Minimal Residual Disease Analysis by Monitoring Immunoglobulin and T-Cell Receptor Gene Rearrangements by Quantitative PCR and Droplet Digital PCR.

Analysis of immunoglobulin and T-cell receptor gene rearrangements by real-time quantitative polymerase chain reaction (RQ-PCR) is the gold standard for sensitive and accurate minimal residual disease (MRD) monitoring; it has been extensively standardized and guidelines have been developed within the EuroMRD consortium ( www.euromrd.org ). However, new generations of PCR-based methods are standing out as potential alternatives to RQ-PCR, such as digital PCR technology (dPCR), the third-generation implementation of conventional PCR, which has the potential to overcome some of the limitations of RQ-PCR such as allowing the absolute quantification of nucleic acid targets without the need for a calibration curve. During the last years, droplet digital PCR (ddPCR) technology has been compared to RQ-PCR in several hematologic malignancies showing its proficiency for MRD analysis. So far, no established guidelines for ddPCR MRD analysis and data interpretation have been defined and its potential is still under investigation. However, a major standardization effort is underway within the EuroMRD consortium ( www.euromrd.org ) for future application of ddPCR in standard clinical practice.

Minimal residual disease in advanced or metastatic solid cancers: The G0-G1 state and immunotherapy are key to unwinding cancer complexity.

In the last decade, a large amount of research has focused on elucidating the mechanisms that account for homing disseminated cancer cells (DCCs) from solid tumours to distant organs, which successively progress to overt metastatic disease; this is currently incurable. A better understanding of DCC behaviour is expected to allow detectable metastasis prevention by more effectively targeting 'metastatic seeds before they sprout'. As DCC biology co-evolved with that of the primary tumour, and due to the many similarities between them, the term 'niche' has been borrowed from normal adult stem cells (ASCs) to define the site of DCC metastatic colonisation. Moreover, heterogeneity, survival, protection, stemness and plasticity as well as the prolonged G0-G1 dormant state in the metastatic niche have been the main aspects of intense investigation. Consistent with these findings, in solid cancers with minimal residual disease (MRD), it has been proposed to prolong adjuvant therapy by targeting specific molecular pathway(s) involving DCC dormancy. However, so far, few disappointing clinical data have been reported. As an alternative strategy, because immune-surveillance contributes to the steady state of the DCC population and likely to the G0-G1 state of cancer cells, we have used prolonged immune-modulatory cytostatic chemotherapy, active immune stimulation with an INF-ß/IL-2 sequence or drugs inhibiting myeloid-derived suppressor cell (MDSC)/Treg-mediated immune suppression. This strategy, mainly aimed at boosting the immune response, is based on recent findings suggesting the downregulation of immune escape mechanisms as well as other principal hallmarks during the G0-G1 state and/or in MRD. Preliminary clinical and/or laboratory data suggest the efficacy of this strategy in gastrointestinal and some endocrine-dependent cancers. Following this, we propose therapeutic schedules to prevent DCC activation and proliferation in solid cancers at a high risk of relapse or as maintenance therapy in metastatic patients after complete response (CR) to conventional treatment.

VS38c and CD38-Multiepitope Antibodies Provide Highly Comparable Minimal Residual Disease Data in Patients With Multiple Myeloma.

OBJECTIVES: To compare flow cytometric minimal residual disease (MRD) data obtained using the EuroFlow approach, including the CD38-multiepitope (ME) antibody or the VS38c antibody. METHODS: We evaluated 29 bone marrow samples from patients with multiple myeloma (MM), of whom 15 had received daratumumab within the past 6 months. We evaluated MRD data and fluorescence intensities. RESULTS: Qualitative MRD data were 100% concordant between the 2 approaches. In MRD-positive samples (n = 14), MRD levels showed an excellent correlation (R2 = 0.999). Whereas VS38c staining was strong in both normal plasma cells and MM cells, independent of daratumumab treatment, staining intensities for CD38 were lower in MM cells compared with normal plasma cells, and on both cell types CD38 expression was significantly reduced in daratumumab-treated patients. CONCLUSIONS: Both CD38-ME and VS38c allow reliable MRD detection in MM patients, but the high expression of VS38c allows easier identification of MM cells, especially in daratumumab-treated patients.

