[Routine high-throughput targeted sequencing of lymphoproliferative diseases : Clinical utility and challenges]. / Routinemäßige zielgerichtete Hochdurchsatzsequenzierung von lymphoproliferativen Erkrankungen : Klinischer Nutzen und Herausforderungen.

In contrast to other tumour entities such as lung carcinoma, melanoma or gynaecological and gastrointestinal tumours, the routine application of mutation analyses using high-throughput sequencing via next-generation sequencing (NGS) has not yet been widely established in haematopathology, especially not in lymphomas.Here we describe our experience with the use and routine implementation of a lymphoma NGS panel primarily developed for research purposes.In addition to a discussion of the steps necessary for transferring such a panel into the routine framework of an accredited institute, we show by the comprehensive workup of 80 investigations and the presentation of several case studies how the panel was able to guide us to the correct diagnosis and how it also provided clinicians with indications for possible tailored therapy options.Even if NGS does not (yet) have to be routinely applied in lymphoma diagnostics for every case, a respectively dedicated NGS panel offers the advantage of having an additional option in the case of difficult differential diagnostic considerations or uncertainties as well as at the request of the treating oncologist to identify potential targets for tailored treatment of the patients.

[Abdominal pain and hypertension in a 55-year-old male patient]. / Abdominale Beschwerden und Hypertonus bei einem 55­jährigen Patienten.

A 55-year-old male patient under permanent testosterone therapy for hypogonadism presented with abdominal pain and increased blood pressure values. In the physical examination a plethora was noted and laboratory examinations revealed polyglobulia. In the subsequent diagnostic process polycythemia vera and cancer could be excluded as the cause. A secondary polyglobulia due to testosterone substitution was diagnosed. Unphysiologically high testosterone levels represent a rare cause of secondary polyglobulia and with an appropriate medical history should be taken into account at an early stage.

[Normal bone marrow and common reactive alterations]. / Normales Knochenmark und häufige reaktive Veränderungen.

Histological examination of bone marrow biopsies is an important and powerful diagnostic tool to assess various hematological and non-hematological disorders. Morphological examination of such biopsies requires knowledge of the composition of normal bone marrow and its variations, such as age-related changes. Diagnostic problems may arise due to poor specimen quality, insufficient sections or stainings and insufficient experience with reactive bone marrow changes which occasionally resemble neoplastic disorders. Reactive bone marrow processes can affect one or more hematopoietic cell lines, lead to disruption of the normal architecture and specifically affect the bone marrow stroma. Optimal bone marrow diagnosis requires adequately stained slides and, when needed, immunophenotyping and molecular examinations. Furthermore, rather than biopsy interpretation of other organs, pathologists routinely need clinical history information for correct interpretation and diagnosis of bone marrow changes. In this article, the normal features of bone marrow as well as the most frequent reactive bone marrow alterations are described.

[Clonal relationship of Hodgkin lymphoma and its recurrence]. / Klonale Verwandtschaft von Hodgkin-Lymphomen und deren Rezidiven.

In this study, we investigated whether recurrences of classical Hodgkin's lymphoma (HL) are true relapses arising from the primary tumour or clonally unrelated secondary neoplasias. Formalin-fixed, paraffin-embedded tissue specimens of eleven patients with recurrent HL were analyzed. Hodgkin and Reed-Sternberg cells were microdissected after immunohistochemical staining for CD30 using laser-capture technique. Immunoglobulin heavy chain (IgH) gene fragment lengths were analyzed applying consensus FR3 and J primers. Two early relapses after the first HL diagnosis were clonally related to the initial tumour, while three of four early recurrences after a first or second relapse were not. Three patients presenting with late relapses had clonally unrelated neoplasms. Therefore, we conclude that recurrent HL may represent a novel neoplasm, a finding which might play a role in clinical decision-making.

Patterns of liver infiltration in lymphoproliferative disease.

