- Minimal residual disease in mantle cell lymphoma: insights into biology and impact on treatment
- Leukemia and Lymphoma - Detection of Minimal Residual Disease | Theodore F. Zipf | Springer
- Immunologic Detection of MRD by MFC
- Minimal Residual Disease Testing in AML: Still a Shifting Target
It is obvious that an excellent MRD approach has to be characterized by a high sensitivity level reached by optimizing the available technologies after complex procedures of standardization and harmonization. Qualitative PCR was the pioneer molecular approach implemented for NHLs in in particular, in FL, the translocation t 14;18 juxtaposes chromosome 14 and 18, posing the BCL2 gene under the control of the heavy chain immunoglobulin enhancer, with an increased BCL2 anti-apoptotic activity. In particular, qualitative assays are performed by a nested PCR using broad and internal primers annealing both the chromosome 14 and MCL is characterized by the translocation between chromosome 11 and 14, that once again juxtaposes the BCL1 gene under the control of the IGH enhancer, thus increasing the BCL1 pro-proliferating action.
Qualitative PCR can be performed using fluorescent primers in order to run the PCR product on a DNA sequencer capillary electrophoresis ; in this way, the clonal or polyclonal pattern will be more visible and easier to be read Figure 1. Figure 1. Moreover, for the IGH -positive cases, the set of the standard curve necessary for MRD quantification could be affected by the entity of tissue tumor infiltration at diagnosis, so making the quantitation not always accurate.
Minimal residual disease in mantle cell lymphoma: insights into biology and impact on treatment
Figure 2. The figure represents a comparison of results coming from two different labs. The tested sample, MRD-positive at the first follow-up, became MRD-negative at the second control, then positive but not quantifiable at the limit of detection , and finally MRD-positive again. Digital droplet PCR technology is based on the sample partition into many thousand droplets, so that one single DNA copy is introduced into a single droplet.
After an end-point amplification phase, the appropriate software counts and quantifies the numbers of droplets containing a positive PCR product. This technology does not require the reference standard curve because, according to the Poisson's statistics, the final quantitative results take into account that it would be possible that in some droplets do not enter any DNA molecule or that in other droplets would be co-present two or more DNA copies 80 Figure 3. Figure 3. The lines identified the threshold amplitudes of positive vs.
The cfDNA sample was tested also for housekeeping gene. Patient was MRD-negative. The amplified region encompassed kb and the average depth of sequencing was x. Of the 3 failed samples, in two cases DNAs were degraded or of low-quantity and in one there was a technical error.
Leukemia and Lymphoma - Detection of Minimal Residual Disease | Theodore F. Zipf | Springer
Consequently, outside this specific setting, the standardization of NGS is still not a reality. Today, commercially kits are also available that can make the use of NGS and the interpretation of its data easier for each laboratory Figure 4. Figure 4. Thus, the first studies concerning MRD in FL were designed to answer two questions: 1 if the contamination of the graft by neoplastic cells could condition the further outcome, and 2 if the MRD status after ASCT could have a prognostic role.
About the first question, different studies showed that contaminated harvests could play a negative impact on progression-free-survival PFS and overall survival OS When Rituximab entered into the clinical practice and RQ-PCR became available, it was clear that Rituximab was able to purge the apheretic products when used either ex vivo or in vivo , and that the infusion of grafts uncontaminated or with a lower degree of contamination was associated to a better outcome The fact that authors did not find a clear prognostic impact of the MRD precociously assessed could depend on the chosen evaluation timing: indeed, the MRD was evaluated by 2 months from the end of treatment, when Rituximab is still in the circulation and could probably falsely clear the molecular marker, especially from the peripheral blood PB.
To the same conclusions went also another trial, the ML, where the role of Rituximab in maintenance was assessed in elderly FL patients after a brief chemo-immunotherapy. In this study, it was evident that 90 Yttrium-Ibritumomab-Tiuxetan was able to perform a good clearance of MRD, because the molecular burden decreased from an initial median value of 2, to 4. At the ASH meeting held in , Dr.
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Pott presented the molecular results from the Gallium trial, where Obinutuzumab was compared to Rituximab in a series of 1, advanced FL patients Obinutuzumab represents a second-generation of anti-CD20 monoclonal antibodies, designed for overcoming the resistance to Rituximab and for exerting a more effective action in B-cell lymphoproliferative diseases.
It is a monoclonal antibody of the IgG1 subclass derived by humanization of the parental B-Ly1 mouse antibody by recombinant DNA technology, carrying a glyco-engineered Fc portion, with higher affinity for FcgRIIIa receptors on immune effector cells, and a stronger antigen binding activity. At the end of induction, The median clearance of molecular disease was 3 logs, a value higher than that observed in the Foll05 or Zeus trials Finally, once again the sustained MRD negativity was predictive of a better outcome either after ASCT in the young cohort or after maintenance in elderly patients Not many data about MRD in T-cell lymphomas have been produced, probably for the poor prognosis of these malignancies.
