Optimización y análisis crítico del estudio de la monotorización de enfermedad mínima residual mediante ASO RQ:PCR en pacientes con mieloma múltiple. Comparación con la citometría de flujo

Tesis por compendio de publicaciones.[EN] In patients with multiple myeloma ( MM) , the quality of response to treatment has been shown to have prognostic value , so that today the complete response (CR ) represents a therapeutic target in all MM schemes . However, we know that the current response criteria are suboptimal , since they are based on low sensitivity techniques such as immunofixation and morphology. Several studies indicate that immunophenotypic and molecular techniques are more sensitive and allow assessing minimal residual disease (MRD ) in bone marrow (BM ) , so that a better quality of response would be associated with longer survival. In the molecular field , strategies based on the use of specific primers and probes to quantify a patient and the tumor mass (ASO RQ- PCR ) has shown clinical value in patients with MM . Since the applicability of the same low, we consider first objective of this thesis improve its applicability , the search for new molecular markers and / or the use of samples enriched in plasma cells by CD138 + selection . Of molecular markers used in MM , VDJH somatic mutations frequently occurs and is detected only DJH in 60 % of patients . Alternatively, the Kappa deleting element rearrangements ( Kde ) are detected in all lymphoproliferative lambda B and 1/3 of the kappa and somatic mutations have not compromise the efficiency of primers and probes . Therefore, as in acute lymphoblastic leukemia , may be successfully used as markers for EMR in MM . This rearrangement studied in 96 patients with MM and found that is detected in 50 % of them , which can be easily sequenced and no somatic mutations. Then analyze its value as an additional marker in MM and saw EMR increases the applicability of these studies in 9% of cases overall and in 20 % of cases of MM lambda group that would be a significant advance . Then investigated using samples enriched MO plasma cells by immunomagnetic selection CD138 + as a material for obtaining molecular markers of EMR by PCR in MM . For this, of the 25 samples analyzed, a part was subjected to CD138 + immunomagnetic selection and the rest , not processed , was used as control. In both sample groups try to identify three molecular targets : VDJH , DJH and Kde . Seen that the use of selected samples increased from 60% to 96 % the proportion of cases with at least one molecular marker is available at the time of diagnosis . Its use , therefore, increase the applicability of such studies. The second objective of the thesis, we propose identifying problems responsible for failures in each of the steps in the molecular study of EMR following the recommendations of the European Group Euro - MRD , standardized but not in MM . Also, take advantage of this prospective study to compare the results of molecular techniques against immunophenotypic , in order to determine which of the two is ideal for monitoring in MM EMR . ASO saw RQ -PCR enabled monitoring of the EMR only in 42 % of patients due to pre- analytical problems responsible for a 28 % loss and post- analytical , 30% of them. In a series of 103 evaluable cases , we found that the quantification of MRD in patients with MM by ASO RQ -PCR allows monitoring the effectiveness of treatment and demonstrated to have prognostic value , stratifying patients into groups with different risk of progression. When we compare both techniques , we found that while the ASO RQ -PCR has a slightly higher sensitivity, the applicability of the CMF ( > 90 % ) is significantly higher. Therefore, the CMF is to be considered the technique of choice for monitoring EMR in MM .[ES] En pacientes con mieloma múltiple (MM), la calidad de la respuesta al tratamiento ha demostrado tener valor pronóstico, de manera que hoy en día la respuesta completa (RC) representa un objetivo en todos los esquemas terapéuticos de MM. Sin embargo, sabemos que los criterios actuales de respuesta son subóptimos, ya que se basan en técnicas de baja sensibilidad, como la inmunofijación y la morfología. Diversos estudios indican que las técnicas inmunofenotípicas y moleculares tienen mayor sensibilidad y permitirían evaluar la enfermedad mínima residual (EMR) en médula ósea (MO), de manera que una mejor calidad de la respuesta se asociaría con mayor supervivencia. En el campo molecular, las estrategias basadas en el uso de primers y sondas específicos de paciente y que permiten cuantificar la masa tumoral (ASO RQ-PCR) han demostrado valor clínico en pacientes con MM. Dado que la aplicabilidad de las mismas en baja, nos planteamos como primer objetivo de esta tesis mejorar su aplicabilidad, con la búsqueda de nuevos marcadores moleculares y/o el uso de muestras enriquecidas en células plasmáticas mediante selección CD138+. De los marcadores moleculares empleados en MM, VDJH presenta con frecuencia mutaciones somáticas y DJH se detecta sólo en 60% de los pacientes. Como alternativa, los reordenamientos de Kappa deleting element (Kde) se detectan en todos los síndromes linfoproliferativos B lambda y en 1/3 de los kappa y no presentan mutaciones somáticas que comprometan la eficiencia de primers y sondas. Por ello, al igual que en leucemia linfoblástica aguda, podrían usarse con éxito como marcadores de EMR en MM. Estudiamos este reordenamiento en 96 pacientes con MM y vimos que se detecta en 50% de los mismos, que se puede secuenciar fácilmente y que no presenta mutaciones somáticas. Analizamos luego su valor como marcador adicional de EMR en MM y vimos que aumenta la aplicabilidad de estos estudios en un 9% de los casos en general y en 20% de los casos de MM lambda, grupo en el que supondría un avance significativo. Investigamos después el uso de muestras de MO enriquecidas en células plasmáticas mediante selección inmunomagnética CD138+ como material para la obtención de marcadores moleculares de EMR mediante PCR en MM. Para ello, de las 25 muestras analizadas, una parte se sometió a selección inmunomagnética CD138+ y el resto, no procesado, se usó como control. En ambos grupos de muestras intentamos identificar 3 dianas moleculares: VDJH, DJH y Kde. Vimos que el uso de muestras seleccionadas incrementa de 60% a 96% el porcentaje de casos con al menos un marcador molecular disponible en el momento del diagnóstico. Su uso, por tanto, incrementaría la aplicabilidad de tales estudios. Como segundo objetivo de la tesis, nos planteamos identificar los problemas responsables de los fallos en cada uno de los pasos del estudio molecular de EMR siguiendo las recomendaciones del grupo europeo Euro-MRD, estandarizadas pero no en MM. Además, aprovechamos este estudio prospectivo para comparar los resultados de las técnicas moleculares frente a las inmunofenotípicas, con el fin de determinar cuál de las dos es la idónea para la monitorización de EMR en MM. Vimos que la ASO RQ-PCR permitía el seguimiento de la EMR sólo en 42% de los pacientes debido a problemas pre-analíticos, responsables de un 28% de las pérdidas y post-analíticos, de un 30% de las mismas. En una serie de 103 casos evaluables, comprobamos que la cuantificación de la EMR en pacientes con MM mediante ASO RQ-PCR permite monitorizar la eficacia de los tratamientos y demuestra tener valor pronóstico, estratificando a los pacientes en grupos con diferente riesgo de progresión. Cuando comparamos ambas técnicas, vimos que mientras que la ASO RQ-PCR tiene una sensibilidad ligeramente más alta, la aplicabilidad de la CMF (>90%) es significativamente superior. Por ello, la CMF ha de considerarse la técnica de elección para la monitorización de EMR en MM