AIMS: Liver involvement is a common finding in patients suffering from lymphoproliferative disease, and histopathological patterns of infiltration vary according to lymphoma subtype. Data correlating the form of liver involvement with distinct lymphoma subtypes is, however, scarce. The aim was to review 89 liver biopsies diagnosed with lymphoma infiltration and evaluate the infiltration patterns. METHODS AND RESULTS: In equivocal cases, additional immunohistochemical and molecular pathology analyses were performed to differentiate between neoplastic and reactive cell infiltrates and to classify the lymphoma subtypes. Diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukaemia (CLL), Hodgkin's lymphoma (HL) and Burkitt lymphoma (BL) were the most prevalent subtypes in our series, which included 14 different lymphoma entities in total. Whereas DLBCL and BL predominantly demonstrated tumour nodules deranging the normal hepatic architecture, CLL and HL mostly showed infiltration of the portal fields. Interestingly, distinct lymphoma entities, particularly marginal zone B-cell lymphomas (MZL) and HL, commonly revealed lympho-epithelial lesions of bile ducts, which were observed in 10% of all investigated cases. Four cases, initially interpreted as T-cell lymphomas, proved to be reactive T-cell lesions. CONCLUSIONS: Distinct lymphoma subtypes show characteristic patterns of liver infiltration. Additional molecular analyses can support diagnosis by verification of clonality or detection of characteristic genetic aberrations.

[Primary cerebellar T-cell lymphoma]. / Primäres zerebelläres T-Zell-Lymphom.

Primary central nervous system T-cell lymphomas are rare and have to be differentiated from reactive lesions. It is therefore essential to use all possible tools to establish the diagnosis, including immunohistochemistry, molecular genetic analysis, and/or cytogenetic methods. In this paper we present the case of a primary cerebellar T-cell lymphoma in a 50-year-old man; a clonal T-cell receptor gene rearrangement was documented. After two cycles of methotrexate therapy the patient developed Pneumocystis carinii-induced pneumonia, dying 10 weeks after his diagnosis. The autopsy did not reveal any residual tumour.

High-throughput sequencing of nodal marginal zone lymphomas identifies recurrent BRAF mutations.

Nodal marginal zone lymphoma (NMZL) is a rare small B-cell lymphoma lacking disease-defining phenotype and precise diagnostic markers. To better understand the mutational landscape of NMZL, particularly in comparison to other nodal small B-cell lymphomas, we performed whole-exome sequencing, targeted high-throughput sequencing, and array-comparative genomic hybridization on a retrospective series. Our study identified for the first time recurrent, diagnostically useful, and potentially therapeutically relevant BRAF mutations in NMZL. Sets of somatic mutations that could help to discriminate NMZL from other closely related small B-cell lymphomas were uncovered and tested on unclassifiable small B-cell lymphoma cases, in which clinical, morphological, and phenotypical features were equivocal. Application of targeted gene panel sequencing gave at many occasions valuable clues for more specific classification.

Human chorionic gonadotropin (hCG) in the male reproductive tract.

