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.

Primary Sjögren's syndrome associated agranulocytosis: a benign disorder?

OBJECTIVE: To report on an uncommon association of agranulocytosis in primary Sjögren's syndrome (SS). METHODS: The clinical, haematological, and immunological features of seven patients with primary SS associated with a chronic (>6 months) agranulocytosis, and the outcome of the patients, were analysed. RESULTS: Patients were white women with an unexplained agranulocytosis. They all had non-erosive arthritis and three had a thrombocytopenia or Evan's syndrome. In three patients, transient or durable expansion of T lymphocytes was present in the peripheral blood or in the bone marrow, but evolved independently from neutrophil counts. There was no paroxysmal nocturnal haemoglobinuria clone or antibodies to neutrophil surface antigens. In vitro bone marrow culture was normal (four patients) or showed a decrease in colony forming unit-granulocyte monocyte (CFU-GM) and colony forming unit-erythroblast (CFU-E) (one patient). Serum levels of soluble Fas ligand (sFasL) were normal, and granulocyte-colony stimulating factor (G-CSF) concentrations were either normal or raised. One patient was treated with steroids associated with intravenous immunoglobulins and achieved a lasting response. Two other patients were treated with steroids and methotrexate, with poor efficacy. Short courses of subcutaneous G-CSF produced a transient and mild response in all three patients. Complete recovery of the neutrophils occurred temporarily during pregnancy in two patients. After a mean follow up of 34.8 months (range 6-139) all patients were alive and none developed serious infections. CONCLUSION: A subset of patients with primary SS and non-destructive arthritis may develop a chronic but well tolerated agranulocytosis that is usually poorly responsive to steroids and oral methotrexate.

Is the thrombopoietin assay useful for differential diagnosis of thrombocytopenia? Analysis of a cohort of 160 patients with thrombocytopenia and defined platelet life span.

BACKGROUND: Thrombopoietin (TPO), the major hormone controlling platelet production, has been measured in thrombocytopenias with discordant results. The aim of our work was to assess the value of the TPO assay for differential diagnosis of thrombocytopenias in a large cohort of patients classified according to the results of their platelet isotopic study. METHODS: We measured TPO (R&D Systems) in serum of 160 thrombocytopenic patients referred to our department for platelet life span isotopic studies. We classified patients as follows: (a) idiopathic or autoimmune thrombocytopenia group (ITP; patients with increased platelet destruction and shortened platelet life span; n = 67); (b) pure genetic thrombocytopenia group (patients with decreased platelet production, normal platelet life span, and without bone marrow aplasia; n = 55); (c) bone marrow aplasia group (BM; patients with decreased platelet production, normal platelet life span, and bone marrow aplasia; n = 13). RESULTS: In patients with pure genetic thrombocytopenia, TPO (median, 55 ng/L) was not different from TPO in patients with ITP (median, 58 ng/L) or controls (n = 54; median, 51 ng/L). Only in patients with bone marrow aplasia was TPO significantly higher (median, 155 ng/L) and negatively correlated to the platelet count (r(2) = 0.5014). CONCLUSIONS: Although the median serum TPO is increased in thrombocytopenia with decreased platelet production from bone marrow aplasia, it does not differentiate patients with pure genetic thrombocytopenia from those with ITP.

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.

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.

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.

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.

Short- and long-term follow-up of thyroid dysfunction after allogeneic bone marrow transplantation without the use of preparative total body irradiation.