Immune Dysfunction as Measured by the Systemic Immune-Inflammation Index is Associated with the Sub-Type of Minimal Residual Disease and Outcome in Stage II Colon Cancer Treated with Surgery alone.

OBJECTIVE: Within 5 years after curative surgery for stage II colon cancer 25% of patients will relapse due to minimal residual disease (MRD). MRD is the net result of the biological properties of subpopulations of primary tumour cells which enable them to disseminate, implant in distant tissues and survive and the immune system's ability to eliminate them. We hypothesize that markers of immune dysfunction such as the systemic inflammation index (SII) are associated with the sub-type of MRD defined by bone marrow micro-metastasis (mM) and circulating tumour cells (CTCs). A higher immune dysfunction being associated with a more aggressive MRD and worse prognosis. METHODS AND PATIENTS: Blood and bone marrow samples were taken to detect CTCs and mM using immunocytochemistry with anti-CEA one month after surgery. The SII, absolute neutrophil, platelet and lymphocyte counts (ANC, APC, ALC) were determined immediately pre-surgery and one month post-surgery. These were compared with the sub-types of MRD; Group I MRD (-); Group II mM positive and Group III CTC positive; cut-off values of SII of >700 and >900 were used. Follow-up was for up to 5 years or relapse and survival curves using Kaplan-Meier (KM) were calculated. RESULTS: One hundred and eighty one patients (99 women) participated, mean age 68 years, median follow up 4.04 years; I: = 105 patients, II: N= 36 patients, III: N=40 patients. The SII significantly decreased post-surgery only in Group I patients. The frequency of SII >700 and >900 was significantly higher in Group III, between Groups I and II there was no significant difference.  The SII was significantly associated with the number of CTCs detected. The 5-year KM was 98% Group I, 68% Group II and 7% Group III. CONCLUSIONS: The results of the study suggest that the severity of immune dysfunction as determined by the SII is associated with differing sub-types of MRD and a worse prognosis; increasing immune dysfunction is associated with a more aggressive CTC positive MRD sub-type; a more severe immune dysfunction is associated with a higher number of CTCs detected.
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Comparison of minimal residual disease detection in multiple myeloma between the DuraClone and EuroFlow methods.

In this study, the minimal residual disease (MRD) levels in patients with multiple myeloma (MM) were assessed by comparing the new 8-color single-tube multiparameter flow cytometry method (DuraClone), which reduces the cost of antibodies and labor burden of laboratories, with the EuroFlow next-generation flow (NGF) method. A total of 96 samples derived from 69 patients with MM were assessed to determine the total cell acquisition number (tCAN), percentages of total and normal plasma cells (PCs), and MRD levels using two methods. We found that the tCAN was significantly higher with EuroFlow-NGF than with DuraClone (median 8.6 × 106 vs. 5.7 × 106; p < 0.0001). In addition, a significant correlation in the MRD levels between the two methods was noted (r = 0.92, p < 0.0001). However, in the qualitative analysis, 5.2% (5/96) of the samples showed discrepancies in the MRD levels. In conclusion, the DuraClone is a good option to evaluate MRD in multiple myeloma but it should be used with caution.

CD19 CAR-T cell treatment conferred sustained remission in B-ALL patients with minimal residual disease.