A Low Frequency of Losses in 11q Chromosome Is Associated with Better Outcome and Lower Rate of Genomic Mutations in Patients with Chronic Lymphocytic Leukemia

[EN] To analyze the impact of the 11q deleted (11q-) cells in CLL patients on the time to first therapy (TFT) and overall survival (OS), 2,493 patients with CLL were studied. 242 patients (9.7%) had 11q-. Fluorescence in situ hybridization (FISH) studies showed a threshold of 40% of deleted cells to be optimal for showing that clinical differences in terms of TFT and OS within 11q-CLLs. In patients with >= 40% of losses in 11q (11q-H) (74%), the median TFT was 19 months compared with 44 months in CLL patients with <40% del(11q) (11q-L) (P<0.0001). In the multivariate analysis, only the presence of 11q-L, mutated IGHV status, early Binet stage and absence of extended lymphadenopathy were associated with longer TFT. Patients with 11q-H had an OS of 90 months, while in the 11q-L group the OS was not reached (P = 0.008). The absence of splenomegaly (P = 0.02), low LDH (P = 0.018) or beta 2M (P = 0.006), and the presence of 11q-L (P = 0.003) were associated with a longer OS. In addition, to detect the presence of mutations in the ATM, TP53, NOTCH1, SF3B1, MYD88, FBXW7, XPO1 and BIRC3 genes, a select cohort of CLL patients with losses in 11q was sequenced by next-generation sequencing of amplicons. Eighty % of CLLs with 11q-showed mutations and fewer patients with low frequencies of 11q-had mutations among genes examined (50% vs 94.1%, P = 0.023). In summary, CLL patients with <40% of 11q-had a long TFT and OS that could be associated with the presence of fewer mutated genes.European Commision (EC). Funding FP7/SP1/HEALTH. Project Code: 30624

