Presence Of Minimal Residual Disease Research Articles

The Hedgehog signal transducer Smoothened and microRNA-326: pathogenesis and regulation of drug resistance in pediatric B-cell acute lymphoblastic leukemia.

PurposeMultidrug resistance (MDR) and the subsequent disease relapse are the major causes of childhood acute lymphoblastic leukemia (ALL) related death. The Hedgehog (Hh) signaling pathway can contribute to cancer MDR. In the current study, Smoothened (Smo) was selected as the experimental target due to its importance in the Hh pathway in order to evaluate its probable role in pediatric B-ALL drug resistance.Patients and methodsThe study included 27 pediatric B-ALL and 16 control bone marrow samples. Quantitative RT-PCR was used to investigate the expression levels of Smo and miR-326 as the key players of the Hh pathway. Western blot analysis was performed. The presence of minimal residual disease was studied using PCR-SSCP. The association between Smo expression and drug resistance was analyzed statistically.ResultsResults showed a significant increase in the Smo expression levels in drug-resistant patients in comparison with drug-sensitive children with B-ALL (P=0.0128, AUC=0.82). A considerable negative association between miR-326 and Smo expression levels was identified (r=−0.624, P=0.002). A binomial test confirmed the regulatory role of miR-326 on the translational repression of Smo (P=0.031). Statistics showed no association between Smo and ABCA2 expression levels. However, a significant positive correlation was observed between the Smo and ABCA3 transcripts in the resistant ALL children (r=0.607, P=0.016).ConclusionData revealed the possible oncogenic impact of Smo on leukemogenesis and drug resistance in pediatric B-ALL. Upregulation of Smo was introduced, for the first time, as a prognostic factor for drug resistance in childhood B-ALL. To the best of our knowledge, this is the first study that shows a positive correlation between Smo and ABCA3 expression levels in pediatric B-ALL, explaining a possible mechanism for the development of drug resistance in this cancer. Moreover, the current project revealed a negative modulatory effect of miR-326 on the expression levels of Smo.

Highlights of This Issue

Treatment options are limited for breast cancer patients with central nervous system (CNS) metastasis. The combination of capecitabine and lapatinib, at conventional doses, has activity against HER2-positive breast cancer brain metastases; however, few patients respond, especially long-term. In this study, Morikawa and colleagues evaluated the safety and toxicities associated with intermittent high dose lapatinib alternating with intermittent fixed dose capecitabine in HER2-positive breast cancer patients with CNS metastasis. This regimen resulted in manageable toxicity and promising efficacy and will inform future phase II/III trials.Hypoxia is a negative prognostic feature in nearly all cancer types and predicts treatment failure. Validated, widely available tests are lacking for clinical use. Salem and colleagues provide the first-in-human evidence that oxygen-enhanced MRI (OE-MRI) can track changes in hypoxia that are induced by radiotherapy. OE-MRI was well-tolerated in patients with non-small cell lung cancer, and radiotherapy-induced hypoxia modification was detected. OE-MRI has potential to select patients for hypoxia-modification, track therapeutic efficacy, and identify tumors that may fail immunotherapy.Immunotherapy has been ineffective for patients with pancreatic ductal adenocarcinoma (PDA). In this study, Seo and colleagues demonstrate that CD8+ T cells are often present in PDA, but are localized to the stroma, rather than immediately adjacent to cancer cells. Using slice culture live microscopy from primary PDA tumors, the authors demonstrate that inhibition of the CXCR4 chemokine receptor led to the migration of clonally expanded CD8+ T cells to the cancer cell-rich regions of the tumors. Subsequent PD-1 blockade activated effective cancer cell killing. These results provide a new basis for the rational selection of combination immunotherapy for PDA, a disease with limited treatment options.Relapse remains a major issue for patients with multiple myeloma due to the presence of minimal residual disease (MRD) posttherapy. Bispecific T cell-engager antibody therapy has been effective in the elimination of MRD in other hematological malignancies. Zuch de Zafra and colleagues describe AMG 424, a novel bispecific T-cell–recruiting antibody targeted at CD3 and CD38, the multiple myeloma surface cell marker. AMG 424 effectively killed CD38-positive cells in vitro and showed antitumor activity in mouse and nonhuman primate models. AMG 424 is a promising candidate for multiple myeloma patients and is currently in clinical development.

