Qualitative and Quantitative Molecular Follow up of Minimal Residual Disease after Non Myeloablative Allografting for Multiple Myeloma.
Authors: Bruno B. , Ladetto M. , Astolfi M. , Veneziano L. , Cimolin L. , Galletto E. , Pagliano G. , Rotta M. , Mordini N. , Giaccone L. , Omedè P. , Battaglio S. , Storb R. , Boccadoro M. 
Published: October 2019

Abstract
New allogeneic transplant protocols with non myeloablative conditioning regimens for treatment of multiple myeloma (MM) have been developed in the attempt to reduce the transplant related toxicity associated with myeloablation. Preliminary data have been encouraging with remarkable clinical response rates (Maloney et al, Blood 2003). However, data on the achievement of molecular remission, prerequisite for eventual cure, are still lacking. We implemented a tandem transplant approach consisting of high dose melphalan (200 mg/sqm) with autografting followed by non myeloablative low dose (2.0 Gy) total body irradiation and G-CSF mobilized PBSC infusion from HLA-identical siblings. The curative potential relies exclusively upon a potent graft versus myeloma (GVM) effect through donor T cells. At diagnosis, patient specific clonal markers were generated based upon the rearrangement of the immunoglobulin heavy chain (IgH) genes and used for nested polymerase chain reaction (PCR) detection of minimal residual disease after transplant. Molecular remission was defined as the disappearance of the molecular marker post transplant in both bone marrow and blood. The sensitivity of the nested PCR-based assay was 1 in 100000 cells. A patient specific marker was generated in 11/15 (73%) patients who entered the study. After a median follow up of 16 months (range 5–50), molecular follow up post transplant showed that 3/11 (27%) reached molecular remission at 1, 3 and 7 months post allografting, respectively. Of the remaining 8 patients, 3/8 and 5/8 reached clinical complete remission, defined as the disappearance of the monoclonal paraprotein by immunofixation, and partial remission, respectively. However, minimal residual disease by nested PCR could be detected at all timepoints. The molecular remissions have been durable at 7, 30, and 48 months post transplant, respectively. In 1 case the remission was achieved and sustained in the absence of graft versus host disease (GVHD) which is consistent with the notion that GVHD is not essential for GVM. Furthermore, in 4/11 patients real-time quantification of IgH rearrangements was performed on genomic DNA samples using tumor specific primers and consensus probes. All patients showed a considerable tumor burden reduction post autografting. Samples from two patients became negative by real time PCR at 3 months post allografting, but became PCR-negative by nested PCR at 3 and 7 months, respectively. This discrepancy is explained by the greater sensitivity of nested PCR and the larger amount of IgH copies which are expected in cDNA compared to genomic DNA. The remaining two patients only obtained a clinical partial response throughout the study period. This report indicates that the tandem auto-allo transplant approach can lead to molecular remission in MM. Prospective quantitative monitoring of disease response may be helpful to design individual additional immunotherapeutic manoeuvres, such as donor lymphocyte infusions, to enhance GVM. Longer follow up on a larger series of patients is needed to determine the frequency and durability of molecular remissions.