Depth of Response in Multiple Myeloma: A Pooled Analysis of Three PETHEMA/GEM Clinical Trials

[EN] Purpose—To perform a critical analysis on the impact of depth of response in newly diagnosed multiple myeloma (MM). Patients and Methods—Data were analyzed from 609 patients who were enrolled in the GEM (Grupo Español de Mieloma) 2000 and GEM2005MENOS65 studies for transplant-eligible MM and the GEM2010MAS65 clinical trial for elderly patients with MM who had minimal residual disease (MRD) assessments 9 months after study enrollment. Median follow-up of the series was 71 months. Results—Achievement of complete remission (CR) in the absence of MRD negativity was not associated with prolonged progression-free survival (PFS) and overall survival (OS) compared with near-CR or partial response (median PFS, 27, 27, and 29 months, respectively; median OS, 59, 64, and 65 months, respectively). MRD-negative status was strongly associated with prolonged PFS (median, 63 months; P < .001) and OS (median not reached; P < .001) overall and in subgroups defined by prior transplantation, disease stage, and cytogenetics, with prognostic superiority of MRD negativity versus CR particularly evident in patients with high-risk cytogenetics. Accordingly, Harrell C statistics showed higher discrimination for both PFS and OS in Cox models that included MRD (as opposed to CR) for response assessment. Superior MRD-negative rates after different induction regimens anticipated prolonged PFS. Among 34 MRD-negative patients with MM and a phenotypic pattern of bone marrow involvement similar to monoclonal gammopathy of undetermined significance at diagnosis, the probability of “operational cure” was high; median PFS was 12 years, and the 10-year OS rate was 94%. Conclusion—Our results demonstrate that MRD-negative status surpasses the prognostic value of CR achievement for PFS and OS across the disease spectrum, regardless of the type of treatment or patient risk group. MRD negativity should be considered as one of the most relevant end points for transplant-eligible and elderly fit patients with MM

Next Generation Flow for highly sensitive and standardized detection of minimal residual disease in multiple myeloma

[EN]Flow cytometry has become a highly valuable method to monitor minimal residual disease (MRD) and evaluate the depth of complete response (CR) in bone marrow (BM) of multiple myeloma (MM) after therapy. However, current flow-MRD has lower sensitivity than molecular methods and lacks standardization. Here we report on a novel next generation flow (NGF) approach for highly sensitive and standardized MRD detection in MM. An optimized 2-tube 8-color antibody panel was constructed in five cycles of design-evaluation-redesign. In addition, a bulk-lysis procedure was established for acquisition of ⩾107 cells/sample, and novel software tools were constructed for automatic plasma cell gating. Multicenter evaluation of 110 follow-up BM from MM patients in very good partial response (VGPR) or CR showed a higher sensitivity for NGF-MRD vs conventional 8-color flow-MRD -MRD-positive rate of 47 vs 34% (P=0.003)-. Thus, 25% of patients classified as MRD-negative by conventional 8-color flow were MRD-positive by NGF, translating into a significantly longer progression-free survival for MRD-negative vs MRD-positive CR patients by NGF (75% progression-free survival not reached vs 7 months; P=0.02). This study establishes EuroFlow-based NGF as a highly sensitive, fully standardized approach for MRD detection in MM which overcomes the major limitations of conventional flow-MRD methods and is ready for implementation in routine diagnostics.This work has been supported by the International Myeloma Foundation-Black Swan Research Initiative, the Red Temática de Investigación Cooperativa en Cáncer (RTICC); grant SA079U14 from the Consejería de Educación, Junta de Castilla y León, Valladolid, Spain and; grant DTS15/00119 from Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain