B-361 Method Validation of Human Chorionic Gonadotropin (hCG) and a-Fetoprotein (AFP) in Cerebrospinal Fluid (CSF)

Our study objective was to validate 2 individual methods to measure α-fetoprotein (AFP) and human chorionic gonadotropin (hCG) in cerebrospinal fluid (CSF) on the Roche cobas® 6000 analyzer. A 3-year retrospective chart review of CSF samples analyzed for AFP and hCG was also conducted. Serum samples with high concentrations of AFP or hCG were added to aliquots of pooled CSF. Precision, linearity, detection limit, recovery, carryover, stability, and interference studies of the AFP and hCG+β assays were performed. Within-day and day-to-day assay imprecision for AFP and hCG assays were <5% at all concentrations tested. The linear range of the AFP assay was established as 1.0-1100 μg/L, and limit of quantification (LOQ) was <1.0 μg/L. The linear range of the hCG assay was established as 1.0-9500 IU/L and LOQ 0.7 IU/L. There was no demonstrable matrix effect, and neither assay was affected by the presence of hemolysis or xanthochromia. AFP in CSF was stable at room and refrigerated temperatures for up to 48 h at concentrations of 19 and 306 μg/L but increased by 24 h at a concentration of 908 μg/L. AFP in CSF was stable frozen (-20 °C) for up to 7 days. hCG in CSF at all concentrations tested was stable at room, refrigerated, and frozen temperatures for up to 7 days. The Roche cobas 6000 AFP and hCG+β assays accurately quantify AFP and hCG in CSF, facilitating rapid and accurate diagnosis as well as monitoring of intracranial germ cell tumors.

The measurement of human chorionic gonadotropin (hCG) in cerebrospinal fluid (CSF) is useful for the differential diagnosis of suprasellar lesions. However, the concentrations that prove diagnostic for neurohypophyseal germinoma have not been well defined. In addition, the immunoassays used for such measurements are the same as those applied in serum, and few studies have been performed regarding the validation of such techniques in CSF. The present study aims to apply the Elecsys(®) hCG + β immunoassay from Roche Diagnostics to measure hCG in CSF, as a useful tool in the diagnosis of neurohypophyseal germinomas in children and young adults. Validation of the immunoassay involved calculation of the functional sensitivity and reference values for hCG in CSF in 35 controls in the absence of pregnancy, trophoblastic disease or tumour pathology. For the clinical application study, three patients diagnosed with neurohypophyseal germinoma have been reviewed. The functional sensitivity obtained was 0.4 IU/L. The reference values for hCG in CSF ranged from undetectable values to 0.7 IU/L. The hCG concentrations in CSF in the three studied patients, with confirmed diagnosis of neurohypophyseal germinoma, were 21.1, 32.6 and 23 IU/L, respectively. The Elecsys® hCG + β immunoassay from Roche Diagnostics can be used to detect hCG in CSF with high precision. According to our results, CSF-hCG concentrations that exceed the established reference interval (undetectable values to 0.7 IU/L) in the presence of suprasellar lesions and hypophyseal stalk thickening must be considered pathological, establishing the need to exclude the presence of germinoma.

Re-evaluation of the significance of cerebrospinal fluid human chorionic gonadotropin in detecting intracranial ectopic germinomas

Background. The measurement of human chorionic gonadotropin (hCG) in cerebrospinal fluid (CSF) is important for the diagnosis of intracranial or intraspinal trophoblastic tumours. The current study was performed to establish reference values for hCG in CSF and to explore the relationship of CSF hCG and serum hCG in patients who are not pregnant or do not have trophoblastic tumours. Material and methods. CSF samples were obtained from 369 inpatients admitted because of various neurological diseases, excluding pregnancy, trophoblastic tumours and other malignant tumours. In 271 of the 369 patients, paired samples of CSF and serum were obtained. Both CSF hCG and serum hCG were measured. The 97.5th percentile and maximum value of CSF hCG were obtained. The CSF hCG and serum hCG concentrations in each of the 271 paired samples were compared. Results. The 97.5th percentile and maximum value of CSF hCG concentration for overall participants were 1.00 and 5.00 IU/L, respectively. The CSF hCG concentration was found to be higher than the simultaneous serum hCG concentration in 81.9% (222/271) of the participants. Conclusions. The reference value determined in this study of CSF hCG in men is significantly lower than that usually used in clinical practice. A CSF hCG concentration higher than the simultaneous serum hCG concentration but lower than the upper reference limit does not necessarily suggest abnormal intrathecal hCG-secretion.

