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The recent World Health Organization classification recognizes different subtypes of acute myeloid leukemia (AML) according to the presence of several recurrent genetic abnormalities. Detection of these abnormalities and other molecular changes is of increasing interest because it contributes to a refined diagnosis and prognostic assessment in AML and enables monitoring of minimal residual disease. These genetic abnormalities can be detected using single RT-PCR, although the screening is still labor intensive and costly. We have developed a novel real-time RT-PCR assay to simultaneously detect 15 AML-associated rearrangements that is a simple and easily applicable method for use in clinical diagnostic laboratories. This method showed 100% specificity and sensitivity (95% confidence interval, 91% to 100% and 92% to 100%, respectively). The procedure was validated in a series of 105 patients with AML. The method confirmed all translocations detected using standard cytogenetics and fluorescence in situ hybridization and some additional undetected rearrangements. Two patients demonstrated two molecular rearrangements simultaneously, with BCR-ABL1 implicated in both, in addition to RUNX1-MECOM in one patient and PML-RARA in another. In conclusion, this novel real-time RT-PCR assay for simultaneous detection of multiple AML-associated fusion genes is a versatile and sensitive method for reliable screening of recurrent rearrangements in AML., (Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

The most frequent KIT mutations reported in core-binding factor acute myeloid leukemia are point mutations and insertions/deletions in exons 17 and 8. The vast majority of KIT mutation detection procedures are time-consuming, costly, or with a high lower limit of detection. High-resolution melting (HRM) is a gene scanning method that combines simplicity and rapid identification of genetic variants. We describe an HRM method for the simultaneous screening of exons 8 and 17 KIT mutations and report the results obtained in 69 core-binding factor acute myeloid leukemia patients. Mutation detection was compared with sequencing as the gold standard. The HRM method used high-resolution melting master reagents (Roche) and the LightCycler 480 (Roche) platform. HRM was reproducible, showed a lower limit of detection of 1%, and discriminated all patients with mutated KIT from controls without false positive or false negative results. Additionally, most of the mutations were differentiated from the other mutations. KIT mutations were present in 15.9% of patients, showing a higher incidence in inv(16) (25.8%) than in t(8;21) (7.9%). The presence of a KIT mutation was associated with a high white blood cell count, and adult patients with an exon 17 mutation had a higher incidence of relapse. These findings verify that HRM is a reliable, rapid, and sensitive method for KIT mutation screening. Furthermore, our study corroborates the unfavorable prognosis associated with exon 17 KIT mutations.

A previous report of the Programa de Estudio y Tratamiento de las Hemopatías Malignas (PETHEMA) Group showed that a risk-adapted strategy combining all-trans retinoic acid (ATRA) and anthracycline monochemotherapy for induction and consolidation in newly diagnosed acute promyelocytic leukemia results in an improved outcome. Here we analyze treatment outcome of an enlarged series of patients who have been followed up for a median of 65 months. From November 1999 through July 2005 (LPA99 trial), 560 patients received induction therapy with ATRA plus idarubicin. Patients achieving complete remission received 3 courses of consolidation followed by maintenance with ATRA and low-dose chemotherapy. The 5-year cumulative incidence of relapse and disease-free survival were 11% and 84%, respectively. These results compare favorably with those obtained in the previous LPA96 study (P = .019 and P = .04, respectively). This updated analysis confirms the high antileukemic efficacy, low toxicity, and high degree of compliance of a risk-adapted strategy combining ATRA and anthracycline monochemotherapy for consolidation therapy.

Background: Molecular analysis of minimal residual disease is only applicable in acute myeloblastic leukemia (AML) patients with genetic markers (20-30%). This study analyzes the feasibility of the real-time quantitative polymerase chain reaction (RQ-PCR) assay to detect mutant nucleophosmin (NPM1) during follow-up in AML patients. Moreover, we compare the NPM1 results with those of WT1 expression to MRD assessment., Methods: The study includes 97 samples from 24 AML patients with type A NPM1 mutation at diagnosis. MRD was evaluated simultaneous by RQ-PCR assay to detect NPM1-mutated and WT1 expression., Results: The expression levels of WT1 and NPM1 in 93 paired samples showed a strong positive correlation (r=0.81; p<0.0001). However, the kinetics of disappearance were different, WT1 decreased rapidly after induction but maintained these residual levels after treatment in patients in complete remission, whereas NPM1 experienced a mild reduction after induction but was undetectable in long survivor patients., Conclusions: This study shows the feasibility of the RQ-PCR assay to monitor MRD in AML patients carrying NPM1 mutations and its advantage over RQ-PCR assay for WT1. Owing to NPM1-mutated is specific of leukemic cells and shows higher levels at presentation.

Background: beta-Thalassemia is one of the most common single gene disorders and is widely distributed in the Mediterranean region. The deltabeta-thalassemias are a rare group of disorders, however in Spain the Spanish deltabeta0 thalassemia that removes 114 kb of beta-globin cluster is quite frequent., Methods: To establish a molecular diagnosis of beta-thalassemia in a second-trimester fetus from a couple who are beta-thalassemia carriers, a rapid real-time PCR method was performed to detect major mutations prevalent in people of the Mediterranean basin (IVS I-1, IVS I-6, IVS I-110, CD-6, CD-37, and CD-39). The Spanish deltabeta0 deletion was detected using PCR with deletion-specific primers., Results: The father was diagnosed as a carrier for the IVSI-6 (T>C) mutation and the mother as carrier for the Spanish deltabeta0 deletion. The fetus was a compound heterozygote for both those mutations. The phenotype of compound heterozygosity for deltabeta/beta-thalassemia can range from mild thalassemia intermedia to thalassemia major. After confirming these results in the DNA from amniocentesis obtained at the 20th week of gestation, the couple decided to terminate the pregnancy., Conclusions: This paper reports the first molecular characterization of Spanishdeltabeta0-thalassemia associated with beta-thalassemia IVSI-6, and confirms the importance of prenatal genetic testing for single gene disorder such as beta-thalassemia.

All-trans-retinoic acid (ATRA) increases the efficacy of chemotherapy when used for induction and maintenance treatment of acute promyelocytic leukemia (APL), but its role in consolidation is unknown. Since November 1996, 426 patients with newly diagnosed APL have received induction therapy with ATRA and idarubicin. Before November 1999 (LPA96 study), consolidation therapy consisted of 3 courses of anthracycline monochemotherapy. After November 1999 (LPA99 study), patients with intermediate and high risks of relapse received consolidation therapy with ATRA and increased doses of anthracyclines. Of the 384 patients who achieved complete remission (90%), 382 proceeded to consolidation therapy. Seven patients died in remission (1.8%). The 3-year cumulative incidence of relapse for patients in the LPA96 and LPA99 studies was 17.2% and 7.5%, respectively (P =.008). Patients treated with ATRA in consolidation therapy showed an overall reduction in the relapse rate from 20.1% to 8.7% (P =.004). In intermediate-risk patients the rate decreased from 14.0% to 2.5% (P =.006). This improved antileukemic efficacy also translated into significantly better disease-free and overall survival. A risk-adapted strategy combining anthracycline monochemotherapy and ATRA for induction and consolidation therapy of newly diagnosed APL results in improved antileukemic efficacy and a high degree of compliance.

The potential role of unrelated donor cord blood transplantation (UD-CBT) in adults remains unclear. This study reports the results of UD-CBT in 22 adults with hematologic malignancies following conditioning with thiotepa, busulfan, cyclophosphamide, and antithymocyte globulin in 21, with thiotepa, fludarabine, and antithymocyte globulin in 1, and graft-versus-host disease (GVHD) prophylaxis with cyclosporine and prednisone. Median age was 29 years (range, 18-46 years), and median weight was 69.5 kg (range, 41-85 kg). HLA match was 6 of 6 in 1 case, 5 of 6 in 13 cases, and 4 of 6 in 8 cases. Median number of nucleated cells infused was 1.71 x 10(7)/kg (range, 1.01 x 10(7)/kg to 4.96 x 10(7)/kg). All 20 patients surviving more than 30 days had myeloid engraftment, and only 1, who received the lowest cell dose, developed secondary graft failure. Median time to reach an absolute neutrophil count of at least 0.5 x 10(9)/L was 22 days (range, 13-52 days). Median time to platelets numbered at least 20 x 10(9)/L was 69 days (range, 49-153 days). Seven patients (32%) developed acute GVHD above grade II, and 9 of 10 patients at risk developed chronic GVHD, which became extensive in 4 patients. Twelve patients remained alive and disease-free 3 to 45 months after transplantation. Disease-free survival (DFS) at 1 year was 53%. Age strongly influenced DFS (P =.01). For patients aged 30 years or younger, the DFS at 1 year was 73%. These preliminary results suggest that UD-CBT should be considered a reasonable alternative in young adults with hematologic malignancy and no appropriate bone marrow donor.

