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Background and Objectives: The detection of CBFbeta/MYH11 transcripts by RT-PCR has became a valuable and widely used technique in the accurate cytogenetic and molecular classification of acute myeloid leukemia (AML), but the clinical value of RT-PCR for monitoring minimal residual disease (MRD) during follow-up remains unclear., Design and Methods: We analyzed the factors predicting relapse and the value of MRD monitoring by RT-PCR in a series of 16 patients with CBFb/MYH11-positive AML (15 M4Eo; 1 M4). Fifteen were newly diagnosed cases (CR1) and one was studied after first relapse (CR2). Eight patients had clinical relapse at 6 to 19 months from the achievement of CR., Results: Presenting WBC count had a significant prognostic influence on disease-free survival (p=0.001). All four patients with a WBC count >100x10(9)/L relapsed, while only four additional relapses occurred among the eleven patients who had an initial WBC count below 100x10(9)/L. With regards to molecular monitoring, all relapses but one occurred in patients who showed persistent RT-PCR positivity during hematologic remission. By contrast, conversion to a repeatedly PCR-negative status was observed in the seven patients who remained in CR1 after a median follow-up of 48 months (range 31-79 months), as well as in the transplanted patient who was monitored in CR2. In these patients a PCR-positivity could be detected up to 24 months after diagnosis (median time to conversion to PCR-negative: 8 months)., Interpretation and Conclusions: In conclusion, marked hyperleukocytosis (>100x10(9)/L) confers poor prognosis to the patient with CBFbeta/MYH11-positive AML. In addition, slow kinetics of molecular remission was observed in this subset of AML, but the CBFb/MYH11 fusion transcript is no longer detectable in long-term survivors, indicating that molecular remission is an important therapeutic goal.

Background: Molecular assay commonly used to detect the PML/RAR alpha rearrangement in acute promyelocytic leukemia (APL) has the limited sensitivity in comparison with the higher sensitivity of RAR alpha/PML detection. This prompted us to perform both assays in parallel to monitor a group of APL., Patients and Methods: The study included 56 APL patients mainly treated according with the PETHEMA LPA-96 protocol. The PML/RAR alpha was detected according with Biondi's et al method and the RAR alpha/PML following the Grimwade's et al RT-PCR method (Human Press Inc.)., Results: RAR alpha/PML rearrangement was detected in 90% (20/22) of the patients at diagnosis positives for PML/RAR alpha. RAR alpha/PML was detected in 74% (14/19) of post-induction samples versus 37% (7/19) of positives for PML/RAR alpha. Likewise RAR alpha/PML rearrangement was detected in some post-consolidation samples (2/11) that all were PMI/RAR alpha negatives. In patients in maintenance regimen a greater proportion of RAR alpha/PML positives (6/28) versus PML/RAR alpha (2/28) were observed. In a patient in complete remission RAR alpha/PML preceded the positivity of PML/RAR alpha and persisted after PMI/RAR alpha negativization. The results of the patients monitored since the diagnosis showed that RAR alpha/PML revert to negative one month after PML/RAR alpha negativization., Conclusions: RAR alpha/PML rearrangement is not expressed in the totality of the APL patients, but in only a 90% of them. RAR alpha/PML rearrangement was detected in a greater proportion of samples than PML/RAR alpha. RAR alpha/PML rearrangement lasted longer than PML/RAR alpha after treatment.