The persistence or recurrence of minimal residual disease (MRD) after chemotherapy predicts relapse of B-cell acute lymphoblastic leukemia (B-ALL). CD19-directed chimeric antigen receptor T (CD19 CAR-T) cells have shown promising responses in B-ALL. However, their role in chemotherapy-refractory MRD-positive B-ALL remains unclear. Here we aimed to assess the effectiveness and safety of CD19 CAR-T cells in MRD-positive B-ALL patients. From January 2018, a total of 14 MRD-positive B-ALL patients received one or more infusions of autogenous CD19 CAR-T cells. Among them, 12 patients achieved MRD-negative remission after one cycle of CAR-T infusion. At a median follow-up time of 647 days (range 172-945 days), the 2-year event-free survival rate in MRD-positive patients was 61.2% ± 14.0% and the 2-year overall survival was 78.6 ± 11.0%, which were significantly higher than patients with active disease (blasts ≥ 5% or with extramedullary disease). Moreover, patients with MRD had a lower grade of cytokine release syndrome (CRS) than patients with active disease. However, the peak expansion of CAR-T cells in MRD positive patients showed no statistical difference compared to patients with active disease. Five patients received two or more CAR-T cell infusions and these patients showed a decreased peak expansion of CAR-T cell in subsequent infusions. In conclusion, pre-emptive CD19 CAR-T cell treatment is an effective and safe approach and may confer sustained remission in B-ALL patients with chemotherapy-refractory MRD. The trials were registered at www.chictr.org.cn as ChiCTR-ONN-16009862 (November 14, 2016) and ChiCTR1800015164 (March 11, 2018).

Predictive value of tumor-infiltrating lymphocytes for response to neoadjuvant chemotherapy and breast cancer prognosis.

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) are predictive for the response to neoadjuvant chemotherapy (NAC) of breast cancer. However, little is known about the predictive value of TILs for axillary lymph node involvement after NAC. METHODS: We analyzed 282 breast cancer patients who were operated following NAC and curative surgery from 2008 to 2018. TILs were assessed in core needle biopsies before NAC, and the biopsies were divided into three groups: low (0%-10% immune cells in stromal tissue within the tumor), intermediate (11%-59%), and high (≥60%). The patients were followed for an average of 63 months (range, 2-116 months). We analyzed retrospectively the predictive value of TILs for the response to NAC, including pathological complete response (pCR) and axillary lymph node involvement (positive lymph node ratio (LNR; the ratio of the number of nodes involved to the total number of nodes dissected)). The prognostic values of TILs and LNR were assessed. RESULTS: A pCR was achieved in 27 of 188 patients (14.4%) in the low-TIL group, in 14 of 57 patients (24.6%) in the intermediate-TIL group, and in 13 of 37 (35.1%) in the high-TIL group (p = .007). Among patients who underwent axillary lymph node dissection after NAC, patients with high TILs had lower LNR (p = 0021) compared with the other groups. Kaplan-Meier analysis showed that overall survival (OS; p < .001) and disease-free survival (p < .001) were significantly longer for patients with low LNR (≤0.2). TILs were positively correlated with disease-free survival (p = .028), but TILs did not correlate with OS (p = .171). Moreover, by multivariable analysis, LNR independently affected disease-free survival (p < .001). CONCLUSIONS: TILs may be predictive for pCR rate, postoperative residual lymph node involvement, and disease-free survival of breast cancer patients. High TILs may suggest favorable outcomes.

14-Color single tube for flow cytometric characterization of CD5+ B-LPDs and high sensitivity automated minimal residual disease quantitation of CLL/SLL.

INTRODUCTION: The diagnosis of CLL/SLL relies on flow cytometric immunophenotyping. Increasing emphasis is being placed on precise detection of the minimal residual disease. Following antigen recommendations of ERIC and ESCCA's Harmonization Project, we validated a 14-color assay for the characterization CD5+ lymphoproliferative neoplasms and CLL MRD with a sensitivity of at least 10-4 . METHODS: The assay was designed based on ERIC/ESCCA recommended antigens with the addition of CD40 for alternate gating when CD19 expression is reduced. Lower limit of quantitation/lower limit of detection, assay procedural precision, linearity, and limit of blank were established. Then, 52 CD5+ B-cell lymphoproliferative neoplasms (41 CLL/11 non-CLL) and 29 normal samples were used for parallel evaluation. Automated cluster identification and quantitation of CLL clones in MRD setting was performed using Barned-Hutt SNE. Separation analysis between CLL and non-CLL phenotypes was performed by PCA and bh-SNE. RESULTS: Separation ratios for each antigen exceeded ERIC/ESCCA guidelines. Precision was <20% at LLOQ (0.01%). The limit of blank was <10/500,000 cells. Concordance between the 14-color and legacy assay (Deming regression y = 1.01x, r2  = .99) was seen. All 20 samples with MRD levels 0.5%-0.006% (median 0.04%) showed an abnormal cell cluster by bh-SNE, with concordant results between manual and automated quantitation (y = x, r2  = 1). CLL cases clustered together and away from mantle cell lymphoma by bh-SNE and PCA with outlier atypical phenotype CLL cases posing diagnostic challenges by both manual and automated analysis. CONCLUSION: The 14-color CD5+ LPD assay provides a robust standardization platform for MRD and disease characterization using both manual and automated analysis.

