Comparison of seven serum thyroglobulin assays in the follow-up of papillary and follicular thyroid cancer patients.

BACKGROUND: Serum thyroglobulin (Tg) is the marker of differentiated thyroid cancer after initial treatment and TSH stimulation increases its sensitivity for the diagnosis of recurrent disease. AIM: The goal of the study is to compare the diagnostic values of seven methods for serum Tg measurement for detecting recurrent disease both during L-T4 treatment and after TSH stimulation. METHODS: Thyroid cancer patients who had no evidence of persistent disease after initial treatment (total thyroidectomy and radioiodine ablation) were studied at 3 months on L-T4 treatment (Tg1) and then at 9-12 months after withdrawal or recombinant human TSH stimulation (Tg2). Sera with anti-Tg antibodies or with an abnormal recovery test result were excluded from Tg analysis with the corresponding assay. The results of serum Tg determination were compared to the clinical status of the patient at the end of follow-up. RESULTS: Thirty recurrences were detected among 944 patients. A control 131I total body scan had a low sensitivity, a low specificity, and a low clinical impact. Assuming a common cutoff for all Tg assays at 0.9 ng/ml, sensitivity ranged from 19-40% and 68-76% and specificity ranged from 92-97% and 81-91% for Tg 1 and Tg2, respectively. Using assays with a functional sensitivity at 0.2-0.3 ng/ml, sensitivity was 54-63% and specificity was 89% for Tg1. Using the two methods with a lowest functional sensitivity at 0.02 and 0.11 ng/ml resulted in a higher sensitivity for Tg1 (81% and 78%), but at the expense of a loss of specificity (42% and 63%); finally, for these two methods, using an optimized functional sensitivity according to receiver operating characteristic curves at 0.22 and 0.27 ng/ml resulted in a sensitivity at 65% and specificity at 85-87% for Tg1. CONCLUSION: Using an assay with a lower functional sensitivity may give an earlier indication of the presence of Tg in the serum on L-T4 treatment and may be used to study the trend in serum Tg without performing any TSH stimulation. Serum Tg determination obtained after TSH stimulation still permits a more reliable assessment of cure and patient's reassurance.

Modulation of IL-8, IL-1 beta, and G-CSF secretion by all-trans retinoic acid in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a homogeneous subgroup of acute myeloid leukemias (AML) characterized by the presence of the t(15;17) translocation and the resulting PML/RAR alpha fusion proteins. To date APL is the only AML which is sufficiently sensitive to all-trans retinoic acid (ATRA) differentiating effect. We have recently reported that APL express and secrete hematopoietic growth factors (HGF) such as IL-1 beta, TNF alpha, and IL-6. In vivo ATRA alone allows achievement of complete remission in APL patients. One of ATRA therapy's drawbacks is the increase of peripheral blast cells often associated with the ATRA leukocyte activation syndrome. To determine if this specific side-effect was linked to an increase of HGF release by APL cells, we studied the modulation of cytokine production by APL cells, we studied the modulation of cytokine production by APL samples (n = 12) before and after incubation with ATRA. ATRA failed to modulate TNF alpha, IL-6 or GM-CSF secretion levels; however, IL-8 levels decreased in 11 cases, and in four cases up-regulation of IL-1 beta and G-CSF protein expression was observed. These modulations were found to be linked to ATRA sensitivity as ATRA failed to modulate cytokine production in non-APL cells (n = 8). Interestingly, the increase of IL-1 beta and G-CSF production in the presence of ATRA was highly correlated to an increase in APL cell count in vitro and in vivo hyperleukocytosis, resulting in fatal outcome. IL-1 beta, TNF alpha, IL-6, and IL-8 are known to be implicated in leukocyte activation. The results of this study suggest that ATRA-induced hyperleukocytosis and ATRA leukocyte activation syndrome in APL may be inherent to the secretion of specific hematopoietic growth factors by the APL cells.

Induction of high-affinity GM-CSF receptors during all-trans retinoic acid treatment of acute promyelocytic leukemia.

Differentiation of normal myeloid cells is accompanied by the increase of high-affinity GM-CSF receptors necessary for progenitor proliferation/differentiation and mature neutrophil function. All-trans retinoic acid (ATRA) induces terminal differentiation of acute promyelocytic leukemia cells (AML3 subtype). We report in this study that AML3 cells, like other AML subtypes, harbor high-affinity GM-CSF R (n = 138.3 +/- 69.3 sites/cell, Kd = 76.9 +/- 68.8 pM). In all cases, incubation with ATRA induces either an increase in the number of affinity of GM-CSF R (n = 212.7 +/- 116.2 sites/cell, Kd = 43.2 +/- 22.5 pM). The data presented show that modulation of GM-CSF receptors cells is correlated to the degree of ATRA-induced granulocytic differentiation but not to increased cell growth.

Percentage of free serum prostate-specific antigen: a new tool in the early diagnosis of prostatic cancer.

Prostate-specific antigen (PSA) is a protease able to bind to serum antiproteases as alpha 1 antichymotrypsin (ACT). Free PSA (FPSA) corresponds to the fraction of total PSA (TPSA) which is unbound to ACT. Specific detection of the FPSA seems to be a valuable tool in the distinction between prostatic cancer (PCa) and benign prostatic hyperplasia (BPH). Our aim was to evaluate retrospectively the FPSA/TPSA ratio in comparison to TPSA or FPSA determination, using two new immunoradiometric assays (PSA-RIACT and FPSA-RIACT, CIS bio international, Gif Sur Yvette, France) in the early diagnosis of PCa. 256 men, with TPSA levels between 0.7 and 44.7 ng/ml (median age = 69 years), including 164 sera obtained from patients with BPH and 92 sera from patients with untreated PCa were assayed. All diagnoses were histologically confirmed and patients tested before any adjuvant treatment. The evaluation of the median FPSA/TPSA ratio in the two groups showed significantly different values (BPH group: 24.2%, PCa group: 12.1%, P < 0.0001). By R.O.C. (Receiver-Operating-Characteristics) analysis, we show that the FPSA/TPSA ratio is the method of choice for discriminating BPH and PCa, since the area under curve is the greatest for the FPSA/TPSA ratio curve, as compared to the TPSA or FPSA curves (P < 0.0001). The best accuracy (number of true positive + true negative/total = 82.4%) was obtained with a FPSA/TPSA ratio < or = 15% with high odds ratio (20.5; confidence interval (CI): 11.2; 37.7). Of interest, similar results were also confirmed even in the subpopulation with serum TPSA levels between 2.5 and 10 ng/ml (161 patients including 99 BPH and 62 PCa). We thus confirm that combined serum measurement of FPSA and TPSA is of particular interest in the early diagnosis of PCa for patients with non-suspicious digital rectal examination and a TPSA value between 2.5 and 10 ng/ml. In those patients, biopsy should be reserved to the cases with FPSA/TPSA below 15%, which allows significant odds ratio (12.8; CI: 5.2; 31.4). Otherwise, to avoid the risk of missing any PCa, usual follow-up with combined TPSA and FPSA determination would be required with the same criteria of biopsy (i.e. FPSA/TPSA ratio < or = 15% when TPSA value is between 2.5 and 10 ng/ml; or TPSA > 10 ng/ml).

From guidelines to hospital practice: reducing inappropriate ordering of thyroid hormone and antibody tests.

OBJECTIVE: Because of major technical improvements and conscious care about cost effectiveness, limiting the inadequate use of thyroid biological tests appears to be a major issue. DESIGN: To (i) estimate the ordering prevalence of each thyroid test, (ii) assess the prevalence of relevant thyroid tests, and (iii) evaluate the impact of expressing justification for tests during a 2-month intervention period on these prevalences. METHODS: During a prospective 2-month survey (June-July 1997), all the request forms were divided into four groups of prescription: (1) investigation of thyroid function, (2) taking drugs affecting the thyroid, (3) monitoring of nodule and cancer, and (4) investigation of thyroid autoimmunity. Their appropriateness was thus determined according to consensus in our hospital and previously published recommendations. Results were compared with those of retrospective similar 2-month periods in 1996 and 1998. Combinations of thyroid function tests and thyroid antibodies were analyzed during the 1996, 1997 and 1998 periods. RESULTS: The overall estimated rate of appropriate ordering between 1996 and 1997 increased from 42.5% to 72.4% (P<10(-4)), with a significant improvement in each group of main diagnosis referral, except in group 3 where suitability was always over 85%. However, in group 4, appropriateness remained low (36%). Combinations of thyroid tests revealed an increase in single TSH order forms and single autoantibodies to thyroperoxidase (TPOAb) ones, while TSH+free thyroxine+free tri-iodothyronine and TPOAb+ autoantibodies to thyroglobulin ones decreased significantly. Interestingly, all these changes were maintained 1 year later (June-July 1998) even though physicians were not aware of this new study. CONCLUSIONS: Persistent change in medical practice was thus assessed.

All trans retinoic acid abrogates spontaneous monocytic growth in juvenile chronic myelomonocytic leukaemia.

INTRODUCTION: All trans retinoic acid, the active metabolite of vitamin A, exerts profound effects on cell differentiation. On normal myeloid progenitors, retinoids switch the differentiation program of granulo-macrophagic progenitors towards the granulocytic lineage and consequently reduce CFU-M colony formation. Bone marrow and peripheral blood mononuclear cells from children with Juvenile Chronic Myelomonocytic Leukaemia show typical spontaneous monocytic growth. We questioned whether in this disease, retinoids could switch myelomonocytic growth and inhibit the abnormal CFU-M colony proliferation. METHODS: Ten JCML samples were studied in the presence of ATRA in methyl cellulose colony assay, before (CFU-C) or after (pre-CFU) liquid suspension culture. RESULTS: In vitro characteristics of JCML such as spontaneous monocytic growth in the absence of growth factor was noted in all patients. In the presence of leucocyte-conditioned medium, nine samples showed only CFU-M growth and one sample CFU-GM growth. Incubation with ATRA inhibited CFU-M colony formation in nine cases. Enhancement of granulocytic differentiation (CFU-G) was noted in nine cases. ATRA also inhibited CD34+ JCML monocytic growth and GM-CSF hypersensitivity. CONCLUSION: These data suggest that, in JCML progenitors, retinoid pathways are functional and inhibition of immature monocytic progenitors cells may be achieved with retinoids, without impeding granulocytic cell growth.

Evaluation of total PSA assay on vitros ECi and correlation with Kryptor-PSA assay.

BACKGROUND: An increasing number of multiparametric immuno-analysers for PSA assays are available. As different immuno-assays may vary in their analytical quality and their accuracy for the follow-up of patients, expertise is necessary for each new assay. METHODS: The PSA assay on the Vitros-ECi analyser has been evaluated and compared with the PSA assay from the Kryptor analyser. RESULTS: Variation coefficients were 0.91 to 1.98% for within-run assays, and 4.2% to 5.4% for interassay (PSA levels = 0.8 microgram/L to 33.6 micrograms/L). Dilution tests showed 93 to 136% recovery until 70 micrograms/L PSA. Functional sensitivity was estimated at 0.03 microgram/L. Equimolarity of the test was confirmed. Correlation of PSA levels measured with Vitros-ECi and Kryptor analysers displayed a correlation coefficient r2 of 0.9716. The half-lives and doubling times of PSA were similar using both methods. CONCLUSION: Vitros-ECi PSA assay meets the major criteria for the management of prostate cancer patients.

