Reliable quantification of hematopoietic chimerism after allogeneic transplantation for acute leukemia using amplification by real-time PCR of null alleles and insertion/deletion polymorphisms.

Increasing mixed chimerism (MC) after allogeneic stem cell transplantation (SCT) has been associated with a high risk of relapse in acute leukemia. We evaluated a new method for chimerism detection, based on the quantitative real-time PCR (qrt-PCR) amplification of null alleles or insertion/deletion polymorphisms (indels). All qrt-PCR assays with null alleles and indels attained a sensitivity of at least 10(-4), as well as good intra- and interassay concordance, and a high accuracy in experiments with cell mixtures. Informativeness was found in 80.3% of the 61 donor/recipient pairs tested. Nonrelapsed patients showed a progressive decrease in peripheral blood chimerism to values below 0.01% (complete chimerism (CC)). Bone marrow chimerism failed to reach CC more than 4 years after SCT. Increasing MC was observed prior to relapse in 88.2% of patients. Compared with conventional PCR amplification of variable number of tandem repeats, qrt-PCR predicted a significantly higher number of relapses (88.2 vs 44.4%) with a median anticipation period of 58 days. In conclusion, chimerism determination by qrt-PCR amplification of null alleles and indels constitutes a useful tool for the follow-up of patients with acute leukemia after SCT, showing better results than those obtained with conventional PCR.

[Evaluation of clinical competence of family medicine trainer]. / Evaluación de la competencia clínica de tutores de residentes de medicina familiar y comunitaria.

OBJECTIVES: We show the first experience of the application of an objective and structured clinical evaluation (OSCE) procedure to family medicine trainers, that has been carried out in Andalucia. The objective is to use a competence evaluation instrument that, in the short term, will be used not only for trainer accreditation but also for other public sanitary professionals. PARTICIPANTS: Tutors of family and community medicine residents. DESIGN: Observational descriptive. SETTING: Educational unity of family medicine. PRINCIPAL MEASUREMENTS: The competencial components to be assessed are the following: anamnesis, physical exploration, communication, technical skill, management, family attention y preventive activities. The clinical situations were selected using the following priority criteria: prevalence, clinical gravity, prevention and early diagnosis importance, case complexity, doctor's capacity of evaluation and simplicity. RESULTS: Thirteen family medicine trainers took part in the OSCE. Their average age was 42.8 +/- 3.6 years. The test had an overall reliability coefficient (Cronbach's alpha) of 0.73. The overall mean score of the participants was 73 +/- 6.2. The best results about the competencial components were family attention, communication and technical skill. CONCLUSIONS: The OSCE can be a convenient tool for family medical trainer evaluation, helping to orientate their education in the weak points and, in the near future, it can also be used as an instrument do accredit family medicine trainers.

Transcriptional silencing of the Dickkopfs-3 (Dkk-3) gene by CpG hypermethylation in acute lymphoblastic leukaemia.

DKK-3: is a newly characterised mortalisation-related gene and an antagonist of the Wnt oncogenic signalling pathway whose expression is decreased in a variety of cancer cell lines, suggesting that the Dkk-3 gene, located at chromosome 11p15.1, functions as a tumour suppressor gene. Although 11p15 is a 'hot spot' for methylation in acute lymphoblastic leukaemia (ALL), the role of Dkk-3 abnormalities has never been evaluated in this disease. We analysed CpG island methylation of the Dkk-3 promoter in six ALL cell lines and 183 ALL patients. We observed Dkk-3 hypermethylation in all cell lines and in cells from 33% (60/183) of ALL patients. Moreover, Dkk-3 methylation was associated with decreased Dkk-3 mRNA expression and this expression was restored after exposure to the demethylating agent 5-AzaC. Clinical features did not differ between hypermethylated and unmethylated patients. Estimated disease-free survival (DFS) and overall survival at 10 and 11 years, respectively, were 49.8 and 45.6% for normal patients and 10.5 and 15.1% for hypermethylated patients (P=0.001 and 0.09). Multivariate analysis demonstrated that Dkk-3 methylation was an independent prognostic factor predicting DFS (P=0.0009). Our data suggest that Dkk-3 methylation occurs at an early stage in ALL pathogenesis and probably influences the clinical behaviour of the disease.