Abstract 119: The influence of CD44/HA signaling on CML survival and resistance to tyrosine kinase inhibitor treatment

Abstract The development of tyrosine kinase inhibitors (TKIs) such as imatinib has greatly improved the prognosis and survival of patients diagnosed with chronic myeloid leukemia (CML). However, many CML patients relapse due to the presence of minimal residual disease and the development of TKI resistance. It is unknown exactly what causes the relapse, but based on research from our laboratory we hypothesize that hyaluronic acid (HA) produced by fibroblasts in the microenvironment of the bone marrow provide for a protective environment preventing the tumor cells from therapy-induced cell death or senescence. The purpose of this study is to determine the influence that HA and its binding to its receptor (CD44) has on CML survival and resistance development. Initially, we examined the effect that stromal cell derived and/or exogenously added HA has on CML resistance to imatinib. Cell survival, apoptosis, HA production, and signaling events elicited by the interactions of HA with CD44 was examined using methods such as ELISA, western blotting, and MTT analysis. Furthermore, we examined the potential therapeutic use of compounds known to alter HA production and/or signaling for the treatment of CML. Our study demonstrates increased cell survival when the tumor cells are co-cultured with fibroblasts or have exogenous HA added to them after imatinib treatment. Additionally, we show that knocking out hyaluronic acid synthases reduces this protective effect. This data supports our hypothesis and we believe it could help lead to improved therapeutic treatment for CML. Note: This abstract was not presented at the meeting. Citation Format: Kylie Humphries, Olga Uchakina, Bryan Hostetler, Robert McKallip. The influence of CD44/HA signaling on CML survival and resistance to tyrosine kinase inhibitor treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 119.

Abstract 422: Duplex sequencing for MRD detection in acute myeloid leukemia

Abstract The purpose of this study is to develop a universally applicable Duplex Sequencing (DS) assay for extremely sensitive detection of minimal residual disease (MRD) in acute myeloid leukemia (AML). The presence of MRD plays a critical role in AML recurrence and mortality, and is an important prognostic marker to guide clinical care and clinical trial risk stratification. Unfortunately, current MRD detection methods, including light microscopy, cytogenetics, qPCR, and flow cytometry have shortcomings in terms of sensitivity, general applicability, and/or inter-laboratory reproducibility. Duplex Sequencing eliminates technical artefacts observed with other sequencing technologies and delivers an unprecedented level of accuracy and sensitivity, on widely available NGS platforms. Here, we report validation of our DS MRD mutation detection assay, with a background mutation level more than 100-fold below single-stranded tag-based error correction (SSCS). Our complete DS MRD probe panel targets mutation hotspots or full coding sequences in 29 genes recurrently mutated in AML, representing mutations found in approximately 90% of patients. Supplementing with a targeted RNA-seq panel for recurrent gene fusions would approach 100%. To simulate low frequency mutations in MRD, we generated 2 DNA dilution series: either AML patient DNA or cell line DNA with known AML mutations that were serially diluted into normal human DNA. Replicate libraries were prepared with custom targeted probe panels and sequenced to over 50,000x or over 1,000,000x total Duplex depth (original Duplex DNA molecules sequenced per site), respectively. Expected mutations were detected down to MAFs of 1.9x10-5 and 6.0x10-6. Background mutation levels of 5.7x10-7 and 5.8x10-7, were respectively seen in each series, and likely reflect the true age-associated biological background, rather than technical noise. SSCS artefacts in these samples were approximately 100x higher than the frequency of DS mutations, and obscured expected mutations in the lower dilutions. We are further validating our method in both retrospective and prospective studies. In retrospective samples from a completed clinical trial, we are comparing the sensitivity and accuracy of DS vs. multicolor flow cytometry for MRD with known clinical outcomes. The relative prognostic value of DNA from blood vs. bone marrow will be quantified with DS as well. We will also perform DS on prospective samples to examine the prognostic value of specific genes and MAFs to eliminate uninformative variants, which will also allow accurate quantification of low level mutations in non-leukemic clones that accumulate with aging and can confound detection of MRD. Elimination of sequencing artefacts is critical for the application of NGS assays to MRD detection, especially when expected mutations are unknown, and DS is an important new tool in high sensitivity cancer diagnostics. Citation Format: Jacob E. Higgins, Lindsey N. Williams, Christopher S. Hourigan, Jerald P. Radich, Jesse J. Salk. Duplex sequencing for MRD detection in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 422.