[Current data on GM-CSF (Granulocyte-Macrophage Colony Stimulating Factor) in acute myeloid leukemia]. / Données actuelles sur les récepteurs du GM-CSF (Granulocyte-Macrophage Colony Stimulating Factor) dans les leucémies aiguës myéloïdes.

GM-CSF (Granulocyte-Macrophage Colony Stimulating Factor) activates neutrophil, eosinophil, granular, and macrophage precursors through binding to specific receptors. GM-CSF receptor is a member of the "cytokine receptor superfamily", which displays a particular transmembrane structure. It is expressed in small amounts on normal mature blood or medullary cells, with a high affinity. On acute myeloid leukemia blasts (18 patients), our results agree with the review of the literature: GM-CSF receptors are in small amounts, of two types (high and low affinity), with no relation to the FAB classification of leukemias.

[Screening for cancer of the prostate using prostate-specific antigen]. / Dépistage du cancer de la prostate par l'antigène spécifique de prostate.

Systematic screening for prostate cancer was carried out in 600 men over 50 years of age by the industrial medicine departments of four big companies in the Paris region. The exploratory methods included prostate specific antigen (PSA) and prostatic acid phosphatase (PAP) assays, rectal palpation and two-dimensional transrectal ultrasonography. Biopsy of the prostate was performed either when the PSA level was above 5 ng/ml or when rectal palpation gave suspicious results, or when ultrasonography showed abnormal images. A total of 93 biopsies were performed, and 18 cases of cancer were detected. Eleven of these 18 patients underwent radical prostatectomy. The PSA assay, with an accepted limit of 5 ng/ml, detected 17 out of 18 cancers but was not very specific. The PAP assay had low sensitivity (only 5 positive results). Combined PAP assay and rectal palpation provided high sensitivity and good specificity. Transrectal ultrasonography was helpful only to determine the site of biopsy and the distribution of the lesions.

Kinetic asymmetry of renal Na+-L-lactate cotransport. Characteristic parameters and evidence for a ping pong mechanism of the trans-stimulating exchange by pyruvate.

Brush border vesicles prepared from horse renal cortex were used to study the kinetic properties of the Na+-L-lactate carrier on the outer and inner faces of the membrane. Two methods were applied for these measurements (in the absence of an electrical gradient): a direct method using influx and efflux kinetics, and an indirect method applied to trans-stimulated influx kinetics using membrane vesicles preloaded with various pyruvate concentrations (the latter enabled us to observe simultaneously the inner and outer carrier properties). Kinetic parameters obtained by the first method have shown that under sodium lactate chemical gradient, the carrier efficiency (estimated by the ratio of k = Vm/Km) is higher for the influx than efflux, a mechanism indicating a kinetic asymmetry of the transport. This difference remains at chemical equilibrium of solute concentration. The similarity of outer and inner affinity of sodium permits one to conclude that the kinetic asymmetry of the sodium lactate transport is related to the lactate-carrier interaction and not to that of the sodium-carrier. The second method using the pyruvate trans-activation effect (under sodium chemical equilibrium) has shown an affinity of lactate (Kt(out) = 1.1 mM), about 15 times higher for the carrier in the extracellular orientation than that of pyruvate for the carrier in the intracellular orientation (Kt(pyr) = 36 mM). This method has demonstrated a ping pong mechanism for the trans-activation exchange which accounts for a selective pore carrier model like a gated channel. These asymmetric properties are related to the AS glide sequential model (A and S being Na+ and lactate, respectively) proposed previously for the Na-L-lactate cotransport and to a different accessibility of the organic solute but not of the sodium on the two membrane faces.

Radioimmunoassay of erythropoietin: analytical performance and clinical use in hematology.