We studied the incidence and potential prognostic value of thyroid abnormalities after allogeneic bone marrow transplantation (BMT) without total body irradiation (TBI) conditioning. 77 consecutive patients who received a chemotherapy-alone-based conditioning regimen pretransplant were included. Free serum thyroxine (FT4), free serum triiodothyronine (FT3) and serum thyrotropin (TSH) levels were assayed before and 3 and 14 months after BMT. Patients were classified in three categories: normal thyroid profile if FT3 and FT4 were within the normal range and TSH was normal or low, peripheral thyroid insufficiency (PTI) if TSH was >4 mIU/l, or an 'euthyroid sick syndrome' (ETS) if FT3 and/or FT4 were low and TSH was normal or low. The incidence of thyroid dysfunction at 3 months was 57%, and 29% at 14 months. This was mostly due to the occurrence of ETS which was more frequent at 3 months (48%, 29/61) than at 14 months (19%, 9/48). Furthermore, at 3 months, survival was significantly lower in the ETS group (34.5%) than in the euthyroid group (96.2%), or in the PTI group (83.3%) (P < 0.0001). PTI was observed even in the absence of TBI in 11 patients (14%) and was equally distributed at 3 months (n = 6) and 14 months (n = 5). In conclusion, thyroid dysfunction is not a rare complication even without pretransplant TBI conditioning regimen. Hypothyroidism prevalence was 10%, and ETS, which was more frequently observed, displayed a dismal predictive value at 3 months.

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.

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).

Tumour-associated trypsin inhibitor and renal cell carcinoma.

In the absence of a specific marker for renal cell carcinoma (RCC), we evaluated the tumour-associated trypsin inhibitor (TATI) in patients with RCC. Between November 1990 and November 1993, 63 patients with RCC and 23 patients with benign renal disease underwent a competitive radioimmunoassay of TATI. The cutoff value was defined on a series of serum samples of 96 healthy subjects (normal n < 20 micrograms/l, then 25 micrograms/l after April 1993). We related the value of TATI to the tumour stage and compared the sensitivities of TATI and other markers (CEA, CA 15-3, CA 125, CA 19-9, ferritin). In 24 patients the TATI assay was repeated 3-12 months after radical nephrectomy. 15 patients with benign disease had a normal value of TATI (specificity: 65%). 44 of the 63 patients had a value of TATI above the cutoff point (sensitivity: 69%). Sensitivities of CEA, CA 15-3, CA 125, CA 19-9 and ferritin were 5, 10, 13, 5, 35%, respectively. The TATI value was correlated with the stage of the disease. Among the 15 patients without metastasis, the mean preoperative value was 112 micrograms/l (14-760) versus 46 micrograms/l (24-180) postoperatively. In the 9 patients with metastasis, the preoperative mean value was 100 micrograms/l (20-434) versus 240 micrograms/l (22-544) postoperatively. TATI showed a better sensitivity than other markers for RCC but its specificity is limited. Nevertheless it can be useful for a postoperative follow-up. TATI remains one of the best serum markers for RCC.

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.

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.

Hematopoietic growth factor expression and ATRA sensitivity in acute promyelocytic blast cells.

Acute promyelocytic leukemia (APL) is a homogeneous subgroup of acute myeloid leukemias (AMLs) characterized by the presence of the t(15,17) translocation and the resulting promyelocytic myeloid leukemia/retinoic acid receptor alpha (PML/RAR alpha) fusion proteins. To date APL is the only AML that is sufficiently sensitive to all-trans retinoic acid's (ATRA) differentiating effect. In vivo ATRA alone achieves complete remission in most APL patients. However, failure or partial responses are observed and the molecular basis of the absence of ATRA response in these patients has not been determined. To gain insights in the cell growth and differentiation of APL cells, expression of hematopoietic growth factors (HGF) shown to be produced by leukemic cells (interleukin-1 beta [IL-1 beta], IL-6, tumor necrosis factor alpha (TNF alpha), granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage colony-stimulating factor [GM-CSF], and IL-3) was studied in 16 APL samples. Twelve APL cases expressed IL-1 beta, IL-6, and TNF alpha, but not G-CSF, GM-CSF, and IL-3. These cases achieved complete remission with ATRA therapy. The four remaining patients (either TNF alpha negative or G-CSF, GM-CSF or IL-3 positive) did not achieve complete remission with ATRA. In all cases, in vivo response to ATRA therapy was correlated to the in vitro differentiation effect of all-trans retinoic acid 10(-6) mol/L. Thus, ATRA differentiation induction was strongly correlated to the HGF expression (P < .0001). These results suggest that the presence or absence of HGF's expression by APL cells may contribute to the therapeutic effect of ATRA in this disease.

[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.