Small molecular fluorescence dyes for immuno cell analysis.

Many diseases, including cancers, AIDS, diabetes, asthma, Parkinson's, and lymphoma, are associated with the immune cell responses of patients suffering from them. Identifying the underlying immune response in such diseases is critical to correctly diagnose their root cause and determine the correct medications to target that root cause for personal therapy and immunotherapy. This work focuses on small molecular CF dyes to conjugate with antibodies, such as CD4 and CD19, for their application in flow cytometry. The CF dyes enable the expansion of flow cytometry reagent panels to support high dimensional flow cytometry analysis of the resulting emissions of 30-40 fluorescent colors, a record in flow cytometry. The CF dyes can be used along with existing flow cytometry dyes to provide a quick, accurate, and cost-effective method for the diagnosis and immunology treatment of diseases such as minimal residual disease (MRD) after cancer therapy. The CF dyes will also be an effective tool for the clinical studies of immune response to SARS-CoV-2 and the related vaccine development.

Autologous tumor cell vaccine induces antitumor T cell immune responses in patients with mantle cell lymphoma: A phase I/II trial.

Here, we report on the results of a phase I/II trial (NCT00490529) for patients with mantle cell lymphoma who, having achieved remission after immunochemotherapy, were vaccinated with irradiated, CpG-activated tumor cells. Subsequently, vaccine-primed lymphocytes were collected and reinfused after a standard autologous stem cell transplantation (ASCT). The primary endpoint was detection of minimal residual disease (MRD) within 1 yr after ASCT at the previously validated threshold of ≥1 malignant cell per 10,000 leukocyte equivalents. Of 45 evaluable patients, 40 (89%) were found to be MRD negative, and the MRD-positive patients experienced early subsequent relapse. The vaccination induced antitumor CD8 T cell immune responses in 40% of patients, and these were associated with favorable clinical outcomes. Patients with high tumor PD-L1 expression after in vitro exposure to CpG had inferior outcomes. Vaccination with CpG-stimulated autologous tumor cells followed by the adoptive transfer of vaccine-primed lymphocytes after ASCT is feasible and safe.

Validation of a modified pre-lysis sample preparation technique for flow cytometric minimal residual disease assessment in multiple myeloma, chronic lymphocytic leukemia, and B-non Hodgkin lymphoma.

BACKGROUND: Minimal residual disease (MRD) assessment of hematopoietic neoplasia below 10-4 requires more leukocytes than is usually attainable by post-lysis preparation. However, not all laboratories are resourced for consensus Euroflow pre-lysis methodology. Our study aim was to validate a modified pre-lysis protocol against our standard post-lysis method for MRD detection of multiple myeloma (MM), chronic lymphocytic leukemia (CLL), and B-non Hodgkin lymphoma (B-NHL), to meet demand for deeper MRD assessment by flow cytometry. METHOD: Clinical samples for MRD assessment of MM, CLL, and B-NHL (50, 30, and 30 cases, respectively) were prepared in parallel by pre and post-lysis methods for the initial validation. Total leukocytes, MRD, and median fluorescence intensity of antigen expression were compared as measures of sensitivity and antigen stability. Lymphocyte and granulocyte composition were compared, assessing relative sample processing stability. Sensitivity of the pre-lysis assay was monitored post validation for a further 18 months. RESULTS: Pre-lysis achieved at least 10-4 sensitivity in 85% MM, 81% CLL, and 90% B-NHL samples versus 24%, 48%, and 26% by post-lysis, respectively, with stable antigen expression and leukocyte composition. Post validation over 18 months with technical expertise improving, pre-lysis permitted 10-5 MRD assessment in 69%, 86%, and 82% of the respective patient groups. CONCLUSION: This modified pre-lysis procedure provides a sensitive, robust, time efficient, and relatively cost-effective alternative for MRD testing by MFC at 10-5 , facilitating clinically meaningful deeper response assessment for MM, CLL, and B-NHL. This method adaptation may facilitate more widespread adoption of highly sensitive flow cytometry-based MRD assessment.