Preliminary independent reports of the Italian GIMEMA and the Spanish PETHEMA trials for newly diagnosed acute promyelocytic leukemia (APL) indicated a similarly high antileukemic efficacy in terms of complete remission and disease-free survival rates. To better investigate these studies and the prognostic factors influencing relapse risk, this study analyzed the updated results of 217 patients with PML/RAR alpha-positive APL enrolled in GIMEMA (n = 108) and PETHEMA (n = 109). All patients received identical induction (AIDA schedule) and maintenance. For consolidation, GIMEMA patients received 3 courses including idarubicin/cytarabine, mitoxantrone/etoposide, and idarubicin/cytarabine/thioguanine, whereas PETHEMA patients received the same drugs and dose schedule of idarubicin and mitoxantrone with the omission of nonintercalating agents. Depending on whether molecular relapses were classified as censored or uncensored events, the 3-year Kaplan-Meier estimates of relapse-free survival (RFS) for the combined series were 90 +/- 2% and 86 +/- 2%, respectively. Minor differences observed between the 2 patient cohorts were negligible. Multivariate regression analysis of RFS showed that initial leukocyte (WBC) and platelet counts were the only variables with independent prognostic value. The resulting predictive model for RFS demonstrated its capability of segregating patients into low-risk (WBC count 40 x 10(9)/L), intermediate-risk (WBC count 10 x 10(9)/L) groups, with distinctive RFS curves (P <.0001). The conclusions are that omission of nonanthracycline drugs from the AIDA regimen is not associated with reduced antileukemic efficacy and a simple predictive model may be used for risk-adapted therapy in this disease. (Blood. 2000;96:1247-1253)

The Spanish PETHEMA group designed a protocol for newly diagnosed PML/RARalpha-positive acute promyelocytic leukemia (APL) in which induction and consolidation followed the original AIDA regimen, except for the omission of cytarabine and etoposide from consolidation. Induction consisted of 45 mg/m(2) all-trans retinoic acid (ATRA) daily until complete remission (CR) and 12 mg/m(2) idarubicin on days 2, 4, 6, and 8. Patients in CR received 3 monthly chemotherapy courses: idarubicin 5 mg/m(2)/d x 4 (course no. 1), mitoxantrone 10 mg/m(2)/d x 5 (course no. 2), and idarubicin 12 mg/m(2)/d x 1 (course no. 3). Maintenance therapy consisted of 90 mg/m(2)/d mercaptopurine orally, 15 mg/m(2)/wk methotrexate intramuscularly, and, intermittently, 45 mg/m(2)/d ATRA for 15 days every 3 months. Between November 1996 and December 1998, 123 patients with newly diagnosed PML/RARalpha-positive APL from 39 centers were enrolled. A total of 109 patients achieved CR (89%; 95% confidence interval [CI], 83 to 95), 12 died of early complications, and the remaining 2 were resistant. Consolidation treatment was associated with very low toxicity and no deaths in remission were recorded. Molecular assessment of response by reverse transcriptase-polymerase chain reaction (RT-PCR) showed conversion to PCR-negative in 48 of 99 (51%) and 82 of 88 patients (93%) after induction and consolidation, respectively. The 2-year Kaplan-Meier estimates of overall survival and event-free survival were 82% +/- 4% and 79% +/- 4%, respectively. For patients who achieved CR, the 2-year disease-free survival (DFS) was 92% +/- 3%. These data indicate that a significant reduction in toxicity might be obtained in APL using a less intensive consolidation without apparently compromising the antileukemic effect. These results also suggest a minor role for cytarabine and etoposide in the treatment of newly diagnosed PML/RARalpha-positive APL patients.

