Role of MTHFR (677, 1298) haplotype in the risk of developing secondary leukemia after treatment of breast cancer and hematological malignancies.

Therapy-related myelodysplasia and acute myeloid leukemia (t-MDS/AML) is a malignancy occurring after exposure to chemotherapy and/or radiotherapy. Polymorphisms involved in chemotherapy/radiotherapy response genes could be related to an increased risk of developing this neoplasia. We have studied 11 polymorphisms in genes of drug detoxification pathways (NQO1, glutathione S-transferase pi) and DNA repair xeroderma pigmentosum, complementation group (3) (XPC(3), X-ray repair cross complementing protein (1)), Nijmegen breakage syndrome (1), excision repair cross-complementing rodent repair deficiency, complementation group (5) and X-ray repair cross complementing protein (3) and in the methylene tetrahydrofolate reductase gene (MTHFR(2), 677C>T, 1298A>C), involved in DNA synthesis. The analyzed groups were a t-MDS/AML patients group (n=81) and a matched control group (n=64) treated similarly, and they did not develop t-MDS/AML. We found no significant differences when the groups were compared globally. However, when analysis was carried out according to the primary neoplasia involved, a significant association was observed between the MTHFR haplotype (single nucleotide polymorphisms 677 and 1298) and the risk of developing t-MDS/AML in the breast cancer patients group (P=0.016) and cyclophosphamide-treated hematological disease group (P=0.005). Risk haplotype was different for each case, corresponding to the 677T1298A haplotype after breast cancer treatment and the 677C1298C haplotype after hematological malignancy treatment. We postulate that such differences are related to variations in chemotherapy schemes between hematological and breast cancers and their differential interaction with the MTHFR route.

Outcome of patients with acute promyelocytic leukemia failing to front-line treatment with all-trans retinoic acid and anthracycline-based chemotherapy (PETHEMA protocols LPA96 and LPA99): benefit of an early intervention.

To determine prognosis of acute promyelocytic leukemia (APL) failing to front-line therapy with all-trans retinoic acid (ATRA) and anthracyclines, outcome of 52 patients (32 M/20 F; age: 37, 3-72) included in PETHEMA trials LPA96 and LPA99 who presented with either molecular failure (MOLrel, n=16) or hematological relapse (HEMrel, n=36) was analyzed. Salvage therapy consisted of ATRA and high-dose ara-C-based chemotherapy (HDAC) in most cases (83%), followed by stem-cell transplantation (autologous, 18; allogeneic, 10; syngeneic, 1). Fourteen patients with MOLrel (88%) achieved second molecular complete response (molCR), whereas 81% HEMrel patients responded to second-line treatment, with 58% molCR. After median follow-up of 45 months, four MOLrel and 18 HEMrel patients, respectively, experienced a second relapse. Outcome after MOLrel compared favorably to HEMrel, with longer survival (5-year survival: 64+/-14 vs 24+/-8%, P=0.01) and lower relapse risk (5-year relapse risk: 30+/-13 vs 64+/-9%; P=0.044). Additionally, age

Aromatase activity and estradiol in human breast cancer: its relationship to estradiol and epidermal growth factor receptors and to tumor-node-metastasis staging.

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.

Prolonged molecular remission after PML/RAR alpha-positive autologous peripheral blood stem cell transplantation in acute promyelocytic leukemia: is relevant pretransplant minimal residual disease in the graft?

The contribution of residual malignant cells contaminating the autologous graft with the occurrence of post-transplant relapse in acute myeloid leukemia (AML) is still unclear. The presence of a specific molecular marker (the PML/RAR alpha rearrangement) in acute promyelocytic leukemia (APL) offers the opportunity to investigate better the pathogenesis of disease recurrence after transplant. We report an APL patient who received high-dose chemotherapy and peripheral blood stem cell (PBSC) autograft in second hematologic remission. Two leukaphereses that tested PML/RAR alpha positive by RT-PCR were obtained during the post-reinduction hematopoietic recovery, while the patient also tested PCR positive in the BM, and was reinfused after myeloablative chemotherapy (BUCY4), when the patient had spontaneously converted to PCR negative in the marrow. At present, he remains in continuous molecular and hematologic remission 22 months after PBSC transplantation. This is the second report of an APL patient who was transplanted in molecular remission with a PML/RAR alpha-positive PBSC autograft. As in the previous report, the prolonged clinical and molecular remission experienced post-transplant suggests that autologous PBSC infusion is still worthy of consideration for patients with APL in spite of the detection of PML/RAR alpha-positive cells in the PBSC collections. Possible underlying mechanisms and the potential role of molecular monitoring of the graft, as well as the host, before and after transplant, in patients with APL undergoing autologous HSCT are also discussed.