Tratamiento antiagregante en la trombocitemia esencial asociada al embarazo / Anticoagulant therapy in pregnancy associated essential thrombocytaemia

La trombocitemia esencial y embarazo es una asociación infrecuente. Nuestra paciente estaba en estudio por trombocitosis cuando quedó embarazada. Presentaba antecedentes de malos resultados obstétricos, como una gestación interrumpida previa del primer trimestre y una amenaza de aborto actual. Tras sopesar riesgos-beneficios nos decantamos por una actitud intervencionista desde el punto de vista terapéutico, prescribiendo antiagregantes plaquetarios a dosis bajas, combinando 100 mg de aspirina hasta la semana 36 de gestación, sustituyéndola por 2.500 unidades de dalteparina diarias hasta el parto. El embarazo y parto evolucionaron favorablemente sin ningún tipo de complicación (AU)

[Evolution of the communication profile of family medicine residents]. / Evolución del perfil comunicacional de los médicos residentes de medicina de familia.

AIMS: To study the development of patient relation skills, as used during interviews with patients for health problems that are common within their specialty, in family medicine residents during the third year of their residency program. METHODS: Quasi-experimental (before-after), national-level, multicenter study. The participants were 193 third-year residents in family medicine at 8 training units who were trained between 1996 and 1999. During this period all residents participated in the usual training and clinical activities included in the National Plan for this specialty. The GATHA-RES questionnaire was used to evaluated six clinical scenarios in video recordings of encounters with standardized patients (3 at the start of the third year and 3 at the end of the third year). Descriptive, bivariate and multivariate statistical analyses were used. RESULTS: A total of 1,024 interviews were analyzed. The time spent with each patient decreased significantly at the end of the residency program; the duration of the visit was directly proportional to the score on the GATHA-RES questionnaire (p < 0.05). Improvements were seen in formal and organizational aspects of the interview. In contrast, skills related with the exploration of personal and contextual aspects of the problem, and negotiating skills, were worse at the end of the study. The variables that best predicted residents' communicational profile were age (inverse relation), duration of the interview, training of the tutor in clinical interviewing, and teaching unit. CONCLUSIONS: Residents learn to shorten the duration of the visit to the detriment of communication skills that are basic to appropriate care for their patients' health problems. These results suggest the need for substantial changes in the training of family medicine residents in Spain.

E1A3 as a unique, naturally occurring BCR-ABL transcript in an indolent case of chronic myeloid leukaemia.

A woman with Ph-positive chronic myeloid leukaemia (CML) with an atypical e1a3 BCR-ABL hybrid gene is described. To our knowledge, this is the first report of this transcript type as a unique naturally occurring BCR-ABL fusion in a CML patient. This case was characterized by a low leucocyte count and a very indolent course without treatment. Because the deletion of ABL exon 2 sequences results in deletion of an essential part of the ABL SH3 domain, our case suggests that this ABL SH3 domain is not absolutely necessary for efficient induction of a myeloproliferative disease in the context of BCR-ABL/p190.

[Validity and reliability of an instrument to assess the clinical interviews of residents in family and community medicine: the GATHA-RES questionnaire]. / Validez y fiabilidad de un instrumento para la valoración de la entrevista clínica en médicos residentes de medicina de familia: el cuestionario GATHA-RES.

OBJECTIVE: To check the validity of content, the internal consistency and the intra-observer reliability of a questionnaire to evaluate the doctor-patient communication of family medicine residents. DESIGN: Observation study, to validate a measurement instrument. SETTING: Primary care. Family and community medicine teaching units. STUDY POPULATION: For the validity analysis: 25 family and community medicine residents. For the reliability analysis: 48 doctors in the same speciality. MEASUREMENTS AND INTERVENTIONS: The questionnaire was constructed on the basis of a version (GATHA-BASE) composed of 42 items selected by a panel of 60 general practitioners. For content validity, 68 clinical encounters with simulated patients, video-recorded and evaluated, were used. The questionnaire's validity content was studied through a factorial analysis. To measure its internal consistency, Cronbach's alpha coefficients were calculated. Intra-observer reliability of the GATHA-RES version was evaluated through the kappa indexes and the intra-class correlation coefficients. RESULTS: We obtained a version of the GATHA-RES with 27 items. The factorial analysis revealed that there were 9 factors (<>, <>, <>, <>, <>, <>, <> and <>) which showed close correlation with the theoretical and formal contents of the original questionnaire (originally grouped in three sections: attitudes, communication tasks and skills). All the intraclass correlation coefficients had figures > or = 0.90. CONCLUSIONS: The GATHA-RES questionnaire is a valid and reliable instrument that can be used for evaluating the communication skills of general practitioners in training.

