A Deep Molecular Response (DMR), defined as a BCR::ABL1 transcript at levels ≤ 0.01% by RT-qPCR, is the prerequisite for the successful interruption of treatment among patients with Chronic Myeloid Leukemia (CML). However, approximately 50% of patients in Treatment-Free Remission (TFR) studies had to resume therapy after their BCR::ABL1 transcript levels rose above the 0.1% threshold. To improve transcript detection sensitivity and accuracy, transcript levels can be analyzed using digital PCR (dPCR). dPCR increases BCR::ABL1 transcript detection sensitivity 10–100 fold; however, its ability to better select successful TFR patients remains unclear. Beyond the role of the immune system, relapses may be due to the presence of residual leukemic stem cells (LSCs) that are transcriptionally silent. Flow cytometry can be used to identify and quantify circulating bone marrow Ph+ LSCs CD34+/CD38− co-expressing CD26 (dipeptidylpeptidase-IV). To date, the significance of circulating Ph+ LSCs in TFR is unclear. The aim of this work is to compare and examine the values obtained using the three different methods of detecting minimal residual disease (MRD) in CML at RNA (RT-qPCR and dPCR) and LSC (flowcytometry) levels among patients in TFR or exhibiting a DMR. The twenty-seven patients enrolled received treatment with either imatinib (12), dasatinib (6), nilotinib (7), bosutinib (1), or interferon (1). Twelve patients were in TFR, while the rest exhibited a DMR. The TFR patients had stopped therapy for less than 1 year (3), <3 years (2), 6 years (6), and 17 years (1). Blood samples were collected and tested using the three methods at the same time. Both d-PCR and LSCs showed higher sensitivity than RT-qPCR, exhibiting positive results in samples that were undetectable using RT-qPCR (17/27). None of the patients tested negative with d-PCR; however, 23/27 were under the threshold of 0.468 copies/μL, corresponding to a stable DMR. The results were divided into quartiles, and the lowest quartiles defined the lowest MRD. These data were strongly correlated in 15/27 patients, corresponding to almost half of the TFR patients. Indeed, the TFR patients, some lasting up to 17 years, corresponded to the lowest detectable DMR categories. To the best of our knowledge, this is the first attempt to analyze and compare DMRs in a CML population using standard (RT-qPCR) and highly sensitive (dPCR and LSCs) methods.

Background: We previously showed that peptides encompassing the unique b3a2 or b2a2 breakpoint amino-acid sequence of oncogenic p210 induced peptide-specific T-cell responses in chronic myeloid leukemia (CML) patients. Methods: From 2007 to 2011, two multicenter peptide vaccine phase II studies, GIMEMA CML0206 and SI0207, enrolling overall 109 CML patients (68 b3a2 and 41 b2a2) with persistence of molecular disease during imatinib treatment, were carried out. Peptide vaccination schedule included the following: “immunization phase” (six vaccinations every 2 weeks); “reinforcement” phase (three monthly boosts) and “maintenance” phase (two boosts at 3-month intervals). GM-CSF (granulocyte-macrophage-colony-stimulating factor, sarmograstim) served as the immunological adjuvant. Results: The short-term results (at completion of vaccine protocol—12 months) and long-term follow-up are reported. All patients completed the vaccination schedule with no toxicity. After vaccinations, the BCR::ABL1 peptide-specific CD4+ T-cell response was documented in 80% of patients. In the short term, 30% of patients achieved a reduction in BCR::ABL1, while the majority showed stable molecular disease with fluctuations. The median follow-up since diagnosis and last vaccination are 18 and 10 years, respectively, with an overall survival (OS) rate at 18 years of 89%. In addition, 97/109 (89%) patients are alive, while 12/109 (11%) died of CML-unrelated reasons. Overall, 18/109 (16.5%) patients are in treatment-free remission (TFR) for a median time of 48 months. Conclusions: The long-term results of p210 peptide vaccinations in CML patients with persisting disease during imatinib treatment showed its feasibility, safety, absence of off-targets events, high OS and not negligible rate of successful TFR. Active immunotherapeutic approaches in CML patients with low disease burden, eventually employing newer vaccine strategies such as mRNA vaccines, may be reconsidered.

• A higher GTL ratio improves daratumumab response and prolongs progression-free survival in patients with MM. • BM-derived granulocytes are involved in the therapeutic activity of daratumumab.