We report here the performance of a recently commercialized radioimmunoassay kit for determining erythropoietin (EPO) in serum or plasma. The lower detection limit of the method was 3 U/L. Precision, analyzed by the variation coefficients between different assay runs and in the same experiment, was always less than 10%; accuracy was assessed by recovery and dilution tests. In anemic patients (hematocrit 18-39%), the concentration of EPO was logarithmically related to hematocrit. A relatively large dispersion of the results was noted, as reported by others with various RIAs. Patients with severe renal failure demonstrated a very low EPO value, whatever the degree of their anemia. In some chronic anemias resulting from malignancy, EPO concentrations were also relatively low. In the polycythemia vera group, the EPO mean was below normal for greater than 95% of the patients, whatever their clinical stage (first evaluation, relapse, or remission). In contrast, 91% of the patients with pure erythrocytosis had a normal or increased EPO value, even when the etiology was unknown. Measurement of EPO concentration may be useful for the clinical differentiation of myeloproliferative disorders and, subsequently, for their prognosis and choice of treatment.

[Detection of a soluble serum antigen by a new monoclonal antibody ED8 in patients with breast cancer]. / Détection par un nouvel anticorps monoclonal ED8 d'un antigène soluble dans le sérum de malades atteintes de cancer du sein.

A murine hybridoma, secreting monoclonal antibody ED8, was generated by immunization with the breast cancer cell line H466B. ED8 reacts with a 300 kDa antigen present on H466B cell surface. The antigen can be detected in serum from breast cancer patients, using ELISA and immunoblotting.

Granulocytic colony-stimulating factors (G-CSF and GM-CSF) in the treatment of adult acute myeloid leukemia.

Large randomized trials have shown that granulocytic colony-stimulating factors (G-CSF and GM-CSF) may be interesting in vivo in promoting neutrophil recovery after high-dose myelosuppressive therapy in some clinical settings. However, any beneficial effect on survival was not yet demonstrated. Granulocytic CSFs act as growth and viability factors on myeloid leukemic cells and their use in acute myeloid leukemias (AML) is theorically not without risk. Paradoxically, these CSFs will maybe be usefull in the future as a part of AML treatment. First, the high early mortality rate in elderly AML patients after intensive chemotherapy should allow a demonstration of a CSF-induced improvement of survival. Secondly, GM-CSF should increase the efficacy of cell cycle dependent cytotoxic drugs based on recruitment of quiescent leukemic cells. Third, granulocytic CSFs should be used to induce programmed cell death in myeloid leukemic cells.

Radioimmunoassay of immunoreactive erythropoietin as a clinical tool for the classification of polycythaemias.

Radioimmunoassay of erythropoietin (EPO) has been used for evaluating its clinical usefulness in distinguishing polycythaemia vera (PV) from pure erythrocytosis (PE). A normal log distribution (13.40 mU/ml +/- 2.45) was observed in the 66 reference samples. Similar results were observed in 29 pure thrombocytaemias (13.23 +/- 5.19). In PV patients, whether in clinical remission or in active phase, the EPO titer was lower than normal values (respectively 7.32 +/- 3.63 and 6.59 +/- 2.75), without any correlation with the haematocrit (44 to 51% and 52 to 71%). In the anaemic cases (excessive therapy, spent phase, myelofibrosis), a slight excess of EPO titer was observed, but less than expected when taking the hematocrit into account. In contrast, pure erythrocytosis is a very heterogeneous group, with the highest EPO values in the well-defined secondary cases, and normal or slightly excessive values in most of the cases, but with some low values similar to those observed in PV cases. In term of PV diagnosis, a 90% predictive value is observed when a "cut-off" value of 11 mU/ml is chosen. When the "cut-off" is 16 mU/ml, no PV case was observed beyond this value. We conclude that EPO assay is useful in myeloproliferative diseases from a practical point of view.

Chronic myelomonocytic leukemia: from biology to therapy.