Background Human chorionic gonadotropin (hCG) detection in cerebrospinal fluid (CSF) can provide additional value in the diagnosis of germinoma. However, matrix effects can influence the results when alternative sample types are used. Therefore, modified-cleared/approved methods, which are standard methods used outside their intended scope, are of interest. The aim of the present study was to establish a model to validate modified-approved methods in agreement with the College of American Pathologists (CAP) accreditation requirements.Methods Concentrations of hCG in CSF were determined by means of electrochemiluminescence immunoassay using a Roche Cobas e 602 immunoassay analyzer. Based on the intended use, the following performance characteristics were evaluated: precision, the limit of quantitation (LoQ), and the analytical measurement range (AMR). The reference interval (RI) was also established. For the clinical application study, CSF and serum hCG were measured in 10 patients diagnosed with germinoma.Results The intra- and inter-assay precisions at two levels (10, 250 IU/L) were 0.64 and 0.57% and 4.26 and 3.54%, respectively. The LoQ for hCG was determined to be 0.25 IU/L. The AMR was set from 0.2 to 1,200 IU/L. The RI for hCG in CSF was below 0.40 IU/L. The CSF hCG levels of 10 patients were all above 0.4 IU/L before therapy.Conclusion Modified-approved methods were validated and showed that the quality specifications of the medical laboratory have a positive value in the clinical context. The illustration of quantification of hCG in CSF resulted in compliance with the CAP accreditation requirements.

Human chorionic gonadotropin and α-fetoprotein in cerebral spinal fluid: Method validation and retrospective review

Surgical Options for Pineal Region Tumors

Intracranial germ cell tumors (GCTs) are rare central nervous system neoplasms predominantly occurring in pediatric and young adult patients. GCTs predominantly arise in the brain midline, most frequently at the pineal region. The WHO classification defines five histopathological GCT subtypes: germinoma, mature and immature teratoma, yolk sac tumor, choriocarcinoma, and embryonal carcinoma, which are divided into germinomas and non-germinomatous germ cell tumors (NGGCTs). Tumor markers include human chorionic gonadotropin (HCG) and alpha fetoprotein (AFP) in blood serum and cerebrospinal fluid, the elevation of which typically suggests choriocarcinoma and yolk sac tumor components, respectively. Cases with negative tumor markers are diagnosed based on histopathology on surgically obtained specimens. Most germinomas are highly chemo- and radiosensitive, with non-responders accounting for less than 10%. NGGCTs are frequently resistant to standard adjuvant therapies, and sometimes require more intensive treatment, such as myeloablative chemotherapy followed by autologous bone marrow stem cell transplantation or multiple surgical resections at recurrence. Surgical resection is reserved for mature teratoma, which is curable with resection alone, and for cases with poor response to induction chemotherapy. Although approximately 60% of GCTs harbor mutations in either MAPK or PI3K pathways, no targeted therapy has been registered to date. Overall survival at 10 years for germinoma and NGGCT is approximately 90–95% and 75–90%, respectively. Long-term survivors are at risk of secondary malignancy, cardiovascular disease, and cognitive decline due to therapeutic sequelae. Progress in treatment options will include targeted therapy, immunotherapy, and dose reduction of radiation.

Chapter 46 - Germ Cell Tumors

Central Nervous System Tumors in Children.