Normal hypothalamic-pituitary testicular and prostatic functions are essential for maintenance of male fertility, whereby glycoprotein hormones (GPH) as well as androgens are major endocrine and local regulators. We have investigated whether the GPH human chorionic gonadotropin (hCG) and the free alpha and beta subunits thereof are produced in the target organs themselves and potentially act as auto/paracrine modulators of fertility. Immunofluorometric assays (IFMAs) based on our panel of highly selective monoclonal antibodies, immunohistochemistry (IHC), confocal laser scanning microscopy (CLSM) and 1- and 2D gel electrophoreses with subsequent western blotting have been utilized for the detection of hCGalpha, hCGbeta and its metabolite hCGbeta core fragment (cf) in human testis, prostate and seminal plasma. Both organs synthesize hCGalpha and hCGbeta, which are subsequently detectable at high concentrations in seminal plasma of healthy probands (n=17): hCGalpha 2630+/-520 ng/mL (mean+/-S.E.M.), hCGbeta 2+/-0.28 ng/mL, hCGbetacf and hCG 0.19+/-0.039 ng/mL. These parameters significantly exceed physiological values, e.g. ten thousand-fold in the case of hCGalpha, in serum of young men (n=20): hCGalpha 0.142+/-0.054 ng/mL (mean+/-S.E.M.), hCGbeta 0.05 ng/mL and hCG 0.004+/-0.003 ng/mL. Levels of these markers were not correlated with sperm counts. Of all body fluids including those of pregnant women seminal plasma is the richest physiological source for genuine free i.e. non-dissociated GPHalpha (M(r,app) 23k) which may even appear as di- or tetramers. Its concentration is similar to that observed in maternal serum (weeks 10-12 of gestation) and in extra-embryonic coelomic fluid. In contrast to those fluids where ratios of free subunits to hCG are in the range of 1:100 highly inverse ratios in the range of 10.000:1.000:1 were observed for hCGalpha:hCGbeta:hCG in seminal plasma. hCGalpha is not derived from heterodimeric GPH suggesting hCG-independent functions of hCGalpha and hCGbeta in male and female fertility.

Quality assurance trials for Ki67 assessment in pathology.

Ki67 is a broadly used proliferation marker in surgical pathology with an obvious need for standardization to improve reproducibility of assessment. Here, we present results of the so far only existing round robin tests on Ki67, organized annually in Germany, Austria, and Switzerland from 2010 to 2015 with up to 160 participating laboratories (QuIP). In each quality assessment trial, eight probes from each breast cancer, neuroendocrine tumor, and malignant lymphoma were compiled on a tissue microarray (TMA). TMAs were stained in the participants' laboratories with antibodies and procedures also applied in their daily routine. Participating pathologists were expected to assign Ki67 values to one of four different categories for each tumor type. All local stainings and evaluations were reassessed by the organizing panel and compared to a preset standard. On average, 95% of participants reached the benchmark of over 80% concordance rates with the Ki67 category pre-established by the panel. Automatization and type of antibody did not affect the success rate. Concordance rates differed between tumor entities being highest in each tumor type with either very high or very low labeling indices. Lower rates were seen for intermediate Ki67 levels. Staining quality improved during the observation period as did inter-observer concordance with 85% of participants achieving excellent agreement (kappa > 0.8) in the first year and over 95% in 2015. In conclusion, regular external quality assurance trials have been established as a tool to improve the reproducibility and reliability of the prognostic and predictive proliferation marker Ki67.

Epidemiology of non-Hodgkin lymphomas in Tyrol/Austria from 1991 to 2000.

AIMS: To analyse the entity specific incidence and disease specific survival (DSS) of non-Hodgkin lymphomas (NHLs) in Tyrol/Austria, 1991-2000. METHODS: Data from 1307 NHLs (excluding primary cutaneous lymphomas and monoclonal gammopathies of undetermined significance) were obtained. Current status was available for all patients. Except for 29 cases of small lymphocytic (CLL/SLL), lymphoblastic leukaemia (ALL), and myeloma (MM), which were diagnosed cytologically, diagnoses were reclassified on paraffin wax embedded archival material according to new World Health Organisation criteria. Sex specific age adjusted standardised incidence rates were computed using Segi's population weighting. Annual incidence changes were calculated by weighted least square regression analysis. Survival was estimated by the Kaplan-Meier method and compared by log rank test. RESULTS: NHL more frequently affected men (male/female ratio, 1.52). Mean age of occurrence was 61 and 66 years for men and women, respectively. The incidence rate of 14.3 remained constant. There was a significant increase in diffuse large B cell lymphoma (DLBCL) and decrease in CLL/SLL in men, and a decrease in MM in women. Overall DSS was 64% during the mean follow up (43 months). Age, T-NHL, lambda light chain restriction in MM, and male sex in CLL/SLL were associated with poor prognosis. In B-NHL, DSS decreased in the following order: hairy cell leukaemia, marginal zone lymphoma, follicular lymphoma, Burkitt lymphoma, ALL, DLBCL, CLL, MM, and mantle cell lymphoma. CONCLUSIONS: The incidence of NHL in Tyrol has changed in the past decade, with a significant increase in DLBCL, decrease in CLL/SLL in men, and decrease in MM in women.