Background. New drugs with or without autologous stem cell transplantation (ASCT) can induce deep CR responses. MRD can be now considered in the response evaluation by the IMWG and many studies propose it as surrogate for survivals. Multiparametric flow cytometric assays have now been replaced by advanced assays that permit to assess simultaneously more than 8 markers in a single tube. In particular, Euro-flow consortium has developed NGF, a novel high sensitive and standardized approach for MM MRD evaluation that is based on the use of 2 single 8-color tubes, containing all the markers needed to distinguish normal vs MM PCs. However, it is necessary to work on fresh samples and to acquire 10⁷ cell/sample, so to have the possibility to evaluate the Limit Of Quantification (LOQ) and the Limit Of Detection (LOD). The LOQ is calculated as 50 clonal plasmacells among 10⁷ nucleated cells; the LOD as 20 clonal plasmacells among 10⁷ nucleated cells. Aim. DART4MM is a single arm, multicenter, prospective study that evaluate Daratumumab effect on MM patients who already achieved VGPR/CR but MRD positive by NGF after a first line therapy (ASCT, VMP) (Gozzetti et al. IMW 2019). The purpose was to analyze 10.000.000 cells for MRD evaluation and reach at least 10^-6 level. Patients and Methods. Next generation flow (NGF) is centralized and measured at Siena University Hospital with two 8 colors tubes panel developed by the EuroFlow Consortium (BD OneFLOW Tm PCSTe BD OneFLOW Tm PCD. BD BioSciences) with detection of MRD with a sensitivity (≥ 1 in 105 /10-6). Daratumumab 16 mg/kg administered at weekly intervals for 8 weeks, then every 2 weeks for an additional 8 weeks, will be given to 50 MM patients who achieved a VGPR or more defined as per IMWG criteria and MRD-positivity (by NGF). Daratumumab starts at least 12 weeks from ASCT and at least 4 weeks after VMP. Free light chain (FLC) and CT/PET are evaluated at time 0 and every 6 months. NGF is done on marrow aspirate at time 0, at 2 months and every 6 months for 2 years. Primary endpoint is achievement of MRD negativity at 6 months: if patients are MRD negative after 6 months of therapy, treatment is stopped. Otherwise treatment will continue every 4 weeks up to 2 years. Rapid infusion was allowed from the third dose (cycle 1, day 15) if no serious IRR was seen in the previous infusion (second). The infusion rate was calculated to deliver 20% of the dose over 30 min (200 mL/hr), and then the rate was increased to deliver the remaining 80% over 60 min (450 mL/hr). This resulted in a 90 min estimated infusion time (total volume 550 mL). Results. Recruitment started at the end of December 2018. 70 patients were screened until July 2020 at 5 centers in Italy. At least 10 million cells were analyzed for sensitivity at flow for each sample. 31/70 (44%) resulted MRD positive and eligible. M/F =15/16, median age was 61 (range 48-68).Three patients were excluded from the protocol because of consent withdraw. Previous therapy were single ASCT (21 patients), double ASCT 3 patient, VMP (3 patients), KRD (1 patient). ISS stage was I in 8 patients, II in 9 patients, and III in the other 6 patients. Cytogenetics/FISH analysis at diagnosis was done in 25/28 patients : it was negative for 17p deletion, t(14q) and 1q amplification in 16 patients, 2 had t(4;14) , 5 had t(11;14), 2 had del 17p, 1 del 13q, +11 in 2. Grade 2 reaction (moderate infusion-related reactions) during first daratumumab infusion was seen in 10/28 (35%) patients and promptly resolved with corticosteroids administration and temporary infusion interrumption. More than 200 rapid infusions were given to 16 patients. No serious adverse event was registered. 22/28 (79%) patients completed 8 weeks of treatment (2 months) and evaluated MRD. 17/28 (60%) completed 6 months of therapy. MRD negativity was reached at 6 months in 9/17patients (53%). Interestingly 9/13 (62%) patients treated previously with ASCT were MRD negative (10^-6) after 6 months of Dara and stopped treatment. 12 patients reached 12 months of follow up: 2/12 patients are still MRD negative at 10^-7 (6 lost MRD negativity). Conclusions. Follow up will continue with marrow evaluation for MRD every 6 months until 2 years. Having at disposition high quality BM samples for MRD evaluation can ameliorate our assays, even to 10^-6 or 10^-7 and it is crucial to have a good coordination between clinicians and laboratories so to improve the accuracy, sensitivity, and specificity of MM MRD detection in MM patients.