Dear editor, For many years, in case of suspicion of a germ cell tumor (GCT), human chorionic gonadotropin (HCG), its beta subunit and alpha-fetoprotein (AFP) have been used as specific markers in the blood and/or cerebrospinal fluid (CSF) for clinical decision-making [4]. A positive test is an indication for treatment, without requiring histological confirmation [5]. We report a first description of CSF HCG elevation in a non-cystic suprasellar craniopharyngioma, contradicting this dogma. A 52-year-old male with a history of arterial hypertension and tinnitus complained of a decrease in left-side visual acuity over a few weeks, associated with polyuro-polydipsia syndrome. MRI examination revealed a suprasellar lesion of 16×18×16 mm, with a moderate hyper-T2 signal and an iso-T1 signal with homogeneous gadolinium enhancement. The lesion did not have a cystic component (see Fig. 1). Spinal MRI was negative, and the cerebral CT scan did not show any calcification of the tumor. Hormone analysis revealed a pituitary deficiency. Biological analysis of the CSF performed as part of the etiological assessment revealed an elevation of HCG to 10.8 IU/l (normal levels <5 IU/l) and of free beta-HCG to 1.1 ng/ml (normal levels <0.1 IU/l). Elevation of AFP was not detected in the blood or CSF. No abnormal cells were found in the CSF. No elevation of these markers was found in the blood. Given the many atypical features in our patient, we decided to perform a robot-assisted stereotaxic biopsy. The final diagnosis was papillary craniopharyngioma and excluded germinoma. Due to the high risk of surgical treatment, and after a multidisciplinary discussion, the tumor was treated by radiotherapy. Six months after that, the patient recovered his visual field loss, but the hypopituitarism remained. The MRI showed a volumetric reduction of the tumor and a decrease in contrast enhancement. This report points out two issues: First, some authors [2, 3, 6] have suggested that many cystic pineal, sellar or suprasellar lesions, including cystic craniopharyngioma, are able to produce theses markers through their wall (descriptions of HCG in benign pineal cyst contents and walls). This observation of a solid craniopharyngioma demonstrates for the first time that the tumor itself is able to secrete low levels of HCG and β-HCG that may be detectable in the CSF. CSF AFP elevation was also detected in two cases of craniopharyngiomas, one of which was proven to be a malignant form [2]. So far, no blood elevation of these markers has been reported in craniopharyngiomas. Second, therefore, physicians involved in brain management need to be aware that HCG or AFP CSF positivity in a patient with a suprasellar tumor is not a specific GCT signature and is not enough information for making therapeutic decisions. Our clinical case underlines the importance of multidisciplinary discussions and the necessity to perform a biopsy in patients showing isolated suprasellar lesions (bipolar pineal and suprasellar locations remain suggestive of germinoma), positive for CSF markers but with atypical P. Bourdillon (*) : E. Jouanneau Department of neurosurgery A, Hopital Neurologique et Neurochirurgical Pierre Wertheimer, Hospices Civils de Lyon, 59 bd Pinel, 69500 Bron, France e-mail: pierre.bourdillon@neurochirurgie.fr

170 patients with testicular germ cell tumours (88 seminomas and 82 non-seminomas) were examined with immunologic techniques for the presence of the tumour markers alpha-fetoprotein (AFP) and human chorionic gonadotropin (HCG) in tumour tissue and preoperative serum samples. Patients with pure seminomas had AFP negative tumour tissue and normal levels of serum AFP, whereas 13% had HCG demonstrated in the tumour tissue, mainly in syncytiotrophoblast-like cells (STLC), and 9% had raised serum HCG. 55% of patients with HCG positive seminomas had raised serum HCG. HCG positive seminomas did not occur in higher frequency in metastatic disease than in localized. 65% of patients with non-seminomas had AFP positive tumour tissue and 66% had raised serum AFP. 85% of the former group had raised serum AFP and 83% of the latter had AFP demonstrated in the tumour tissue. 69% of the patients with raised serum AFP had a positively stained yolk sac tumour (YST) component, while 15% had positively stained tumour components other than YST, inclusive teratoma components. Although 71% of patients with metastatic disease had raised serum AFP, AFP positive tumours with or without raised serum AFP did not occur with higher frequency in metastatic than in localized disease at the time of diagnosis. 46% of patients with non-seminomas had HCG positive tumours and 29% had raised serum HCG. 61% of the former group had raised serum HCG and 96% of the latter had HCG demonstrated in the tumour tissue. HCG positive tumours with or without raised serum HCG did not occur more frequently in metastatic than in localized disease at the time of diagnosis. 28% of patients with non-seminomas had raised serum AFP as well as HCG, whereas 23% had neither AFP nor HCG in tumour tissue and serum. A search for new tumour markers in this rather large marker negative group of patients is recommended.