High NPM1 mutant allele burden at diagnosis correlates with minimal residual disease at first remission in de novo acute myeloid leukemia.

Acute myeloid leukemia (AML) with mutated NPM1 is a newly recognized separate entity in the revised 2016 WHO classification, and is associated with a favorable prognosis. While previous studies have evaluated NPM1 in a binary fashion, we recently demonstrated a significant independent negative prognostic effect of high NPM1 mutant allele burden (VAF) at diagnosis in a cohort of de novo AML patients. Although the importance of minimal residual disease (MRD) monitoring in NPM1-mutated AML has been well characterized, the potential relationship between diagnostic allele burden and MRD is unknown. We retrospectively evaluated for MRD at first remission (CR1). We used either next-generation sequencing (NGS) [n = 71], and/or immunohistochemistry (IHC) for mutant NPM1 (NPM1c) [n = 60], in a subset of patients from our recently examined cohort. We identified a statistically significant positive correlation between the VAF at diagnosis, and at CR1 (Spearman r = 0.4, P = .006), and enrichment for MRD in high diagnostic VAF patients (P = .05), as previously defined. IHC-positivity also correlated significantly with a higher median diagnostic NPM1 VAF (0.42 vs 0.39, P = .02), and with the VAF at CR1 (Spearman r = 0.7, P = .003). In multivariable analyses, both high diagnostic VAF (P = .003) and MRD (P = .02) were independent predictors of shorter event-free survival (EFS). Our findings suggest a relationship between the NPM1 mutant allele burden at diagnosis, and the presence of MRD at first remission. Our findings support IHC as a potentially useful adjunctive tool for disease monitoring.