High predictive value of epidermal growth factor receptor phosphorylation but not of EGFRvIII mutation in resected stage I non-small cell lung cancer (NSCLC).

AIMS: Overexpression and mutation of epidermal growth factor regulator (EGFR) are frequently found in the carcinogenesis of non-small cell lung cancer (NSCLC). Because targeting of this receptor has proven therapeutic efficacy, studying EGFR has become a matter of particular scientific interest. The present study analysed the EGFR receptor, rate of EGFRvIII mutations, and rate of activated phosphorylated EGFR (pEGFR) by immunohistochemistry on cryostat sections. METHODS: Surgically obtained tumour specimens of a series of 78 NSCLC patients and 66 adjacent tumour free specimens were examined immunohistochemically using monoclonal antibodies to stain EGFR, pEGFR, and EGFRvIII. RESULTS: EGFRvIII and pEGFR expression was found in 42% and 26% of the tumours respectively and both were increased significantly compared with tumour free samples. EGFR, pEGFR, and EGFRvIII expression did not correlate with any of the previously tested markers (c-erbB-2, c-erbB-3, p53, ki-67, and microvessel density). Similar distributions of immunohistochemical profiles were seen, regardless of histological subtype, age, or sex. In stage I patients, EGFR phosphorylation at tyrosine residue 845 proved to be an independent prognostic factor. CONCLUSION: Because pEGFR correlated with poor prognosis, it can be speculated that it plays a crucial biological role in the pathogenesis of NSCLC.

Human chorionic gonadotropin (hCG) and its free subunits in hydrocele fluids and neoplastic tissue of testicular cancer patients: insights into the in vivo hCG-secretion pattern.

To obtain insight into the secretion pattern of human chorionic gonadotropin (hCG) and its free subunits, hCG alpha and hCG beta, in vivo, we analyzed hydrocele fluids of 13 patients with testicular cancer and correlated the respective values to those of cubital vein and testicular vein serum. As a control population, patients with nonmalignant hydroceles (n = 11) were studied. Analyses were performed with a set of highly sensitive and specific time-resolved fluoroimmunoassays based on our own panel of monoclonal antibodies. In the collective of testicular cancer patients, increased hydrocele levels of either hCG or free hCG alpha or free hCG beta were observed in 77, 54, and 92% of cases; the corresponding percentages for cubital vein serum were 62, 23, and 31%. The cubital vein ratio of hCG:hCG alpha (546:1) and hCG:hCG beta (51:1) decreased to 64:1 and to 7:1 in the hydrocele fluids. Surprisingly, hydrocele fluids of five patients with pure seminoma, who were negative for the three markers in the periphery, revealed an elevation of free hCG beta in all cases, while hCG alpha and holo-hCG were elevated twice. Final proof that hCG beta and hCG alpha are indeed produced by these previously termed "marker negative" seminomas has been achieved by reverse transcriptase-polymerase chain reaction with primers specific for the alpha-subunit and the four most abundantly transcribed hCG beta genes 3, 5, 7, and 8. From these data, we conclude that: (alpha) seminomatous and nonseminomatous testicular cancers, irrespective of histology, secrete hCG and its free subunits; (b) the amount of free subunits being secreted in vivo by these tumors has been underestimated; and (c) the classification in marker-positive and marker-negative testicular cancer should be reconsidered.

Distinct genetic evolution patterns of relapsing diffuse large B-cell lymphoma revealed by genome-wide copy number aberration and targeted sequencing analysis.