The inv(16) and t(16;16) characterize a subgroup of acute myelomonocytic leukemia (AML) with distinct morphological features and a favorable prognosis. Both cytogenetic abnormalities result in a fusion of CBF beta at 16q22 and MYH11 gene at 16p13, whose detection by PCR and fluorescence in situ hybridization (FISH) is useful for diagnosis and monitoring of the disease. Variant translocations of inv(16)/t(16;16) are very rare and whether they are also associated with a favorable prognosis is unknown. We report a patient presenting with typical AML-M4Eo and a three-way translocation of inv(16) involving 16p13, 16q22, and 3q22. FISH studies on bone marrow (BM) chromosomes using CBFB and MYH11 DNA probes revealed a fusion of CBFB and MYH11 on 16q of the der(16), as well as a signal from MYH11 on 16p but not from CBFB; normal signals for both probes were present on the normal 16. Neither of these labeled probes was on the der(3), but the translocation between the der(3) and der(16) was confirmed by using a chromosome 16 painting probe. Molecular analysis of BM cells using RT-PCR identified a CBFB-MYH11 fusion transcript type D. After achieving complete remission, the patient relapsed. We conclude that FISH and PCR are feasible tools to distinguish cases with variant abnormalities of inv(16) from cases with other chromosome 16 abnormalities. Variant abnormalities of inv(16) may be not associated with favorable prognosis.

Aims: The sensitivity and specificity of a reverse transcription PCR method (RT-PCR) to detect cytokeratin K19 (CK19) expression was evaluated with the purpose of assessing its capability to detect the presence of breast cancer tumour cells in peripheral blood progenitor cell collection that had to be reinfused to breast cancer patients submitted to intensive chemotherapy as haematopoietic support., Patients and Methods: Two breast cancers as positive samples were used and 34 samples of mononucleated blood cells as negative controls: 18 peripheral blood samples from normal subjects, 14 from different types of leukaemias (M3, M4Eo, M2, etc.) and two from two patients with Hodgkin's lymphoma. The method studied is a nested RT-PCR that amplifies the CK19 expression from the sample RNA extracted following the method of phenol-chloroform., Results: The right performance of the method is demonstrated by observing the detection of CK19 transcripts in the breast cancer RNA and by obtaining good blank results both with non transcribed RNA and with DNA. Moreover, the method has an excellent sensitivity such as to allow the detection of CK19 transcripts in a 10(-6) dilution of cDNA reverse transcribed from 1 microgram of breast cancer RNA. The CK19 transcripts were also detected in the 64% of RNA obtained from the mononucleated blood cells controls, although the percentage of positivities was lower (47%) in the RNA from peripheral blood samples. Nevertheless it should be remarked that the levels of CK19 expression in the blood mononucleated cells is almost negligible since it used to extinguish at 1:5 cDNA dilution., Conclusions: The method studied is specific and has a high sensitivity that explains the detection of CK19 illegitimate expression approximately a half in mononucleated blood cells negative controls. However, the levels of CK19 expression in mononucleated blood cells were almost negligible and it used to extinguish at 1:5 cDNA dilution, therefore it could be concluded that the method might be useful to detect breast cancer occult tumours cells in mononucleated blood cell collection, always provided that a lower amount of cDNA is taken, thus decreasing to nil almost the false positive samples and keeping always a good sensitivity.

Aims: This study describes a molecular method of reverse transcription polymerase chain reaction (RT-PCR) to detect the rearrangement PML/RAR alpha in acute promyelocytic leukaemia (APL) in order to assess its specificity and sensibility, and to evaluate its utility in the characterization of APL patients., Patients and Methods: Between January and June of 1995, 64 samples of bone marrow and peripheral blood stem cells (PBSC) cytapheresis were studied. There were 58 APL samples (23 patients: 10 samples obtained with disease activity, 43 samples in complete remission (CR) and 5 PBSC samples) and 6 control samples, of non-APL hematological neoplasms (3 other AML, 1 CML, 1 ALL, and 1 MDS). On the RNA obtained from the isolated mononuclear cells of each sample a conserved region of the PML/RAR alpha fusion gene was amplified by using a RT-PCR with specific primers., Results: The sensitivity assays were performed by diluting PML/RAR alpha positive RNA samples into RNA of controls. The RT-PCR assay was capable to detect the PML/RAR alpha until an 1/1000 dilution in negative control RNA. Nine out of 58 APL samples failed in the amplification of the control gene, and were considered non-evaluable. None of the 6 control samples showed PML/RAR alpha rearrangement. Nine out of 10 APL samples with disease activity were positive for the presence of PML/RAR alpha (the non-positive sample was a non-evaluable one). Six out of 43 APL samples in CR showed the rearrangement, 3 of them corresponding to 2 patients who posteriory relapsed 12 and 19 months after 1st CR. The other 3 positive samples came from other 3 APL patients (24 months in 3rd CR, 14 months in 1st CR and early CR), who remained in CR at the end of the study. No relapse could be noted in patients with negative PCR samples. PML/RAR alpha was not found in any of the 5 APL PBSC samples studied., Conclusions: The RT-PCR method described here seems to be highly specific as it only detects this rearrangement in LPA patients. Furthermore, the presence of PML/RAR alpha in CR patients could be related to relapse. For all these reasons, this molecular method shows great usefulness and can be advocated, not only for assessing diagnosis, but for as monitoring minimal residual disease in the post remission follow up.