Hypermethylation of the calcitonin gene in acute lymphoblastic leukaemia is associated with unfavourable clinical outcome.

We analysed calcitonin (CALC1) gene hypermethylation using semiquantitative differential polymerase chain reaction in 105 patients with adult (n = 49) and childhood (n = 56) acute lymphoblastic leukaemia (ALL), and studied the association of CALC1 hypermethylation with clinical presentation features and disease outcome. We also investigated the possible relationship between CALC1 methylation status and expression of the cell cycle inhibitor gene p57KIP2. We observed CALC1 hypermethylation in bone marrow cells from 43% (45 out of 105) of ALL patients. Clinical, molecular and laboratory features did not differ significantly between hypermethylated and hypomethylated patients, only T-cell lineage was associated with hypermethylation (14% vs. 47%, P = 0025). Complete remission rate was similar in both groups although hypermethylated patients had a higher relapse rate (68% vs. 19%, P < 0.00001) and mortality rate (55% vs. 36%, P = 0.06) than hypomethylated patients. Estimated disease-free survival (DFS) at 6 years was 66.1% for hypomethylated patients and 5.3% for hypermethylated patients (P < 0,00001). Multivariate analysis from potential prognostic factors demonstrated that CALC1 methylation status was an independent prognostic factor in predicting DFS (P = 0.0001). Separate analysis of adult and childhood ALL patients showed similar results to the whole series. In addition, hypermethylated patients showed downregulation of p57KIP2 expression. Our results suggest that CALC1 gene hypermethylation is associated with an enhanced risk of relapse independently of known poor-prognostic factors and we describe, for the first time, a possible implication of the p57KIP2 gene in the genesis and prognosis of ALL.

Myeloid mixed chimerism is associated with relapse in bcr-abl positive patients after unmanipulated allogeneic bone marrow transplantation for chronic myelogenous leukemia.

BACKGROUND AND OBJECTIVE: Although bcr-abl polymerase chain reaction (PCR) positivity after bone marrow transplantation (BMT) for chronic myelogenous leukemia (CML) is significantly related to relapse, the predictive value of the assay is not very high and therefore most investigators consider that qualitative RT-PCR data alone are too imprecise to enable clinical decisions to be taken in individual cases. To define the clinical outcome of bcr-abl positive patients after unmanipulated BMT better, we sought the origin of hematopoiesis and traced its evolution over time. DESIGN AND METHODS: Forty-nine patients received allogeneic BMT for CML (39 in chronic phase and 10 in accelerated phase/blast crisis). Median follow-up was 61 months (range 4-92). mRNA and DNA were used to assess bcr-abl and chimerism status respectively. Quantitative VNTR-PCR on total cells and lymphoid or myeloid population allowed us to assign and measure the origin of hematopoiesis. RESULTS: Both bcr-abl positivity and the presence of mixed chimerism (MC) were significantly associated with relapse (p = 0.0009 and p < 0.0001 respectively). Relapse was observed in one of 39 patients with complete donor chimerism and in 6 of 9 patients with MC. These six cases showed increasing levels of host hemopoiesis and bcr-abl positivity in the CD15-positive population prior to relapse. The other three cases had decreasing or stable low-level MC which was restricted to the T-cells as well as bcr-abl negativity. INTERPRETATION AND CONCLUSIONS: Whereas the simple detection of bcr-abl fails to identify patients who will relapse with certainty, the assessment of MC by VNTR-PCR does identify patients headed to relapse. Confirmation of myeloid involvement and increasing levels over time further elucidates the clinical outcome of bcr-abl positive patients after BMT.

Molecular analysis of lineage-specific chimerism and minimal residual disease by RT-PCR of p210(BCR-ABL) and p190(BCR-ABL) after allogeneic bone marrow transplantation for chronic myeloid leukemia: increasing mixed myeloid chimerism and p190(BCR-ABL) detection precede cytogenetic relapse.