Recurrences of diffuse large B-cell lymphomas (DLBCL) result in significant morbidity and mortality, but their underlying genetic and biological mechanisms are unclear. Clonal relationship in DLBCL relapses so far is mostly addressed by the investigation of immunoglobulin (IG) rearrangements, therefore, lacking deeper insights into genome-wide lymphoma evolution. We studied mutations and copy number aberrations in 20 paired relapsing and 20 non-relapsing DLBCL cases aiming to test the clonal relationship between primaries and relapses to track tumors' genetic evolution and to investigate the genetic background of DLBCL recurrence. Three clonally unrelated DLBCL relapses were identified (15%). Also, two distinct patterns of genetic evolution in clonally related relapses were detected as follows: (1) early-divergent/branching evolution from a common progenitor in 6 patients (30%), and (2) late-divergent/linear progression of relapses in 11 patients (65%). Analysis of recurrent genetic events identified potential early drivers of lymphomagenesis (KMT2D, MYD88, CD79B and PIM1). The most frequent relapse-specific events were additional mutations in KMT2D and alterations of MEF2B. SOCS1 mutations were exclusive to non-relapsing DLBCL, whereas primaries of relapsing DLBCL more commonly displayed gains of 10p15.3-p12.1 containing the potential oncogenes PRKCQ, GATA3, MLLT10 and ABI1. Altogether, our study expands the knowledge on clonal relationship, genetic evolution and mutational basis of DLBCL relapses.

Transplantation of IL-2-transduced murine bone marrow is associated with dose-dependent toxicity.

OBJECTIVE: The purpose of this study was to investigate the effects of interleukin-2 (IL-2) gene-transduced hematopoietic progenitor cells or cytotoxic function and systemic toxicity following syngeneic bone marrow transplantation. MATERIAL AND METHODS: Marrow of 5-fluorouracil pretreated donor mice were transfected with a retroviral vector containing the murine IL-2 gene and transplanted into lethally irradiated syngeneic hosts. RESULTS: Productive insertion of the IL-2 gene could be demonstrated at various intervals post-transplant without impairment of hematopoietic engraftment. Endogenously augmented IL-2 release resulted in a selective increase in CD4(+), CD8(+), and NK1.1(+) population in spleen and bone marrow, as well as significant cytolytic activity against syngeneic leukemia cells in vitro. Our results also illustrate the interdependence among the magnitude of systemic IL-2 levels, the number of IL-2-transduced cells in the transplant inoculum, and the appearance of systemic toxicity. Infusion of marrow transduced with high-titer, high-expressing IL-2 retrovirus resulted in significant morbidity and mortality in the recipients. Our studies demonstrate that mortality was secondary to severe lymphocytic infiltration of liver and lung, which was associated with increased expression of intercellular adhesion molecule-1 and vascular adhesion molecule-1. Reducing the number of IL-2-transduced cells in the bone marrow inoculum, however, resulted in significantly improved survival with no adverse events being evident during the post-transplant period. CONCLUSION: Delivery of IL-2 to the bone marrow can be achieved by transplantation of genetically modified hematopoietic cells, however, the overall feasibility is strongly influenced by the number of transduced cells in the bone marrow inocolum and/or the expression pattern of IL-2 in vivo.

Hypoxia induces heat shock protein expression in human coronary artery bypass grafts.