PS1413 INCIDENCE OF NEGATIVE MINIMAL RESIDUAL DISEASE IN MULTIPLE MYELOMA PATIENTS AFTER AUTOLOGOUS STEM CELL TRANSPLANT

Background:The survival of multiple myeloma (MM) patients (pts) has improved significantly over the last decade. However, MM remains an incurable disease. Minimal residual disease (MRD) is considered a crucial prognostic factor. Previous studies have demonstrated that the presence of MRD at day 100 (d100) following autologous stem cell transplant (ASCT) independently predicts for both progression‐free and overall survival. Several techniques have been developed to detect MRD, including multiparameter flow cytometry (MFC).Aims:To estimate the incidence of negative MRD (MRD‐) after induction with bortezomib‐based triplets and ASCT. To investigate the correlation between disease features at diagnosis and response to induction therapy, and MRD negativity.Methods:We retrospectively analysed pts diagnosed with MM treated in a single institution with high‐dose therapy and ASCT between July 2016 and November 2018, after receiving induction therapy with VTD or VCD. MRD was assessed by MFC ≈ d100 after ASCT in the bone marrow of all pts, according to recommendations from the EuroFlow consortium. Additional response was assessed according to the IMWG criteria. High‐risk cytogenetic (HRC) abnormalities included del(17p), t(4;14) and t(14;16). Agreement between MRD and other variables was measured by Cohen's kappa test (STATAv.14.0).Results:Forty‐nine (49) MM pts were analysed. Among these, 59,2% (n = 29) were male, with a median age at diagnosis of 64 years (40 ‐ 70). Isotype was IgG in 51% (n = 25), IgA in 22,4% (n = 11), kappa Free Light Chain (KFLC) in 18,4% (n = 9) and lambda FLC (LFCL) in 6,1% (n = 3). By the International Staging System (ISS), 26,1% pts (n = 12) were stage III. HRC were detected in 37,5% (9/24). Median time from diagnosis to ASCT was 12,3 months (5,8 – 115). Nine pts (18,4%) received two lines of therapy prior to ASCT. First‐line induction included VCD (n = 43) and VTD (n = 6). Lenalidomide(Len)‐based regimens were the most common second‐line (7/9; 77,8%).Response prior to ASCT was a complete remission (CR) in 8 pts (16,3%), very good partial remission (VGPR) in 33 pts (67,4%) and partial remission (PR) in 8 pts (16,3%). At d100 after ASCT, thirty pts (61,2%) were MRD‐ (53,3% CR, 43,3% VGPR and 3,3% PR). At this point, in the general population, responses had improved to 40,8% CR (80% MRD‐), 44.9% VGPR (59.1% MRD‐) and 14.3% PR. Kappa coefficient between MRD and ≥VGPR at d100 was 0,32 (fair agreement).The achievement of ≥VGPR prior to ASCT correlated with MRD negativity (OR 6,46; p = 0.035). MRD‐ status was not influenced by age (OR 1.03; p = 0.534), MM subtype, ISS (OR 1,71; p = 0.215), R‐ISS (OR 0.96; p = 0.952), HRC (OR 0.35; p = 0.414), number of prior lines of therapy (OR 1,33; p = 0.711), first‐line regimen (OR 0.28; p = 0.260) or extramedullary disease at diagnosis (OR 1.31; p = 0.771). At a median follow‐up of 13,5 months after ASCT, 6 pts (12,3%) relapsed: 2 presented MRD+ and 4 MRD‐ at d100 (kappa value 0.03 – poor agreement).Summary/Conclusion:In our cohort, nearly 2/3 of pts were MRD‐ after ASCT; there was no significant difference between pts treated with VCD/VTD as first‐line induction. Even pts who required re‐induction with a Len‐based regimen prior to ASCT achieved MRD negativity.As shown in our study, pts achieving VGPR after ASCT may have MRD‐, highlighting the importance of assessing MRD after ASCT in those pts (not only in CR pts). This phenomenon might be explained by the development of oligoclonal bands after ASCT, not related to the original malignant clone.

Evaluation of TH and PHOX2B gene expression by real time PCR for diagnostic of bone marrow metastatic lesion in neuroblastoma patients

Despite on using the use of complex treatment, the survival of high risk neuroblastoma patients doesn’t exceed 50 % with relapse as the main cause of death. The bone marrow (BM) lesion and presence of minimal residual disease at various stages of therapy are key factors of poor outcome and high frequency of relapse. Real time PCR is the most promising method for detecting neuroblastoma cells in BM samples due to the high sensitivity and specificity, which achieved by combination of several molecular markers. In this study we developed a method for evaluation TH and PHOX2B expression for monitoring metastatic BM lesion in neuroblastoma patients. This method has high sensitivity (1·10–4) and specificity to tumor cells. Evaluation of TH and PHOX2B gene expression was performed for 67 children with neuroblastoma at the time of diagnosis. We demonstrated that patients with overexpression of these genes has significantly worse overall and relapse-free survival. So, the developed method can be used in clinical practice for evaluation BM lesion degree as well as for risk group stratification in neuroblastoma patients at the time of diagnosis.Real time PCR is the most promising method for detecting neuroblastoma cells in BM samples due to the high sensitivity and specificity, which achieved by combination of several molecular markers. In this study we developed a method for evaluation TH and PHOX2B expression for monitoring metastatic BM lesion in neuroblastoma patients. This method has high sensitivity (1·10–4) and specificity to tumor cells. Evaluation of TH and PHOX2B gene expression was performed for 67 children with neuroblastoma at the time of diagnosis. We demonstrated that patients with overexpression of these genes has significantly worse overall and relapse-free survival. So, the developed method can be used in clinical practice for evaluation BM lesion degree as well as for risk group stratification in neuroblastoma patients at the time of diagnosis.