We studied 61 patients with primary intracranial germ cell tumors treated between 1954 and 1988 and reviewed previous reports. Our patient series included 48 males and 13 females with an age range of 5 to 41 years (mean, 15 years). Thirty-six tumors arose in the pineal region, 14 were suprasellar, and 11 in the basal ganglia and thalamus. There were 42 germinomas, 5 teratomas, and 14 malignant germ cell tumors (MGT) (8 embryonal carcinomas, 3 yolk sac carcinomas, and 3 choriocarcinomas). The diagnosis was histologically verified in 43 cases (29 operations, 12 autopsies, 2 cytological studies); the remaining 18 tumors were diagnosed on the basis of characteristic clinical and radiological features, tumor markers, and response to radiotherapy.We evaluated these patients' clinical symptoms, radiological images, and tumor markersexisting in both serum and cerebrospinal fluid (CSF) -revealed by immunohistochemistry. These markers included alpha-fetoprotein (AFP), human chorionic gonadotropin (HCG), carcinoembryonic antigen (CEA), and placental alkaline phosphatase (PALP). The levels cf tumor markers reflected tumor state and were well correlated with the histological diagnosis ; i.e., yolk sac carcinoma produced AFP ; choriocarcinoma, HCG ; embryonal carcinoma, AFP and HCG; teratoma, CEA ; and germinoma, PALP.The enzyme activity of PALP in CSF was measured by enzyme antigen immuno-assay (EAIA) in 14 patients in our laboratory. In 4 out of 6 germinoma patients, pre-treatment PALP in CSF exceeded 7 ng/ml and marked reductions occurred with radiation, while pre-and post-treatment PALP in patients with other germ cell tumors lower than 5 ng/ml. These results with EAIA suggested that measurement of PALP levels would be useful in the management of patients with germ cell tumors, particularly germinomas.The therapeutic regimens included surgery, irradiation, and chemotherapy. Germinomas were curable by radiation therapy. Teratomas were cured by microsurgery alone. Patients with radioresistant MGT and distant metastases had a poorer prognosis. Chemotherapy appeared to be a promising approach to treat MGT. Compared to radiation therapy alone, the theoretical advantages of chemotherapy include : 1) tumor specificity ; 2) control of occult distant metastases ; 3) a possible synergistic effect with radiation therapy ; and 4) reduction of the radiation dose and thereby lessening of the potential for late radiation-induced damage to the central nervous system.During the last 3 decades, dramatic improvement in the management and outcome of intracranial germ cell tumors has been accomplished by : 1) the introduction of high-resolution CT and magnetic resonance imaging ; 2) pre- and post-treatment assessment of tumor markers in serum and CSF ; 3) advances in microsurgical and stereotactic surgical techniques ; and 4) new chemotherapeutic agents and regimens. Questions and controversies remain, however. For example, should all pineal germinomas be biopsied or should radiation therapy be initiated without biopsy? How heavily can we rely on the histologic specificity of tumor markers? Is prophylactic spinal irradiation indicated in all cases of germinoma? What chemotherapy regimens are most effective against various types of MGT? Which is preferable as initial treatment of MGT, radiotherapy or chemotherapy? For further improvements in the treatment of primary intracranial germ cell tumors, more extensive investigation of their clinical and biological characteristics is necessary.

To determine medical reference values for tumor markers in cerebrospinal fluid. Concentrations of CEA, CA125, CA15-3, CA19-9, CA72-4, CYFRA21-1, AFP, NSE, SCC and HCG were determined by means of double-antibody sandwich ELISA in 110 patients excluding primary tumors and meningeal carcinomatosis using Roche E170 modular immunoassay analyzer. The determined medical reference values for tumor biomarkers in cerebrospinal fluid were as follows: CEA<0.573 microg/L, CA125<2.591 U/ml, CA15-3<2.045 U/ml, CA19-9<2.272 U/ml, CA72-4<1.252 U/ml, CYFRA21-1<1.44 ng/ml, AFP<0.968 microg/L, NSE<57.666 ng/ml, SCC<0.5 microg/L, HCG<0.769 U/L. There was no correlation between any tumor marker and age (P>0.05). Concentrations of tumor markers were not affected by gender (P>0.05). Medical reference values for CEA, CA125, CA15-3, CA19-9, CA72-4, CYFRA21-1, AFP, NSE, SCC and HCG in cerebrospinal fluid were first determined.

Detection of AFP and HCG in metastatic testicular cancer after treatment with chemotherapy or radiation therapy