Preliminary experience in external quality control of RT-PCR PML-RAR alpha detection in promyelocytic leukemia.

To standardize the results obtained in PML/RAR alpha RT-PCR detection by laboratories of hospitals involved in the Spanish Program for Treatment of Hematological Malignancies (PETHEMA) LPA-96, designed for the treatment of acute promyelocytic leukemia (APL), cDNA samples obtained by reverse transcription of RNA from bone marrow samples of patients with APL were sent to participating laboratories. During the first year of this external quality assessment trial nine samples were tested by a maximum of 12 laboratories. The control gene was satisfactorily amplified in 90% of the samples (62 of 69 samples), supporting the adequacy of the cDNA to be used as control sample. There was an 83% concordance between laboratories for PML/RAR alpha detection with similar results for the type of PML/RR alpha rearrangements. However, 17% disagreement still remained, attributable to low sensitivity or inadequacy of methods followed. The results stressed the need for implementation of an external quality assessment scheme to ensure the standardization of the results.

Epidermal growth factor receptor in human breast cancer: correlation with cytosolic and nuclear ER receptors and with biological and histological tumor characteristics.

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.

Chronic myeloid leukemia with expression of ALL-type BCR/ABL transcript: a case-report and review of the literature.

CML with exclusive expression of ALL-type bcr/abl has only been rarely described. In some cases, the presence of this fusion gene has been associated to a differentiated subtype of CML that share some features with CMML, while in another case this molecular hallmark has been associated to a bad prognosis of the disease with a blast phase as clinical presentation or an early transformation to blast phase. We report a case of a 30-year-old woman who was diagnosed of CML in chronic phase in May 1989. She received treatment first with busulfan, achieving hematological remission and afterwards with interferon and Hydroxiurea. In February 1998, she was admitted at our hospital for an ABSCT. Then, molecular studies were performed. Multiplex PCR revealed the presence of a 481 bp product identified as the ela2 bcr/abl transcript and confirmed by sequencing. After 9 years from diagnosis, the patient remains in hematological remission and in good clinical condition.

Unrelated donor cord blood transplantation in adults with chronic myelogenous leukemia: results in nine patients from a single institution.

The potential role of unrelated donor cord blood transplantation (UD-CBT) in adults is not well established. We report the results of UD-CBT in nine adult patients with chronic myeloid leukemia (CML). The median age was 27 years (range, 19-41 years), and the median weight was 62 kg (range, 45-78 kg). At transplant, six patients were in chronic phase (five in first, and one in second), two in blast crisis, and one in accelerated phase. Eight had received intensive chemotherapy, and three had undergone autologous peripheral blood hematopoietic stem cell transplantation. Four had received interferon with no cytogenetic response, and only three underwent UD-CBT within 1 year of diagnosis. After serological typing for class I antigens, and high-resolution DNA typing for DRB1, the degree of HLA match between patients and cord blood (CB) units was 4/6 in six cases and 5/6 in three cases. The median number of nucleated cells infused was 1.7 x 10(7)/kg (range, 1.2 to 4.9 x 10(7)/kg), and was above 2 x 10(7)/kg in only two cases. All patients received thiotepa, busulfan, cyclophosphamide and anti-thymocyte globulin as conditioning; cyclosporine and prednisone for graft-versus-host disease (GVHD) prophylaxis; and G-CSF from day +7 until engraftment. All seven evaluable cases engrafted. The median time to reach an absolute neutrophil count > or =0.5 x 10(9)/l and > or =1 x 10(9)/l was 22 days (range, 19-52 days) and 28 days (range, 23-64 days), respectively. In the four patients evaluable for platelet recovery time to levels of > or =20 x 10(9) platelets/l, > or =50 x 10(9) platelets/l, and > or =100 x 10(9) platelets/l, these ranged from 50 to 128 days, 60 to 139 days, and 105 to 167 days, respectively. Three patients developed acute GVHD above grade II, and three of the five patients at risk developed extensive chronic GVHD. Four patients, all transplanted in chronic phase, remain alive in molecular remission more than 18, 19, 24 and 42 months after transplantation. These preliminary results suggest that UD-CBT may be considered a reasonable alternative in adults with CML who lack an appropriate bone marrow donor.