The translocation between chromosome 2 and 5 gives origin to the NPM-ALK fusion gene, that can be used as molecular marker for MRD assessment: the Czech group reported that the monitoring of this fusion gene by RQ-PCR was predictive of relapse, that was predicted by the increase of the transcript of 0. Classically, at diagnosis mutational tests are performed on DNA extracted by the neoplastic tissue or bone marrow; nevertheless, DLBCL infiltrates bone marrow quite rarely, and when patients well-respond to treatment no further masses are available for obtaining tumor DNA.
Interestingly, a higher percentage of cases showed a molecular marker in plasma instead of in the peripheral blood mononuclear cells, and the molecular disease resulted twice higher in the plasma. More recently, the same group reported a strict correlation between the cfDNA levels and response to therapy: in patients where cfDNA levels decreased 2 logs after one cycle and 2.
In line with these results, our group recently showed the superiority of cfDNA in respect of circulating cells as source of tumor DNA: indeed, during the surgery for a kidney explant, a DLBCL, not detectable by CT just before procedure, was found in the donor. After further 3 months, the recipient developed an abdominal DLBCL carrying the same IGH rearrangement of the donor 99 , thus supporting the idea that in DLBCL the plasmatic compartment is really predictive and suitable as tool for assessing MRD in the majority of patients, as previously reported From the technical point of view, the problems of adopting molecular techniques for testing B-cell clonality are different: 1 it is possible that some patients could have a unproductive IGH rearrangement or a hypermutation of the variable region of the heavy chain of immunoglobulins VH that makes impossible the right binding of primers; 2 the DNA extracted from plasma could be not enough for molecular analysis; 3 a clone different from the original one can be appear that would be responsible for progression of disease; this eventual clone is not detectable by ASO-PCR set for the specific initial neoplastic clone.
At the end of induction, only one patient remained MRD-positive, and he further relapsed; at the end of treatment, another patient was still MRD-positive, and also this one relapsed. Authors concluded that the low number of patients enrolled in the study was too low for definitively stating that MRD is a relevant prognostic tool also in BL, ma that it was possible to find a role for MRD also in this malignancy In the allogeneic transplantation setting, MRD can be used not only for following the disease and predict the eventual relapse, but also for monitoring the immune reconstitution, and then to proceed with the tapering of immunosuppression or with the donor lymphocytes infusion DLI.
In a series of 59 patients, the authors reported only two relapses in the 32 patients who were MRD-negative, whereas six of the 11 patients remaining MRD-positive after transplant relapsed Two out of seven patients became MRD-negative few weeks after transplant; in one of this cases, the TCR clonal rearrangement suddenly reappeared, followed by a clinical relapse. In the other five cases, all MRD-positive, the tapering of immunosuppression or DLI decreased MRD of more than 1 log, and TCR rearrangement, skewed just after transplantation, became polyclonal , so sustaining the possibility of using MRD after allogeneic transplantation as predictive tool.
As above reported, in the last 20 years many studies demonstrated that MRD play an evident prognostic role, especially in terms of PFS, in NHLs; nevertheless, few studies included MRD as endpoint in their initial design, and also the guidelines edited by the European Society for Medical Oncology ESMO , even if recognizing the role of MRD in NHLs, do not yet include the molecular tests in the necessary and routinary work-up of NHL patients, sustaining the need of a definitive standardization of the molecular tools — Finally, at the ASH meeting a German group presented the results from a trial employing six cycles of Obinutuzumab and Ibrutinib as induction followed by maintenance with the same combination of drugs as first-line treatment for advanced FL patients.
Also this trial was MRD-driven, because after maintenance at the 30th month MRD-positive cases receive further 12 months of Ibrutinib, whereas those MRD-negative start the observation only Results from these interesting and promising MRD-based studies will be available in the next future and they probably will definitively convince the scientific community about the real possibility of exporting MRD in the clinical practice.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. A particular acknowledgment to Prof. Federico, Prof. Luminari who from many years believe in MRD; finally, we thank Dr. Ilaria De Vita for her revision of the English language.
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Immunologic Detection of MRD by MFC
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Minimal Residual Disease Testing in AML: Still a Shifting Target
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Minimal residual disease response at end of induction and during maintenance correlates with updated outcome in the phase III GALLIUM study of obinutuzumab- or rituximab-based immunochemotherapy in previously untreated follicular lymphoma patients. Additional chapters address the use of real-time quantitative PCR RQ-PCR , the emergent method of choice, in patients with acute lymphoblastic leukemia ALL , the evaluation of MRD techniques in clinical trials, and the application of flow cytometry techniques.
Comprehensive and innovative, Leukemia and Lymphoma: Detection of Minimal Residual Disease sets the stage for implementing the new standard detection and quantitation techniques as reliable tools for clinical decision-making, as well as for improved predictions and treatment outcomes. It will certainly be of great help for all those, who are engaged in modern management of leukemias and lymphomas.