle aspiration biopsy (EUS-FNA) in lung cancer staging with promising results. However, no randomised trials have been performed, in which a staging strategy with EUS-FNA performed in all patients is compared with a conventional workup. METHODS: Before surgery (i.e. mediastinoscopy and subsequent thoracotomy) 104 patients from one hospital were randomly assigned to either a conventional workup (CWU), including EUS-FNA only for selected patients, or a strategy where all patients were offered EUS-FNA (routine EUS-FNA) in addition to CWU. Patients were followed up for a median period of 1.3 years (range 0.2-2.4 years). Thoracotomy was regarded as futile if the patient had an explorative thoracotomy without tumour resection or if a resected patient had recurrent disease or died from lung cancer during follow-up. Analysis was by intention to treat. RESULTS: Fifty-three patients were randomly assigned to routine EUS-FNA and 51 patients to CWU. EUS-FNA was performed in 50 patients (94%) in the routine EUS-FNA group and in 14 patients (27%) in the CWU group. In the routine EUS-FNA group five patients (9%) had a futile thoracotomy, compared with 13 (25%) in the CWU group, p = 0.03. CONCLUSION: Addition of routine-EUS-FNA to standard workup in routine clinical practice improved selection of surgically curable patients with NSCLC.

Microinvasive germ cell tumor of the testis.

Microinvasive germ cell tumor (MGCT) consists of a limited number of malignant germ cells in the intertubular tissue of the testis. The cells have large nuclei, prominent nucleoli, abundant clear cytoplasm, and distinct cellular borders in hematoxylin and eosin staining. MGCT can be the first stage of malignancy in the development of testicular germ cell tumor (TGCT). Biopsies from men with maldescended testes have been reported to contain intratubular germ cell neoplasia, unclassified (IGCN) and MGCT in 1.8% of the examined cases (95% CI 0.5-4.6%). MGCT has also been found in testes of subfertile men and in the contralateral testis of patients with TGCT. MGCT is a frequent finding (19%) in the testicular tissue adjacent to an overt TGCT. Men with a high risk of TGCT may gain from screening for precursor lesions of TGCT with ultrasonography of the testes followed by a testicular biopsy if suspicious abnormalities are found: Treatment is high-voltage radiotherapy for intratubular germ cell neoplasia (IGCN), and orchidectomy for MGCT and germ cell tumor in situ, either intratubular seminoma or intratubular embryonal carcinoma. After local treatment, patients with precursor lesions can be followed with a surveillance program. The mRNA levels of invasion-related genes were evaluated based on a DNA microarray data set, and we found two gene abnormalities most relevant for the invasion of malignant germ cells: matrix metalloproteinase 9 (MMP9) and plasminogen activator, urokinase (PLAU) genes were up-regulated in a study comparing tissue samples of TGCT and IGCN.

Endoscopic ultrasound guided biopsy versus mediastinoscopy for analysis of paratracheal and subcarinal lymph nodes in lung cancer staging.

BACKGROUND: Exact mediastinal evaluation of patients with non-small-cell lung cancer (NSCLC) is mandatory to improve selection of resectable and curable patients for surgery. Mediastinoscopy (MS) and endoscopic ultrasound guided fine needle aspiration biopsy (EUS-FNA) are considered complementary, MS covering the anterior- and EUS-FNA the posterior mediastinum. Both methods can reach the paratracheal- and subcarinal-regions, but little is known about which method is most accurate, when compared in patients having both procedures performed. The aim of this study was to assess and compare the diagnostic value of MS and EUS-FNA with regard to mediastinal malignancy in the paratracheal- and subcarinal-regions. METHODS: Sixty patients considered to be potential candidates for resection of verified or suspected NSCLC underwent MS and EUS-FNA. The EUS-FNA diagnoses were confirmed either by open thoracotomy, MS or clinical follow-up. RESULTS: MS and EUS-FNA were conclusive for paratracheal or subcarinal mediastinal disease in 6 and 24 patients, respectively. Two patients with N2 disease diagnosed by EUS-FNA were upstaged to N3 by MS. The sensitivity for lymph node metastases in the right paratracheal region (2/4R) was 67% for EUS-FNA versus 33% for MS (p=0.69). In the left paratracheal region (2/4L) the sensitivity of EUS-FNA was 80% versus 33% for MS (p=0.06). In the subcarinal region (7) the sensitivity of EUS-FNA was 100% versus 7% for MS (p<0.01). The sensitivity for lymph node metastases in region 2/4L and/or 2/4R and/or 7 was 96% for EUS-FNA versus 24% for MS (p<0.01). CONCLUSION: In our hands EUS-FNA was superior to MS in the examination of paratracheal- and subcarinal-regions of patients considered for resection of lung cancer.