We studied lineage-specific chimerism and minimal residual disease (MRD) in sequential posttransplant samples from 55 patients who underwent unmanipulated (n = 44) or partially T-cell-depleted (n = 11) allogeneic bone marrow transplantation (BMT) for chronic myeloid leukemia (CML). Chimerism was assessed by polymerase chain reaction (VNTR [variable number of tandem repeats]-PCR) analysis in highly purified CD19+, CD3+, CD15+, and CD56+ cell fractions, whereas MRD was investigated in whole blood by reverse transcriptase-PCR (RT-PCR) of both p210(BCR-ABL) and p190(BCR-ABL) hybrid transcripts. Of 55 patients, 14 (including 6 T-cell-depleted patients) had cytogenetic relapse at 5-80 months and progressed to hematologic relapse, while 41 patients remained in prolonged cytogenetic remission 12-107 months post-BMT. Before leukemia recurrence, patients in the relapse group showed a consistent evolution pattern sequentially featured by persistent p210(BCR-ABL) positivity, increasing mixed chimerism (MC) in myeloid cells, p190(BCR-ABL) positivity, and, finally, cytogenetic relapse. Myeloid MC preceded cytogenetic relapse by 2-12 months, whereas p190(BCR/ABL) was detected 1-6 months prior to cytogenetic relapse in 11 patients and concomitant with cytogenetic relapse in 3 patients. In the remission group, all patients invariably tested negative for p190(BCR-ABL); 10 patients tested positive for p210(BCR-ABL) at variable time-points but showed persistent full donor chimerism (DC), whereas 31 patients tested p210(BCR-ABL) negative and displayed full DC or transient MC due to the persistence of recipient T cells. Two patients in the relapse group were successfully reinduced into molecular remission with donor lymphocyte infusion. Sequential molecular analysis after such treatment showed the inverse pattern to that observed prior to relapse, ie, progressive disappearance of p190(BCR-ABL) transcripts, conversion of myeloid chimerism to donor type, and, finally, p210(BCR-ABL) negativity. We conclude that lineage-specific chimerism and p190(BCR-ABL) messenger RNA (mRNA) analyses contribute a better characterization of CML evolution after BMT and enable early identification of patients at the highest risk of relapse. (Blood. 2000;95:2659-2665)

Increasing mixed haematopoietic chimaerism after BMT with total depletion of CD4+ and partial depletion of CD8+ lymphocytes is associated with a higher incidence of relapse.

In this study we analysed the incidence and clinical impact of the persistence of host haemopoiesis (mixed chimaerism, MC) after allogeneic BMT in 35 consecutive patients with haematologic malignancies using a total CD4+ cell-depleted graft with an adjusted dose of CD8+ cells (1x10(8)/kg). Chimaerism was assessed by PCR amplification of VNTRs in 30 evaluable patients: 19 non-CML and 11 CML cases which were also evaluated for the BCR-ABL transcript by RT-PCR. All but one had complete engraftment with a donor profile early post-BMT. At the end of the study period, 12 of 30 patients displayed MC (40%). The overall disease-free survival for MC patients was clearly unfavourable when compared to those who exhibited a donor profile (24.7% vs. 100%, P = 0.005). However, we found that only two of five patients with MC in the non-CML group relapsed, whereas a clear correlation could be made between MC and relapse in CML (seven showed MC, preceding cytogenetic or haematological relapse in six of them, which displayed a prior BCR-ABL mRNA positivity). In addition, a quantitative-PCR approach enabled us to demonstrate that increasing amounts of MC are invariably associated with subsequent relapse, whereas a low stable level of host or complete donor haemopoiesis is consistent with clinical complete remission. Although these results suggest that the clinical impact of MC may depend on the underlying disease, it is compatible with the concept that the graft-versus-leukaemia effect against CML is mainly exerted by donor CD4+ lymphocytes. Elimination of this cellular subset may be responsible for the inability of the graft to prevent a progressive increase in the tumor cell burden.

Genetic, phenotypic and clinical features of acute lymphoblastic leukemias expressing myeloperoxidase mRNA detected by RT-PCR.

Myeloperoxidase (MPO) is found in the azurophilic granules of normal myelocytic cells. Cytochemical staining for MPO activity is used clinically to distinguish myeloid from acute lymphoid leukemias (ALL). However, using a highly sensitive RT-PCR technique, it is possible to detect MPO mRNA in otherwise clear ALL. The significance of this finding remains poorly understood. We have extended our observations to a series of 57 patients with the primary diagnosis of ALL (46 patients tested at diagnosis and 11 cases at relapse). We identified 25 cases (43.8%) of MPO mRNA(+)/enzyme(-) ALL (17 B cell and eight T cell lineage). Expression of myeloid antigens (CD13 or CD33) were detected in nine of them, and remarkably, 18 cases (72%) displayed CD34. Of these 25 MPO mRNA(+) leukemias, 10 (40%) are Bcr-Abl positive (with P210 fusion transcript in five patients while the five remaining cases carried P190 transcript). Moreover, 11 of 16 myeloid negative cases were also negative for any type of Bcr-Abl and MLL rearrangement, indicating that MPO mRNA positivity is not either invariably related to that chromosomal abnormality or necessarily associated with the presence of other myeloid differentiation features. Interestingly, six of these 11 cases are T-ALL, suggesting the presence of some overlapping phase for T and myeloid lineage commitment. Taken together, these findings could suggest a separate biological disease with immature origin and bipotential differentiation capability, which involves B and T-ALL subtypes and should lead to new investigations regarding their prognostic impact.