Variant three-way translocation of inversion 16 in AML-M4Eo confirmed by fluorescence in situ hybridization analysis.

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.

Quantitative detection of AML1-ETO rearrangement by real-time RT-PCR using fluorescently labeled probes.

The persistence of the AML1-ETO rearrangement performed by reverse transcription polymerase chain reaction (RT-PCR) has been reported in acute myeloid leukemia (AML) patients in long-term complete remission (CR). This persistence, which is not associated with hematological relapse, limits the clinical use of qualitative RT-PCR. Here, we present a new quantitative real-time PCR method to detect AML1-ETO rearrangement using fluorescently labeled probes. Quantitative detection of AML1-ETO was performed in capillary tubes using two fluorescently labeled probes in the LightCycler equipment. The reliability of the method was checked in twenty-two bone marrow samples and one apheresis sample from eight patients with t(8;21) collected at diagnosis and during follow-up assessment. The regression coefficients obtained for standard curves of AML1-ETO and AML were all greater than 0.98. The sensitivity attained allowed the detection of rearrangements at a dilution of 10(-5) Kasumi-1 cDNA. The intra-assay coefficient of variation was 4% for AML1-ETO, and 7% for AML. The inter-assay coefficient of variation was 19% for AML1-ETO and 12% for AML. A log reduction from two to four in the AML1-ETO/AML ratio was evident after CR. The study of the method and first results obtained in patient samples support that quantitative real-time PCR with hybridization probes is a new reliable and sensitive method to monitor minimal residual disease in AML patients. Moreover, the fluorescent probes with the Light-Cycler technology offer the advantage of a rapid detection.

Salivary corticosteroids in the study of adrenal function.

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.

Estradiol receptors in combination with neu or myc oncogene amplifications might define new subtypes of breast cancer.

Amplifications of neu and c-myc were evaluated in 218 and 145 breast cancers (BC), respectively. Oncogene amplifications were determined for the most part by Southern blot. An association between the proportion of nodes affected and the intensity of neu amplification in estadiol receptor negative (ER-) BC was found (P = 0.028), which was confirmed by the multi-factor analysis of variance (P = 0.05). A significantly greater incidence in neu amplifications among BC with metastases was also found (P = 0.031). A strong association (P = 0.01) between the neu and myc amplification was observed. There is a strong association between myc amplification and ER- BC (P < 0.01). It is concluded that (1) the combination ER- with neu amplification might define a new group of more aggressive BC, as is suggested by their associated nodal involvement; (2) the linkage of myc amplifications with ER- BC and high grade of neu amplification might reflect a trait of tumor aggressivity.

Epidermal growth factor in human breast cancer, endometrial carcinoma and lung cancer. Its relationship to epidermal growth factor receptor, estradiol receptor and tumor TNM.

Epidermal growth factor (EGF) and its receptor (EGFR) were measured in 60 breast cancers (BC), 6 benign mammary tumors (BM), 8 samples of normal breast (NB), 6 endometrial carcinomas (EC) and 30 lung cancers (LC). EGF was measured in plasma, saliva and urine from 20 patients with BC, before and after tumor excision, and in 8 patients with metastatic disease. The median EGF in BM and BC was significantly higher (P < 0.05) than in NB. No significant correlation between EGF and EGFR was found in BC. Neither tumor excision nor the spreading of the disease significantly modified the EGF concentrations in biological fluids. In LC there was an inverse relationship between EGF and EGFR (rs = -0.36; P = 0.09), which disappeared in normal lung. It is concluded that EGF may play a role in malignant transformation; however, the weak correlation between EGF and EGFR lessens the importance of EGF in either autocrine or paracrine stimulation of tumor growth.

Prognostic significance of c-erbB-2/neu amplification and epidermal growth factor receptor (EGFR) in primary breast cancer and their relation to estradiol receptor (ER) status.