T-ALL Minimal Residual Disease Using a Simplified Gating Strategy and Its Clinico-hematologic Correlation: A Single Center Experience from North India.

The presence of minimal residual disease (MRD) is one of the strong predictors of disease outcome in various hematological malignancies including B-ALL and T-ALL, independent of pre-therapeutic risk factors. There is scant Indian data on MRD by flowcytometry in T-ALL including gating strategies, clinical correlation etc. The primary aim of this retrospective observational study was to define the clinico-hematologic characteristics and prognostic significance of patients with ETP/near-ETP versus non-ETP immunophenotype, especially in terms of minimal residual disease at different time points as well as event-free survival (1year). Baseline hematologic characteristics along with post-induction (Day-35) and post-consolidation (Day-78) MRD in bone marrow samples from newly diagnosed T-ALL patients were studied. 14.3% patients had ETP-ALL immunophenotype, 11.4% were near-ETP ALL patients and the remaining 74.5% were of non-ETP subtype. The ETP/near ETP patients was significantly associated with higher risk of MRD positivity ( > 0.01%) at the end of induction in comparison to the non-ETP patients (p = 0.033). Also, these patients showed a trend towards proclivity to anemia (p = 0.06) and higher rates of induction failure (p = 0.07) However, no difference was observed between the two subgroups in terms of age, high TLC, thrombocytopenia, adverse cytogenetics, steroid responsiveness on Day + 8 of induction, MRD-positivity > 0.01% at the end of consolidation and EFS-1year. Through this preliminary study, it can be stated clearly that ETP status is associated with MRD > 0.01% post-induction but has no significant impact on long-term survival of these patients.

Posttransplant maintenance therapy for acute leukemias.

Allogeneic hematopoietic stem cell transplantation cures a significant proportion of patients with hematological malignancies. Unfortunately, leukemia recurrence is the main cause of transplant failure. Risk factors for relapse include presence of minimal residual disease and a variety of well-recognized leukemia prognostic factors. Posttransplant therapy could decrease the risk of leukemia relapse and is under investigation. In this review, we summarize the current research in maintenance therapy for the prevention of acute leukemia recurrence after allogeneic transplant. Epigenetic modifiers, tyrosine kinase inhibitors, and antibody-drug conjugates are among potential maintenance therapies given their side-effect profile and many are currently under investigation. In the posttransplant setting, the phase I/II data are maturing and have been encouraging enough to lead to the initiation of multicenter phase III randomized clinical trials. Currently, there is no standard of care for posttransplant maintenance for acute leukemias. Further investigation into the efficacy and safety of posttransplant maintenance is warranted given phase I/II clinical trials results. Beyond chemo-immunotherapy, the use of targeted cellular therapy, such as chimeric antigen receptor T cells, is a potential therapy as well.

Treatment abandonment in childhood acute lymphoblastic leukaemia in China: a retrospective cohort study of the Chinese Children’s Cancer Group

ObjectivesBefore 2003, most children with acute lymphoblastic leukaemia (ALL) abandoned treatment, with only approximately 30% treated in China. With the development of national insurance for underprivileged patients, we assessed the...

Beyond 5 years: enduring risk of recurrence in oestrogen receptor-positive breast cancer.

Women with early-stage oestrogen receptor (ER)-positive (ER+) breast cancer who receive standard endocrine therapy for 5 years remain at risk of distant recurrence for at least 15 years after treatment discontinuation. The extension of the duration of adjuvant endocrine therapy to 10 years has been shown to reduce the risk of recurrence only in a subset of women and, to date, predictive biomarkers of benefit from therapy do not exist. In this Review, we briefly explore the epidemiology of late recurrence (>5 years after diagnosis) in patients with ER+ breast cancer. The mechanisms underlying this phenomenon remain poorly understood; we discuss the evidence currently available on processes such as alterations of gene expression or specific genomic aberrations and examine several models used for risk prognostication and for estimating the presence of minimal residual disease, as well as the relevance of these prediction tools for clinicians and patients. Our aim is to enable clinicians to make well-informed decisions on whether to extend endocrine therapy for each individual patient.