Immunohistochemical demonstration of vascular endothelial growth factor in vestibular schwannomas correlates to tumor growth rate.

OBJECTIVE: Vascular endothelial growth factor (VEGF) is one of the most potent mediators of angiogenesis, which is a mandatory process during tumor growth. The present objectives were to determine expression of VEGF in vestibular schwannomas by immunohistochemistry and to examine a possible correlation with symptom duration, tumor size, or growth rate. STUDY DESIGN: Retrospective patient file review; immunohistochemistry and light microscopy of vestibular schwannomas removed by surgery. METHODS: Vestibular schwannomas from 18 patients were immunolabelled using a polyclonal antibody against VEGF, followed by light microscopy and blinded semiquantitation of VEGF expression. Fifteen patients had a well-defined tumor growth rate defined by repeated preoperative magnetic resonance imaging scans. RESULTS: All tumors showed expression of VEGF in the Schwann cell cytoplasm, with a more intense staining of the perinuclear region of some cells. The staining intensity varied from tumor to tumor, and semiquantitation revealed a significant correlation between VEGF expression and tumor growth rate, but not symptom duration or tumor size. CONCLUSION: VEGF is expressed in vestibular schwannomas and the level of expression correlates positively with tumor growth rate, but not with tumor size and symptom duration. We conclude that VEGF seems to be a factor involved in the growth of vestibular schwannomas.

[Endoscopic ultrasound-guided biopsy of suspected malignancy in the mediastinum has a major impact on the clinical decision process]. / Endoskopisk UL-vejledt biopsi af malignitetssuspekte processer i mediastinum har stor indflydelse på den kliniske beslutningsproces.

INTRODUCTION: A study was undertaken to evaluate the clinical impact of endoscopic ultrasound-guided, fine needle aspiration biopsy (EUS-FNA) in patients with mediastinal masses suspected of malignancy. MATERIAL AND METHODS: Eighty-four patients were referred for EUS-FNA. In all patients computer tomography (CT) had shown a lesion of the mediastinum suspected of malignancy, which was located adjacent to the oesophagus. The history of each patient up to referral for EUS-FNA was reviewed in order to evaluate the clinical impact of EUS-FNA. A board of thoracic specialists was asked to decide the further course of the patient if EUS-FNA had not been available, and this diagnostic strategy was compared with the actual clinical course after EUS-FNA. RESULTS: For the 79 patients, in whom sufficient verification was obtained, EUS-FNA had a sensitivity of 92%, a specificity of 100%, a positive predictive value of 100%, a negative predictive value of 80%, and an accuracy of 94% for cancer in the mediastinum. As a result of EUS-FNA, a thoracotomy was avoided in 18 of 37 patients (49%) and in 28 of 41 patients (68%) a mediastinoscopy was avoided. The direct result of the cytological diagnosis obtained by EUS-FNA was that a final diagnosis of small cell lung cancer was made in eight patients leading to referral for chemotherapy, and specific therapy could be initiated in another three patients with benign disease (sarcoidosis, mediastinal abscess and leiomyoma of the oesophagus). DISCUSSION: EUS-FNA is a safe and sensitive, minimal invasive method in the evaluation of patients with a solid lesion of the mediastinum, suspected by CT. EUS-FNA has a significant impact on patient management; it should be considered for diagnosing the spread of cancer to the mediastinum of patients with lung cancer in whom surgery is contemplated, as well as for the primary diagnosis of solid lesions located in the mediastinum adjacent to the oesophagus.