The present study attempts to clarify the specific contribution of cathepsin D (CD) and pS2 to the progression of breast cancer (BC) by examining the relationship between these two factors and TNM status, tumour grade, estradiol receptors (ER) and the prognosis factors epidermal growth factor receptor (EGFR) and neu amplification in a group of 270 BC patients. CD and pS2 were determined by an immunoradiometric procedure in tumour cytosols obtained for ER. Neu amplifications were evaluated by dot-blot, in tumour DNA. EGFR was determined in membrane tumour preparations obtained from ER cytosols by a two-point radiometric saturation assay. CD is basically related to bad prognosis factors and has a direct correlation with tumour size (P = 0.025) and EGFR content (P = 0.007) and is associated with the presence of metastases (P = 0.000). pS2 is mostly related to good prognosis factors and showed an inverse correlation with the Scarff-Bloom Index (P = 0.011) and a direct correlation with ER content (P = 0.014). Finally, pS2 and CD also showed a strong mutual association (P = 0.009) and the fact that both correlated with ER content confirms in tumours the experimental finding that they are estrogen-induced proteins.

Purpose: The present report attempts to clarify whether there is a relationship between aromatase activity (ARAC) and estradiol (E2), hormonal receptors, E2 receptor (ER), and epidermal growth factor receptor (EGFR), as well as with tumor stage and histopathology in human breast cancers., Materials and Methods: We studied 225 breast carcinomas, 67 of which were premenopausal and 158 postmenopausal. In each sample, ARAC, EGFR, ER, and E2 were quantified. ARAC was quantified by Thompson and Siiterii's method, EGFR was quantified with a two-point assay method using radioactive iodine (125I)-EGF as ligand, and ER was measured by the Scatchard method using 3H-E2. E2 was quantified by radioimmunoassay in the diethylether tumor extract., Results: ARAC was found in 64% of the cancers studied. There is a strong direct association between ARAC and tumor size in postmenopausal patients (P = .001). In the postmenopausal group, the proportion of ARAC-positive (ARAC+) tumors is significantly higher among ER-positive (ER+) than ER-negative (ER-) ones (P less than .001). ER+ tumors also have significantly higher levels of E2 than do ER- ones (P less than .0001); similarly, ARAC+ tumors have significantly higher levels of E2 than do ARAC- ones (P less than .0001). There is a significant multiple linear correlation between the log of the levels of ARAC, ER, and EGFR and the log of tumor E2 (P less than .0001). The correlation coefficients obtained show that ARAC and ER have a positive effect on tumor E2., Conclusion: The results obtained suggest the importance of tumor ARAC in the tumoral levels of E2 and reinforce the possible biologic significance of tumor ARAC, especially in postmenopausal breast carcinoma patients.