The aim of this study is to evaluate the prognostic significance of c-erbB-2/neu amplification and epidermal growth factor receptor (EGFR) expression in primary breast cancer (BC) and their prognostic implications when combined with estradiol receptor (ER) status. In this work, 825 BCs were studied. Neu amplification was evaluated by dot-blot and EGFR expression was evaluated by ligand binding assay using I125-EGF. Neu, EGFR, estradiol and progesterone receptors (ER and PR) had a marked influence on disease free survival (DFS) in univariate analysis. In node-negative (NO) cases only neu was associated with short DFS (p = 0.005). However, in node-positive (N+) cases both EGFR (p = 0.005) and neu (p = 0.002) influenced DFS. None of the biological markers were significant predictors for overall survival (OS) in NO/BC. On the contrary, in N+/BC, EGFR + (p = 0.003) was associated with short OS. The EGFR + /neu/phenotype represented a sub-group with an even worse prognosis with respect to DFS (p = 0.0034) as well as EGFR + /ER-tumors (p = 0.005). Moreover, neu + /ER-patients also had a high probability of relapse (p = 0.0000) and death (p = 0.006). C-erbB-2/neu, EGFR, histological grade, pN, pT and ER were subjected to a Cox multivariate regression analysis: neu was the most important parameter in predicting recurrence, and EGFR was a significant predictor for OS.

Specific oncological contribution of cathepsin D and pS2 in human breast cancer: their relationship with TNM status, estradiol receptors, epidermal growth factor receptor and neu amplification.

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.

[Monitoring of minimal residual disease with the combined detection of PML/RAR alpha and RAR alpha/PML rearrangements in acute promyelocytic leukemia]. / Monitorización de la enfermedad mínima residual mediante detección conjunta de los reordenamientos PML/RAR alpha y RAR alpha/PML en la leucemia promielocítica aguda.

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.

Wnt signaling pathway is epigenetically regulated by methylation of Wnt antagonists in acute myeloid leukemia.

Activation of the Wnt signaling pathway has been implicated recently in the pathogenesis of leukemia. We studied the function of epigenetic regulation of the Wnt pathway and its prognostic relevance in acute myelogenous leukemia (AML). We used a methylation-specific polymerase chain reaction approach to analyze the promoter methylation status of a panel of Wnt antagonists including sFRP1, sFRP2, sFRP4, sFRP5, DKK1 and DKK3. Aberrant methylation of Wnt antagonists was detected in four AML cell lines and in up to 64% of AML marrow samples. Treatment of the cell lines with 5-aza-2'-deoxycytidine induced reexpression of methylated Wnt antagonists and inactivation of the Wnt pathway by downregulating the Wnt pathway genes cyclin D1, TCF1 and LEF1 and reducing nuclear localization of beta-catenin. In a subgroup of patients 60 years and younger with newly diagnosed AML and intermediate-risk cytogenetics, abnormal methylation of Wnt antagonists was associated with decreased 4-year relapse-free survival (28 vs 61%, respectively, P=0.03). Our results indicate a function of the epigenetic regulation of the Wnt pathway in predicting relapse in a subgroup of AML patients.

[Urinary excretion of 11-deoxy-17-ketosteroids during the normal menstrual cycle (author's transl)]. / Variación de los 11-desoxi-17-cetosteroides a lo largo del ciclo menstrual normal

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.

Basal prolactin and the behaviour of the gonadotrophins, testosterone, androstenedione, estradiol, and the sex-hormone-binding globulin during stimulation with clomiphene in subjects with spermatogenic disorders.

To clarify the significance of clomiphene test in spermatogenic disorders, it was performed on three groups of subjects: 10 with normozoospermia, 29 with oligozoospermia, and 11 with azoospermia. Two basal blood samples were drawn five days apart; prolactin, FSH, LH, estradiol, testosterone, androstenedione and sex-hormone-binding globulin were determined. 100 mg of clomiphene per day were administered for eleven consecutive days; another sample was drawn on the eleventh day and all the basal parameters, except prolactin, were determined. It may be concluded from the results: a) All the parameters studied increased significantly after clomiphene; and b) Testosterone levels obtained after clomiphene, as well as the increases in this hormone during the test and the ratio delta T/delta LH, were significantly lower in the oligozoospermic group. This finding suggests a decrease in the testicular androgenic function of this group.