The cancer-testis gene, NY-ESO-1, is expressed in normal fetal and adult testes and in spermatocytic seminomas and testicular carcinoma in situ.

Cancer/testis genes are potential targets for therapeutic genetic and immunologic approaches, and are highly expressed in a large variety of human cancers. However, they are not expressed in normal tissues, with the exception of the testis. The NY-ESO-1 gene is the most recently identified member of the cancer/testis family and its product is one of the most immunogenic tumor antigens. We used immunohistochemistry to investigate the expression of NY-ESO-1 in healthy human prenatal and adult testes and in 59 human testicular tumors of different subtypes. We found that NY-ESO-1 was expressed from 18 weeks until birth in human fetal testes. In the adult testis, NY-ESO-1 was strongly expressed in spermatogonia and in primary spermatocytes, but not in post-meiotic cells or in testicular somatic cells. NY-ESO-1 was not expressed in the Sertoli cells, Leydig cells, classical seminomas, or nonseminomatous germ cells in the 59 testicular tumors. In contrast, NY-ESO-1 was expressed both in carcinomas in situ, which are the earliest stage of testicular tumors (7 of 15 cases), and in spermatocytic seminomas, which are believed to be derived from spermatogonia or primary spermatocytes (8 of 16 cases). We conclude that NY-ESO-1 is a marker that can be used to follow the early progression of testicular tumorigenesis when the tumors present a similar pattern of expression to the cells from which they originated, although the later tumors cease to express NY-ESO-1.

Serum lactate dehydrogenase isoenzyme 1 in patients with seminoma stage I followed with surveillance.

Serum lactate dehydrogenase isoenzyme I catalytic concentration (S-LD-1) was measured in patients with testicular seminoma clinical stage I followed with surveillance after orchiectomy. The serum samples were obtained before orchiectomy in 110 patients (group A) and soon after orchiectomy in 55 patients (group B). In group A, 60 patients (55%) had elevated S-LD-1 and 10 patients (9%) had elevated serum human chorionic gonadotropin concentrations (S-hCG). In group B, median S-LD-1 was lower than that of group A and decreased with increasing time after orchiectomv (p = 0.001, Jonckheere-Terpstra test, one-sided). After a median follow-up of 5.1 years, 23 patients (21%) in group A had relapses. The patients with elevated S-LD-1 and those with normal S-LD-1 had a similar relapse-free survival (p = 0.79, log-rank test). Thus patients with seminoma stage I had elevated S-LD-1 more often than elevated S-hCG but an elevation in S-LD-1 did not predict a relapse during follow-up with surveillance. Further studies are required to elucidate the value of S-LD-1 in monitoring the surveillance of patients with seminoma stage I.

Immunohistochemical demonstration of tumour associated antigens in carcinoma-in-situ of the testis.

Atypical germ cells or so-called carcinoma-in-situ of the testis are often found in the tissue adjacent to germ cell tumours. The present study was performed to investigate if tumour associated antigens demonstrated immunohistochemically in the tumours could also be demonstrated in the carcinoma-in-situ. Using indirect immunoperoxidase technique 39 orchidectomy specimens were examined for the presence of a series of antigens: alpha-foetoprotein (AFP), alpha1 -antitrypsin (A1 AT), human chorionic gonadotrophin (hCG). specific pregnancy ß-glycoprotein (SP1 ), human placental lactogen (hPL), carcino-embryonic antigen (CEA) and ferritin (FER). FER was demonstrated in the atypical cells in 24/29 cases of carcinoma-in-situ of the testis. HCG was demonstrated in intratubular syncytiotrophoblast-like cells in one case and in atypical germ cells in another specimen. No staining reaction was found for the other antigens investigated Normal germinal epithelium and germinal epithelium in non-malignant pathological changes of the testis were never stained. These findings which indicate that FER may be a possible marker of carcinoma-in-situ of the testis is of utmost interest, however investigation of a larger series is mandatory.