Tumor microenvironment, immune response and post-radiotherapy tumor clearance.

Radiotherapy is the treatment of choice for many cancer patients. Residual tumor leads to local recurrence after a period of an equilibrium created between proliferating, quiescent and dying cancer cells. The tumor microenvironment is a main obstacle for the efficacy of radiotherapy, as impaired blood flow leads to hypoxia, acidity and reduced accessibility of radiosensitizers. Eradication of remnant disease is an intractable clinical quest. After more than a century of research, anti-tumor immunity has gained a dominant position in oncology research and therapy. Immune cells play a significant role in the eradication of tumors during and after the completion of radiotherapy. The tumor equilibrium reached in the irradiated tumor may shift towards cancer cell eradication if the immune response is appropriately modulated. In the modern immunotherapy era, clinical trials are urged to standardize immunotherapy schemes that could be safely applied to improve clearance of the post-radiotherapy remnant disease.

Lineage of measurable residual disease in patients with chronic myeloid leukemia in treatment-free remission.

Approximately half of patients with chronic myeloid leukemia (CML) in sustained deep molecular response who discontinue tyrosine kinase inhibitors (TKIs) remain in treatment-free remission (TFR). Some of these patients have measurable residual disease (MRD) by BCR-ABL1 mRNA testing, and most have detectable BCR-ABL1 DNA by highly sensitive methods. We used fluorescence-activated cell sorting and BCR-ABL1 DNA PCR to investigate the lineage of residual CML cells in TFR. Twenty patients in TFR for >1 year provided blood for sorting into granulocytes, monocytes, B cells, T cells, and NK cells. MRD was identified predominantly in the lymphoid compartment and never in granulocytes. B cells were more often BCR-ABL1 positive than T cells (18 vs 11/20 patients) and at higher levels (median 10-4.9 vs 10-5.7; P = 0.014). In 13 CML patients studied at diagnosis lymphocytes expressing BCR-ABL1 mRNA comprised a small proportion of total leukocytes. These data improve our understanding of TFR biology, since it is now clear that MRD in the blood of TFR patients need not imply the persistence of multipotent CML cells. Lineage-specific assessment of MRD could be explored as a means to improve the prediction of TFR.

Leukemia-associated immunophenotypes subdivided in "categories of specificity" improve the sensitivity of minimal residual disease in predicting relapse in acute myeloid leukemia.

BACKGROUND: The assessment of minimal residual disease (MRD) by flow cytometry (FC) has a prognostic impact in acute myeloid leukemia (AML), despite the low sensitivity in predicting relapse. Nonetheless, the role of leukemic-associated immunophenotypes (LAIPs)-related specificity on the sensitivity of MRD has not been clarified yet. In this respect, we accomplished this study. METHODS: LAIP-frequencies of bone marrow samples from healthy donors and patients after treatment were quantified and subdivided in "categories of specificity" named as: "strong," "good," and "weak." At the following, the diagnostic performance of MRD was investigated in terms of sensitivity, specificity, predictive values, likelihood ratio (LR). RESULTS: "Strong" LAIPs were identified by CD7, CD2, CD4, and CD56 markers while "weak" LAIPs, independently of coexpressed markers, were mainly observed in CD33+ cells. MRD identified patients with significantly low DFS and OS but showed a low sensitivity in predicting relapse. Interestingly, majority of recurrences was noticed in patients with two LAIPs and lacking of "strong" LAIPs or only with one "good" LAIP. Thus, only patients showing one "strong" or two "good" LAIPs were considered suitable for MRD monitoring and selected to be further investigated. In this subset, positive MRD predicted a poor prognosis. Moreover, a higher sensitivity, negative predictive value (NPV) and LR- were observed after comparison with the previous series. CONCLUSIONS: These data highlight the relevant role of LAIP classification in "categories of specificity" in improving the sensitivity of MRD as assessed by FC.