Liquid Biopsy for the Management of Patients with Colorectal Cancer

Background: Liquid biopsy is a collective term that refers to the analysis of tumor-derived biomarkers isolated from biological fluids of cancer patients. Recently, many authors reported the usefulness of liquid biopsy for the management of malignancy. Summary and Key Messages: The peripheral blood of cancer patients is a pool of cells and/or cell products derived from the primary or metastatic tumor, including circulating tumor cells (CTCs), circulating free (cf) DNA or RNA, and exosomes containing proteins, nucleic acids, and lipids. CTCs are tumor cells that can be isolated from peripheral blood. Free circulating DNA with a tumor-specific mutation is called circulating tumor DNA (ctDNA). Some patients who undergo curative surgery experience recurrent disease, which can be due to the presence of minimal residual disease (MRD). Thus, MRD indicates a high risk of relapse. Detection of ctDNA or CTC after surgery is a direct proof of MRD. Molecular volume (e.g., the number of CTCs and level of ctDNA) might reflect tumor burden, thus high molecular volume may indicate poor prognosis. The most notable application of liquid biopsy in cancer is to understand spatial and temporal heterogeneities. Heterogeneity is one of the causes of refractoriness and hampers prediction of chemotherapeutic effect. Emerging mutations that are not present in primary tumors but are found in their metastases can be detected in ctDNA. Some colorectal cancer patients with wild-type RAS do not respond to epidermal growth factor receptor blockade. In a subset of these patients, RAS mutation is detected in ctDNA, indicating heterogeneity.

Prospective Evaluation of Prognostic Relevance of KIT Mutations in Core-Binding Factor Acute Myeloid Leukemia: Results from the JALSG CBF-AML209-KIT Study

Prospective Evaluation of Prognostic Relevance of KIT Mutations in Core-Binding Factor Acute Myeloid Leukemia: Results from the JALSG CBF-AML209-KIT Study

Droplet Digital PCR Monitoring of TP53 Mutated Circulating Tumor DNA Levels in Lymphoma Patients