Epidermal growth factor receptor (EGFr) and cytosolic (cER) and nuclear (nER) estradiol receptors were quantified in 220 primary breast cancers. The EGFr was significantly more frequent (X2 = 5.9; P less than 0.025) and its concentration was significantly higher (P less than 0.001) among ER- tumors than in ER+ tumors. There was a significantly greater proportion (X2 = 6.4; P less than 0.05) of node involvement in EGFr+/ER+ tumors than in EFGr/ER+. Increases in the proportion of EGFr+ in ER- tumors are parallel to Scarff-Bloom scores (X2 = 6.1; P less than 0.05) and there is a significant trend towards increased EGFr concentrations with histologic dedifferentiation. In ER+ tumors the median concentrations of EGFr in the different age groups show linear correlation and follow a parallel profile with the medians of nER. These findings support the hypothesis that considers EGFr as a bad prognosis factor and suggest that EGFr expression and concentration in ER+ tumors might be considered an estrogenic action mediated through the binding of ER to their nuclear acceptors.

Epidermal growth factor receptor (EGFr) and cytosolic (cER) and nuclear (nER) estradiol receptors were quantified in 220 primary breast cancers. The EGFr was significantly more frequent (chi 2 = 5.9; P less than 0.025) and its concentration was significantly higher (P less than 0.001) among ER- tumors than in ER+ tumors. There was a significantly greater proportion (chi 2 = 6.4; P less than 0.05) of node involvement in EGFr+/ER+ tumors than in EFGr-/ER+. Increases in the proportion of EGFr+ in ER- tumors are parallel to Scarff-Bloom scores (chi 2 = 6.1; P less than 0.05) and there is a significant trend (Spearman rs = 0.25; P less than 0.05) towards increased EGFr concentrations with histologic dedifferentiation. In ER+ tumors the median concentrations of EGFr in the different age groups show a linear correlation (LCC = 0.89; P less than 0.05) and follow a parallel profile with the medians of nER. These findings support the hypothesis that EGFr is a bad prognosis factor and suggest that EGFr expression and concentration in ER+ tumors might be considered an estrogenic action mediated through the binding of ER to their nuclear acceptors.

Urinary excretion of 11-deoxy-17-ketosteroids was studied in 20 to 22 women with normal menstrual cycles on the 7, 10, 14 and 20th days of their cycles. The fraction studied showed a significant cyclic variation with a type of pattern for Androsterone and Aetiocholanolone and another one for the Dehydroepiandrosterone. The possibity of one metabolic path for the former metabolites and a different one for the latter, plus its close conection with the functional changes of the ovary, were arrived at.

Salivary corticosteroids (SCC) and plasma corticosteroids (PCC) were studied under basal conditions, after dexamethasone (DXM) and in the ACTH stimulation test in a reference group (RG) of 33 adults, in three groups with non-adrenal pathology and in a group of 4 patients with hypercortisolaemia. SCC and PCC were measured using a non-extraction RIA method using [3H]cortisol. The results for SCC in the RG and in the groups with non-adrenal pathology were similar to those obtained for PCC in terms of percentage of decrease in the circadian rhythm or DXM suppression. However, the responsiveness to ACTH in saliva was twice that obtained in plasma. In patients with hypercortisolism, SCC were in closer agreement with the adrenal hyperfunction than PCC. From the previous results the following conclusions may be drawn: (a) SCC differentiate adrenal gland normal function from hyperfunction as clearly or even better than PCC does; (b) SCC were in a closer agreement with the symptomatology of adrenal hyperfunction than were PCC; and (c) the responses to ACTH obtained with SCC were clearly higher than those obtained with PCC.

The nuclear DNA/cytoplasmic protein ratio was related to the oestradiol receptor content in both cytoplasmic (ERc) and nuclear (ERn) fractions in 165 cases of breast cancer. The patients under study were divided into groups according to whether their biopsies contained functional (ERc/ERn = +/+), abnormal (+/0) or non-detectable receptor (0/0). The mean microgram DNA/mg protein ratio was significantly decreased in both the abnormal and receptor-negative groups, with means of 30.4 and 30.2 respectively, when compared to the mean of 52.8 for the receptor-positive group. When the DNA/protein ratio for each patient was related to age, a discriminant line which clearly differentiated the ER +/+ group from the other two groups was obtained. It may be concluded that an increased DNA/protein ratio is related to the presence of ERn. Furthermore, since the DNA content per cell is higher in ER-negative tumours, an increase in cellularity must exist in ER-positive tumours.