Inflammation induced by incomplete radiofrequency ablation accelerates tumor progression and hinders PD-1 immunotherapy.

Radiofrequency ablation (RFA) promotes tumor antigen-specific T cell responses and enhances the effect of immunotherapy in preclinical settings. Here we report that the existence of remnant tumor masses due to incomplete RFA (iRFA) is associated with earlier new metastases and poor survival in patients with colorectal cancer liver metastases (CRCLM). Using mouse models, we demonstrate that iRFA promotes tumor progression and hinders the efficacy of anti-PD-1 therapy. Immune analysis reveals that iRFA induces sustained local inflammation with predominant myeloid suppressor cells, which inhibit T cell function in tumors. Mechanistically, tumor cell-derived CCL2 is critical for the accumulation of monocytes and tumor-associated macrophages (TAMs). The crosstalk between TAMs and tumor cells enhances the CCL2 production by tumor cells. Furthermore, we find that administration of a CCR2 antagonist or the loss of CCL2 expression in tumor cells enhances the antitumor activity of PD-1 blockade, providing a salvage alternative for residual tumors after iRFA.

Immunophenotyping of Acute Lymphoblastic Leukemia.

Immunophenotyping by flow cytometry is an important component in the diagnostic evaluation of patients with acute lymphoblastic leukemia. This technique further permits the detection of minimal residual disease after therapy, a robust prognostic factor that may guide individualized treatment. We describe here laboratory methods for both the initial characterization of lymphoblasts at diagnosis, and the detection of rare leukemic lymphoblasts after treatment. In addition to antibody combinations suitable for diagnosis and detection of minimal residual disease, we describe procedures for peripheral blood and bone marrow sample preparation, procedures for labeling of cell-surface and intracellular proteins with fluorochrome-conjugated antibodies, and approaches to analysis of immunophenotypic data.

Influence of mutagenic versus non-mutagenic pre-operative chemotherapy on the immune infiltration of residual breast cancer.

Background: Chemotherapeutic agents are often mutagenic. Induction of mutation associated neo-epitopes is one of the mechanisms by which chemotherapy is thought to increase the number of tumor-infiltrating lymphocytes. It is not known, however, whether treatment with various chemotherapeutic agents with different mutagenic capacity induce a significantly different number of stromal tumor-infiltrating lymphocytes (StrTIL) in residual cancer.Methods: One hundred and twenty breast carcinoma cases with residual disease that were treated with one of three types of pre-operative chemotherapy regimens were selected for the study. The percentage of StrTIL was evaluated in pretreatment core biopsies (pre-StrTIL) and post-treatment surgical tumor samples (post-StrTIL). TIL changes (ΔStrTIL) were calculated from the difference between post-StrTIL and pre-StrTIL.Results: When analyzing the pre-StrTIL and post-StrTIL among the three treatment groups, we detected significant StrTIL increase independently of the treatment applied. Based on distant metastases-free survival analysis, both post-StrTIL and ΔStrTIL was found to be independent prognostic factor in HR negative cases. Conclusions: Significant increase of StrTIL in the residual disease was observed in patients treated with the highly (platinum), moderately (cyclophosphamide) and marginally mutagenic chemotherapeutic agents (taxane, anthracycline). Increase in StrTIL in residual cancer compared to pretreatment tumor tissue is associated with improved distant metastasis-free survival in cases with HR negative breast carcinoma.