[The monitoring of BCR-ABL mRNA by reverse transcription PCR in patients undergoing allogeneic bone marrow transplantation for acute lymphoblastic leukemia with a positive Philadelphia chromosome]. / Monitorización del ARNm BCR-ABL mediante retrotranscripción PCR en pacientes sometidos a trasplante alogénico de médula ósea por leucemia aguda linfoblástica con cromosoma Filadelfia positivo.

BACKGROUND: Allogeneic bone marrow transplantation (BMT) has been performed as one mode of cure-oriented therapy for Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL-Ph' positive). However, the clinical significance of the residual BCR-ABL-positive clones after BMT is still controversial. PATIENTS AND METHODS: The BCR-ABL gene (p210 and p190) was prospectively studied by nested RT-PCR in 8 ALL-Ph' positive patients undergoing BMT. RESULTS: All patients received BMT at the time of clinical remission (CR). However, minimal residual disease (MRD) was detected in 7 of them. MRD detected just before BMT seems to be eradicated by BMT protocol. Four patients remained in CR and did not show BCR-ABL transcripts. Other 4 patients, relapsed, demonstrating MRD, which preceded recurrence by a median time of 6 weeks. Three relapsed patients showed p190 transcript and only one, p210 type. CONCLUSIONS: The RT-PCR assay appears to be a useful test for predicting at high risk of relapse after BMT and may identify patients who might benefit from therapeutic interventions. The finding that the expression of p190 BCR-ABL may carry an especially high risk of relapse suggests a different clinical and biologic behaviour between p190 and p210 BCR-ABL.

[Clinical factors that affect the detection of BCR-ABL transcripts after allogenic bone marrow transplantation for chronic myeloid leukemia]. / Factores clínicos que influyen en la detección de transcritos BCR-ABL después del trasplante de médula ósea alogénico por leucemia mieloide crónica.

BACKGROUND: To evaluate the relationship between clinical parameters and the ability to detect BCR-ABL-positive cells in a series of 27 long survivor patients after allogeneic bone marrow transplantation (BMT) for chronic myelogenous leukemia (CML). PATIENTS AND METHODS: A total of 78 samples obtained between 1 and 160 months after BMT were analyzed for the presence of the BCR-ABL transcript detected by the reverse-transcription polymerase chain reaction (PCR) using nested primers. Median follow up was 83 months. RESULTS: 16 patients were persistently PCR-positive and 7 patients persistently PCR-negative. The original transcript became negative in four patients. Only two of the positive cases developed hematologic relapse during the period of study. High white blood cell counts before BMT (17.88 vs 10.12 x 10(9)/l; p = 0.008) and immunosuppressive therapy for chronic graft-versus-host disease (p < 0.05) were associated with an increased ability to detect residual BCR-ABL positive cells. CONCLUSIONS: Our data show that most patient are persistently BCR-ABL positive after BMT for CML. If these findings represents a dynamic balance between the tumour burden prior BMT and the immunological capability of the graft must be confirmed in further studies.

Autologous reconstitution with BCR-ABL-negative haematopoiesis after T cell-depleted allogeneic BMT for CML.

We report a patient with Ph chromosome-positive CML who underwent an HLA-identical T cell-depleted BMT from a sibling donor. DNA polymorphism analysis showed complete donor chimaerism after BMT, followed by mixed chimaerism of granulocytes, natural killer cells and B lymphocytes, with T lymphocytes host-derived at day +120 post BMT. From month +20 haematopoiesis was exclusively of host origin in all cell lineages. RT-PCR was used in order to detect residual disease, but at the time, analysis did not show BCR-ABL transcripts. This case is unusual in that non-malignant stem cells of recipient origin survived the transplant and reconstituted haematopoiesis after BMT. Two years post transplant, no molecular or haematological relapse was documented. The observation that subsequent recipient recovery without molecular relapse implies that, at least in this case, the GVL effect can occur in the absence of donor T cells.