OBJECTIVE: Heat shock proteins (HSPs) are molecular chaperones which are essential for cell survival. Heat shock and hypoxia markedly increase the expression of several HSPs in various tissues, i.e. heart. In our in vitro study, we investigated whether HSPs are inducible in human vessels which are used as coronary artery bypass grafts. METHODS: We used remnants of the saphenous vein and the internal mammary artery from 34 patients undergoing coronary artery bypass surgery. Each vessel was divided into segments, one for control conditions at 37 degrees C (5% CO(2)-95% air), the remaining ones for thermal (30 min at 42 degrees C) or hypoxic treatment (6 h oxygen deprivation with nitrogen). The expression of Hsp60, Hsp72 and Hsp73 was investigated by immunohistochemistry and Western-blot analysis. RESULTS: Compared to controls, segments of the saphenous vein undergoing heat treatment showed significantly increased expression of Hsp72 in the intima (P=0.035) and Hsp73 in the media (P=0.003). In the internal mammary artery, Hsp72 and Hsp73 were expressed in the intima at significantly higher levels (P=0.042 each). A 6 h oxygen deprivation with nitrogen resulted in elevated levels of Hsp60 (media: P=0.048), of Hsp72 (intima: P<0.001 and media: P=0.004) and of Hsp73 (intima: P=0.029) in the saphenous vein. In the internal mammary artery, Hsp73 expression was significantly enhanced (intima: P=0.048 and media: P=0.017). The results were confirmed by Western-blot analysis in representative veins. CONCLUSIONS: These findings demonstrate the common cellular defense mechanism of HSP expression in response to stress in coronary artery bypass grafts. Hypoxia and heat treatment strongly induce Hsp72 and Hsp73 expression in human coronary artery bypass grafts.

Expression of the human chorionic gonadotropin-beta gene cluster in human pituitaries and alternate use of exon 1.

Previous studies have indicated that in addition to other glycoprotein hormones, the pituitary gland produces small amounts of hCG beta, the classical pregnancy and tumor marker. At the gene transcription level, definitive proof for hCG beta messenger ribonucleic acid transcription was still lacking, largely due to the 90% homology to hLH beta at the DNA sequence level, which renders specific hCG detection in the presence of a vast excess of LH difficult. We investigated both the presence of hCG beta messenger ribonucleic acid and the protein itself in normal human female postmenopausal (n = 4) and male pituitaries (n = 2). Reverse transcription-PCR and subsequent restriction enzyme analysis revealed that the hCG beta 3, 5, 7, and 8 genes coding for genuine hCG beta were transcribed in all pituitaries. Additionally, three alternatively spliced gene products derived from hCG beta genes 1 and 2 were detected and verified by single strand sequencing of the complementary DNAs. The most abundant fragment (244 bp) showed a point mutation (T-->A) in the splice donor site for the first intron, resulting in an alternate use of exon 1 and a frame shift in the open reading frame that might give rise to a hypothetical protein, 132 amino acids in length. With regard to protein synthesis, we confirmed the pituitary as the site of production for hCG beta by reverse phase high performance liquid chromatography and subsequent immunoradiometric assays, including a monoclonal antibody directed against the unique C-terminal extension of hCG beta.

Selective growth hormone/placental lactogen gene transcription and hormone production in pre- and postmenopausal human ovaries.

In addition to effects of pituitary-derived gonadotropins, human GH modulates and regulates intraovarian reproductive processes in a dose-dependent manner via the endocrine GHRH/GH/insulin-like-growth-factor I (IGF-I) axis. Based on increasing evidence that ovarian regulation involves a complex system of putative para/autocrine factors, we investigated the possibility of gene-selective intraovarian GH/placental lactogen (PL) hormone production, with emphasis on differences between pre- and postmenopause. Analysis of both premenopausal (n = 8) and postmenopausal (n = 10) ovarian-derived messenger ribonucleic acid by reverse transcription-PCR, which amplifies all major gene products of the five-member GH/PL gene cluster GH-N, GH-V, PL-A/B, and PL-L, revealed specific transcripts in all specimens. Their share in gene selective expression by analytical restriction enzyme digestion was determined. The expression pattern of GH/PL messenger ribonucleic acid shows PL-A/B > GH-N, which sets it apart from those of pituitary and placenta. Local production of the respective protein hormones was verified by two time-resolved immunofluorometric assays for human PL-A/B and GH-N; significant amounts of these hormones were detected in cytosolic extracts of premenopausal (n = 6; 555.5 +/- 171 ng PL-A/B and 0.8 +/- 0.6 ng GH-N/g tissue wet wt) and postmenopausal (n = 6; 5.2 +/- 2.7 ng PL-A/B and 0.9 +/- 0.6 ng GH-N/g tissue wet wt) ovaries. No difference was observed between pre- and postmenopausal ovarian GH-N contents, but PL values were 2-3 orders of magnitude lower in postmenopausal tissue (P < 0.001). Serum levels of healthy premenopausal (n = 21) and postmenopausal (n = 16) women were less than 0.02 ng PL/mL. In summary, ovarian-derived GH-N and PL-A/B synthesis correlates well with the established local cascade of GHRH, GHRH receptor, GH receptor, IGF-I, and IGF-I receptor as a putative para/autocrine regulator of ovarian reproductive function.