INTRODUCTION: Circulating tumor DNA (ctDNA), a portion of circulating cell-free DNA (cfDNA), is released from tumor cells into the circulatory system, which contains mutations corresponding to the patient's tumour alleles. Detection of ctDNA may noninvasively signal the presence of minimal residual disease (MRD) and predict prognosis. In recent years, droplet digital PCR (ddPCR) has emerged as a sensitive and effective tool for detection of point mutations in cfDNA. In this work, by application of ddPCR, we monitored the mutated TP53 ctDNA levels in serial plasma samples of lymphoma patients with identified TP53 hotspot mutations in their tumor biopsies. These results may give clues about the prognostic implications of different mutation locations and therapeutic strategies.METHODS: Lymphoma-focused next-generation sequencing were performed in tumor biopsies from over 200 lymphoma patients. A total of 134 sequential plasma samples from 32 lymphoma patients with TP53 hotspot mutations were subsequently monitored. cfDNA was extracted from a median sample volume of 4.8 ml (range 3.5-7.0 ml) of plasma. Specific MGB probes were designed for each TP53 mutation site and were then validated by tumor biopsies as well as healthy plasma samples as positive and negative controls respectively. Patient samples were considered to be mutation-positive if the mutant concentration in the sample was higher than the 95% confidence interval of the assay-specific false positive rate. The ctDNA sequential samples were quantitatively tracked using the ddPCR system QX200 (Bio-Rad Laboratories).RESULTS: 30 different TP53 mutations in 134 plasma samples from 32 lymphoma patients were monitored. All the mutations are hotspot in tumors according to COSMIC database, and are predicted to make damaging effect on TP53 protein by SIFT prediction. The average TP53 mutation frequency in tumor biopsies is 40.41% (2.30~92.21%). A total number of 30 specific MGB probes were designed for each TP53 mutation site. Their average false positive rate is 0.0004 copies/ul (0~0.002 copies/ul).In our results, 6 out of 20 (30%) patients with mutations in Loop3 and LSH motifs within the DNA binding domain of TP53 had a clearance of ctDNA, while 6 of 12 (50%) patients with mutations outside of the Loop3 and LSH motifs had a clearance of ctDNA. Literatures reported that patients with non-detectable ctDNA in plasma have negative minimal residual disease (MRD). So these results suggested that Loop3 and LSH motifs, which were reported to interact with DNA groove directly, are more critical than other structures.To investigate the association between different Ann Arbor stages and prognosis in TP53 mutated patients, we divided all the patients according to their stages. We found that patients with earlier Ann Arbor stages were more likely to have their ctDNA cleared (stage I/II: 83.33%; stage III/IV: 28%).In addition, we also analyzed the prognostic implication of cytogenetic abnormality. We found that, in patients with del (17p) as well as TP53 mutation, 7 of 8 had persistent detectable TP53 mutated ctDNA, only 1 of 8 (12.5%) had a clearance of ctDNA, While among the patients carrying TP53 mutations but not accompanied del (17p), 11 of 24 (45.83%) patients had a clearance of ctDNA. These results suggested that it is more difficult to achieve remission in patients with no wide-type TP53 allele compared to those have one wide-type TP53 allele.We further evaluated CAR-T cell immunotherapy and chemotherapy alone to 30 refractory recurrent TP53 mutated lymphoma patients. After CAR-T cell immunotherapy, 11 of 21 (52.38%) patients have non-detectable TP53 mutated ctDNA, while only 2 of 9 (22.22%) patients who received chemotherapy alone have non-detectable ctDNA. It suggested that CAR-T cell immunotherapy is more effective than chemotherapy alone to clear TP53 mutated ctDNA.CONCLUSIONS: Circulating tumor DNA is a promising biomarker to noninvasively monitor tumor load and quantify MRD. By applying droplet digital PCR, ctDNA could be measured and monitored sensitively, specifically and fast. In this work, we designed and validated 30 TP53 probes and tracked 134 sequential plasma from 32 patients. We found that it is easier to clear ctDNA for earlier stages of lymphoma patients with TP53 mutations. CAR-T cell immunotherapy is more effective than chemotherapy alone to continuously clear ctDNA.DISCLOSURE: No relevant conflicts of interest to declare. DisclosuresNo relevant conflicts of interest to declare.

Pre-Transplant NPM1 Mutant Transcript Level Is Highly Predictive of Outcome after Allograft and Thresholds Are Dependent on FLT3 ITD Status