This study attempts to clarify the role of c-erbB-2 overexpression in human non-small cell lung cancer (NSCLC) and relate it with the p53 alterations, DNA index (D.I.) and epidermal growth factor receptor (EGFR) content in sixty four patients with NSCLC. c-erbB-2 and EGFR quantification were carried out from tissue homogenates using quantitative ELISA procedures. p53 alterations were determined by immunohistochemical (IHC) detection with the monoclonal antibody DO-7 and analysis for p53 mutations on exons 4 to 8 by single strand conformation polymorphism (SSCP). The D.I. was performed by flow cytometry. c-erbB-2 hyperexpression was found in 13 of 58 LC (22%), and it was closely associated with hyperdiploid tumors (D.I. >1.3; P = 0.00). The p53 abnormalities detected by SSCP were statistically more frequent in hyperdiploid tumors (16/25; P = 0.015) than in diploid ones (8/30). No relationship between the results of IHC p53 and SSCP was found. The patients with c-erbB-2 hyperexpressing tumors were prone to have frequent relapses (P = 0.03), although the patients with hyperdiploid NSCLC are the ones with the highest relapse rate (P = 0.008). From the results obtained in this study the following conclusions can be drawn: (a) c-erbB-2 hyperexpressing NSCLC are associated with abnormalities in other biological markers and with a greater rate of relapses; (b) SSCP seemed to be more specific that IHC to detect p53 molecular abnormalities; and (c) the D.I. is the parameter more tightly related with relapse.

This study attempts of clarify the oncological significance of the p53 molecular abnormalities and p53 expression in lung cancer (LC) and their relationship with flow cytometry (FC) parameters and epidermal growth factor receptor (EGFR). The study includes 65 samples taken from both LC and normal lung (NL). The p53 molecular abnormalities of exons 4-8 were studied by single strand conformation polymorphisms (SSCP) and the loss of heterozygosity (LOH) of exon 4 by the Metzler method. P53 protein was detected by Western blot. EGFR was determined by a radioligand assay using [125I]EGF. The FC parameters S phase fraction (SPF), DNA index (D.I.), G1G0 and growth rate (G2M + SPF) were evaluated from cellular monosuspensions. The LC with SSCP p53 molecular abnormalities have a significantly higher EGFR content (P < 0.001), SPF (P < 0.007), D.I. (P < 0.017) and a lower proportion of G1G0 cells (P < 0.04) than LC with no molecular abnormalities. No relationship between p53 molecular abnormalities and tumor TN or evolutive events was found. Neither the relationship between the molecular results and p53 expression detected by Western blot nor that of the p53 expression detected by Western with FC parameters or EGFR could be shown. In NL the growth fraction cells decrease significantly (P < 0.05) with the intensity of p53 expression. The lack of biological functionality of p53 with molecular abnormalities seemed to relate to fast growing LC whereas p53 expression detected by Western seemed more related to the wild type of p53.

Cytokeratin K19 (CK19) expression was evaluated by a reverse transcription PCR method (RT-PCR) in the RNA obtained from peripheral blood stem cell collections (PBSC) from four patients with breast cancers (BC) and 34 mononucleated blood cell (MBC) negative controls (17 PBMC from normal subjects 12 PBSC from different types of leukaemias--M3, M4Eo, M2, etc.--and two from patients with Hodgkin's lymphoma; and three bone marrow (BM) collections). Two BC tissues were taken as positive controls. The method studied (Datta YH, Paul T, Adams PT, Drobyski WR. Sensitive detection of occult breast cancer by reverse transcription polymerase chain reaction. J Oncol 1994;12:475-8) is sensitive enough to allow the detection of CK19 transcripts in a 10(-6) dilution of cDNA reverse transcribed from 1 microgram of BC RNA, but CK19 transcripts were also detected in 64% of the RNA obtained from the MBC controls. However, the amplified product detected in the control samples represents the transcript of the CK19 gene as confirmed by the results of Mae III digestion. It should be pointed out that although the CK19 expression was detected, the levels of expression in PBMC were almost negligible for they disappeared at 1:5 cDNA dilution. Moreover, a direct relationship between the number of BC cells added to PBMC and the increasing dilution levels of the cDNA necessary to prevent CK19 expression was observed. This allows us to conclude that the cDNA dilutions make it possible to distinguish the false from the true positive samples and that, in addition, the cDNA dilutions inform about the degree of BC cell contamination.

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.

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.

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 (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.

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.