Chronic myelomonocytic leukemia represents a distinct myelodysplastic syndrome in which an excess of monocytes is observed both in the blood and bone marrow of the patients. Whereas diagnosis is relatively easy, therapeutic design and efficacy is difficult and no treatment has to date provided complete or significant partial response. In vitro data suggest that the growth and differentiation of myelomonocytic progenitors may be altered inasmuch as monocytic or granulo-macrophagic colonies show spontaneous growth. Different entities may be observed: the childhood form, Juvenile Chronic Myelomonocytic Leukemia (JCML) shows in vitro a typical pattern with constitutive growth of only macrophagic colonies and hypersensitivity to GM-CSF; in the adult form at least two patterns may be observed one close to the JCML form and one more heterogeneous with absence of GM-CSF sensitivity and spontaneous growth of both CFU-GM and CFU-M colonies. Chemotherapy reduces all myeloid colonies in vitro whereas retinoic acid has a selective effect on monocytic colonies with a concomitant increase of CFU-G colonies forwarding an explanation for the correction of pancytopenia observed in some patients. Recent analysis of altered molecular pathways in this disease suggest a common disruption of intracellular signalling pathways namely the Ras pathway and targetting for drugs with may selectively control or inhibit a constitutive activation may forward novel therapeutic perspectives.

Constitutive elevation of serum alpha-fetoprotein in Fanconi anemia.

The diagnosis of Fanconi anemia (FA) is based on the association of congenital malformations, bone marrow failure syndrome, and hypersensitivity to chromosomal breaks induced by cross-linking agents. In the absence of typical features, the diagnosis is not easy to establish because there is no simple and cost-effective test; thus, investigators must rely on specialized analyses of chromosomal breaks. Because we observed elevated serum alpha-fetoprotein (sAFP) levels in FA patients, we investigated this parameter as a possible diagnostic tool. Serum AFP levels from 61 FA patients and 27 controls with acquired aplastic anemia or other inherited bone marrow failure syndromes were analyzed using a fluoroimmunoassay based on the TRACE technology. Serum AFP levels were significantly more elevated (P <.0001) in FA than in non-FA aplastic patients. In the detection of FA patients among patients with bone marrow failure syndromes, this assay had a sensitivity of 93% and a specificity of 100%. This elevation was not explained by liver abnormalities. Levels of sAFP were unchanged during at least 4 years of follow-up, and allogeneic bone marrow transplantation did not modify sAFP levels. Three of 4 FA patients with mosaicism as well as 5 of 6 FA patients with myelodysplastic syndrome were detected by this test. Heterozygous parents of FA patients had normal sAFP levels. Measurement of sAFP levels with this automated, cost-effective, and reproducible fluoroimmunoassay could be proposed for the preliminary diagnosis of FA whenever this disorder is suspected.

Intermethod discordance for alpha-fetoprotein measurements in Fanconi anemia.

BACKGROUND: The significantly higher serum alpha-fetoprotein (AFP) in patients with Fanconi anemia (FA) than in non-FA aplastic patients has potential diagnostic utility, but the increase is method-dependent. The aim of this study was to compare five AFP assays on FA and non-FA samples and to investigate possible explanations for FA-specific discrepancies. METHODS: Two methods available in our laboratory (Kryptor and IMx) were compared on 59 FA and 27 non-FA patient samples. Kryptor, Immulite, Elecsys, Immuno-I, and Elsa-2 methods were then compared on 14 FA and 14 non-FA patient samples. The AFP glycosylation profile was analyzed by electrophoretic separation in a lectin-containing gel. RESULTS: With all six methods, AFP values were significantly higher in FA than in non-FA patients, but the diagnostic precision and optimal cutoff values varied. Indeed, two methods reached 100% sensitivity and specificity, but in other methods, one or both of these parameters were significantly <100%. Neither heterophilic antibodies nor a specific glycosylation profile was detected in FA samples. CONCLUSIONS: AFP results are method-dependent in FA. New methods must be evaluated before use in differential diagnosis of aplastic patients.