Eutopic production of human chorionic gonadotropin beta (hCG beta) and luteinizing hormone beta (hLH beta) in the human testis.

The classical pregnancy and tumor marker hCG has long been considered to be only accidentally expressed ectopically, e.g. by tumors. The biological functions of low levels of hCG beta, hCG alpha and holo-hCG in the sera of nonpregnant healthy individuals remained unclear. Immunological analyses by our ultrasensitive time-resolved fluoroimmunoassays revealed a concentration gradient from < 5 pg hCG beta/ml in cubital vein serum versus up to 480 pg hCG beta/ml in the corresponding benign testicular hydrocele fluids. Moreover, hCG beta and its cognate molecule luteinizing hormone beta (LH beta) were present in cytosolic extracts of normal human testes. Both hCG beta and hLH beta are eutopically produced as proven by RT-PCR and subsequent Southern and dot blot analyses. Thus, the view of a purely systemic hormonal function of hLH, and of hCG during pregnancy needs a reassessement as hCG beta and hLH beta are synthesized in the human testis and autocrine/paracrine actions seem to be likely.

Serum glycoprotein hormones and their free alpha-subunit in a healthy elderly population selected according to the SENIEUR protocol. Analyses with ultrasensitive time resolved fluoroimmunoassays.

The SENIEUR protocol was elaborated by a working party of European Community's Concerted Action Programme on Aging (EURAGE) to define strict admission criteria for 'healthy' elderly subjects and young controls for immunogerontological studies. This protocol, which is based on case history, laboratory values and drug consumption, intends to limit the influence of underlying disease and/or medication in order to allow analyses of the aging process per se. In a group of 38 male and 37 female individuals we determined the impact of age and classification according to the SENIEUR protocol on luteinizing hormone (LH), follicle stimulating hormone (FSH), human chorionic gonadotropin (hCG) and free glycoprotein hormone alpha-subunit serum values. Analyses were performed by a set of ultrasensitive time-resolved immunofluorometric assays (IFMA) using our own panel of monoclonal antibodies (MCA). HLH and hFSH, but also hCG and free alpha serum levels increased highly significantly with age in the female population (P < 0.001). In males hFSH, hLH hCG and the free alpha-subunit increased with age. However, only the rise of hFSH and of Free alpha was statistically significant (P < 0.01). The influence of the SENIEUR status on the respective hormone serum levels was determined using two factor analysis of variance, which revealed no statistically significant difference (P > 0.01) between SENIEUR and NON-SENIEUR individuals for all four analytes in both sexes. We conclude that the age related increase of hLH, hFSH, hCG and free alpha is an intrinsic age-dependent phenomen and is not modified by or due to underlying disease or medication as demonstrated by analyses of SENIEUR individuals. Since SENIEUR and NON-SENIEUR individuals had comparable hormone values, a randomly chosen, 'apparently healthy' population seems to be sufficient for physiological studies on serum GPH levels. Lastly, these age related hormonal changes in an extremely well defined healthy population underline the need for age adjusted 'normal' hormone values as elaborated in this communication.