Background. A Deep Molecular Response (DMR), defined as BCR-ABL1 p210 transcript at levels <= 0.01% by QPCR, is the prerequisite for a successful interruption of treatment in patients with CML. However, approximately 50% of patients in Treatment Free Remission (TFR) studies had to resume therapy after BCR-ABL1 p210 transcript levels rose above the 0.01% DMR threshold. To improve transcript detection sensitivity, transcript levels were analyzed using D-PCR. D-PCR increased BCR-ABL1 transcript detection sensitivity by 10-100 fold, however its ability to better select successful TFR patients remains unclear. Beyond the role of the immune system, relapse may be due to the presence of residual leukemic stem cells (LSCs) that are transcriptionally low or silent, insensitivity to tyrosine kinase inhibitors (TKIs) or other CML-causing transcripts not easily detectable by BCR-ABL1 PCR techniques Another approach is to use flow cytometry to detect and quantify bone marrow Ph+ LSCs CD34+/CD38− that co-express CD26 (dipeptidylpeptidase-IV) although its meaning in TFR is unclear. Aim. To compare and examine values of the three different methods of detecting minimal residual disease (MRD) in CML at RNA and LSC levels in patients in TFR or DMR. Patients and Methods. The twenty-seven patients in this study received treatment with either Imatinib (12), Dasatinib (6), Nilotinib (7), Bosutinib (1) or Interferon (1). Twelve patients were in TFR, while the rest were in DMR. TFR patients had stopped therapy for less than 1 year (3), 250,000 ABL copy numbers). D-PCR analysis used TaqMan-MGB probes targeting the BCR-ABL1 transcript. A custom assay was designed and produced with a FAM-label, basing on the sequence of routinely-used probes. BCR-ABL1 quantifications were performed analyzing 50ng of cDNA on a QuantStudio 3D Digital PCR System (ThermoFisher Scientific). BCR-ABL1 transcript values using dPCR were expressed as number of copies/ul. The secondary analysis were carried out by AnalysisSuite Cloud Software (ThermoFisher Scientific). To detect peripheral blood circulating CD34+/CD38-/CD26+ LSCs, cells were incubated with anti-CD45 (BD Biosciences), anti-CD34 (581), anti-CD38 (HIT2) and anti-CD26 (M-A261) (BD Pharmigen). Acquisition and analysis were performed by FACSCanto II flow cytometer (BD Biosciences, NR Nannini). CD45+ cells acquired for each sample ranged from 500,000 to 1,000,000. Isotype controls were included in each staining. Median absolute number of CD26+ cells/μL were calculated as follows: (# WBCs/μL) × (% CD34+/CD38−/CD26+ stained CD45+ cells). Results. TFR status, the type of TKI treatment, QPCR results, dPCR data and LSCs quantification are summarized in Table 1. Both d-PCR and LSCs showed higher sensitivity than QPCR, exhibiting positive results when transcript levels using QPCR were undetectable (16). None of the patients tested negative with d-PCR, however 14/16 were under the threshold of 0.468 copies/uL, corresponding to a stable DMR. LSC levels were negative in 5 patients, 3 of which also tested negative with QPCR. In all patients QPCR ranged from 0-0.0068, d-PCR from 0.073 to 0.943, and LSCs from 0 to 0.156. Results were divided in quartiles, depending on molecular response for QPCR, the 0.468 threshold for dPCR and the distribution for the LSCs. The 2 lowest quartiles defined the lowest detectable DMR. A strong correlation of these data in TFR patients was noted (10/12 concordant) while 8/15 DMR patients were discordant. Conclusions To our knowledge this is the first attempt to analyze and compare DMR in a CML population using standard (QPCR) and highly sensitive (dPCR and LSCs) methods. TFR patients, some lasting up to 17 years, were in the lowest detectable DMR categories. Very little is known about the biology of CML implications of circulating LSCs. Larger studies and dynamic scoring will help define their informative and predictive value.

Minimal residual disease (MRD) evaluation is a recognized endpoint in clinical trials. Both next-generation flow and sequencing could be used as complementary techniques to detect myeloma cells after therapy to measure the depth of response and novel drug efficacy. Anti-CD38 monoclonal antibodies combined with proteasome inhibitors and immunomodulatory drugs have increased the quality of response in myeloma patients, and MRD evaluation is also entering routine clinical practice in many hematological centers. This review analyzes updated results from recent clinical trials utilizing anti-CD38 monoclonal antibodies such as isatuximab and daratumumab in terms of their responses and MRD data. MRD-driven therapy appears promising for the future of MM patients, and emerging minimally invasive techniques to assess MRD are under investigation as novel potential methods to replace or integrate traditional MRD evaluation.