IntroductionRelapse remains the most common cause of treatment failure for patients with acute myeloid leukaemia (AML) who undergo allogeneic stem cell transplantation (SCT) and carries a grave prognosis. Multiple studies have identified the presence of minimal residual disease (MRD) prior to SCT assessed by flow cytometry (FCM) as a strong predictor of relapse: however, little is known about the impact of molecular MRD pre-SCT. Molecular techniques can provide 100-1000 fold greater sensitivity than FCM in NPM1 mutated AML allowing identification of patients with very low level MRD prior to SCT. Peri-transplant management decisions for patients with positive molecular MRD are highly challenging due to the absence of robust outcome data.MethodsBetween 2009-14 the NCRI AML17 study enrolled 3215 adult non-APML patients aged 16-77 eligible for intensive chemotherapy. Central screening for NPM1 mutations was positive in 861/2949 (29%); 530 patients provided serial samples for MRD monitoring. Paired blood (PB) and bone marrow aspirates (BM) were requested after each chemotherapy cycle and then quarterly; additional samples were requested pre- and post-SCT. Samples were analysed by RT-qPCR using a suite of 27 mutation-specific reverse primers. Results were only fed back to clinicians after June 2012 when patients could be treated for confirmed re-emergent or persistent molecular positivity. Post-remission treatment was determined according to the validated NCRI risk score, with poor-risk patients recommended for SCT during first complete remission (CR1). Survival analysis was performed using the logrank test.ResultsIn total 108/530 patients received SCT (CR1 57 (52%), after molecular relapse or progression (MR) 30 (28%), CR2 21 (19%)). Five-year survival post-SCT (5y-OS) was 65% in CR1, and 54% in MR/CR2 (p=0.3). After MR 26/30 patients received chemotherapy prior to SCT. Evaluable pre-SCT PB and BM samples taken within 60 days of SCT were available for 104 and 78 patients (both available n=74).Considering PB samples, 5y-OS was 73% (median OS (mOS) not reached (NR)) for MRD-ve patients (n=74) vs. 30% (mOS 8.1 m) for any PB positivity (n=30) (p<0.0001). Patients with a negative pre-SCT BM had 5y-OS of 79% (mOS NR) vs 47% (mOS 11.9 m) if the BM was positive (p=0.002).Of the 47 patients who received additional chemotherapy for morphologic or molecular relapse or progression, 26 (55%) converted to MRD negativity accounting for 44% of 59 patients who were MRD negative pre-SCT. Ninety-three per cent of 59 pre SCT-MRD negative patients had been MRD negative in the PB after the second cycle of induction (a previously identified marker of favourable outcome).A threshold of 200 mutant NPM1 transcripts/105 ABL copies in the pre-SCT PB sample split patients into 3 groups with 2y-OS of 81% (negative, n=74), 54% (low, n=13) and 9% (high, n=17; p<0.0001). In the BM a threshold of 1000 copies defined 3 groups with a 2y-OS of 86% (negative, n=36), 56% (low, n=32) and 12.5% (high, n=8; p<0.0001).Thirty four patients were positive for FLT3-ITD at diagnosis (5y-OS 55%) and 74 were negative (5y-OS 62%; p=0.3). For patients without FLT3 ITD, negative, low and high levels of MRD in the pre-SCT PB sample were associated with 2y-OS of 79% (n=53), 88% (n=8) and 0% (n=9) (p<0.0001). For BM samples, 2y-OS was 78% (n=28), 62% (n=24) and 0% (n=5; p=0.0001). For patients with FLT3 ITD, corresponding 2y-OS for PB samples were 89% (n=21), 0% (n=5) and 25% (n=8; p=0.0005); for BM samples 2y-OS was 83% (n=9), 38% (n=8) and 25% (n=4; p=0.02).We assigned 80 patients to high and low risk groups based on FLT3 ITD status and pre-SCT MRD level in PB and BM. High-risk patients (FLT3 ITD and any detectable level of MRD in the PB or BM, or FLT3 ITD negative with > 200 copies in the PB or > 1000 copies in the BM) had 3y-OS of 14% (n=27, mOS 6.5 months) compared with 80% for low-risk patients (n=53, p<0.0001) (fig 1).ConclusionsPatients who test negative for NPM1 mutant transcripts immediately before SCT have a favourable outcome which is also observed in FLT3 ITD negative patients who are MRD positive at levels which do not exceed 200 copies in the PB or 1000 copies in the BM. FLT3 ITD mutated patients with any level of MRD prior to SCT, and FLT3 ITD negative patients with transcript levels above these thresholds, have a very high risk of relapse and may benefit from further chemotherapy or FLT3 inhibition prior to or after SCT; studies to investigate this are urgently needed. [Display omitted] DisclosuresHills:Daiichi Sankyo: Consultancy, Honoraria. Cavenagh:Celgene: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau. Russell:Daiichi Sankyo: Consultancy; Pfizer: Consultancy, Honoraria, Speakers Bureau; Jazz Pharma: Speakers Bureau.

Outcomes of Allogeneic Stem Cell Transplantation for AML and MDS Based on Pre-Transplant MRD Status By Next-Generation Sequencing

BackgroundAfter induction therapy for acute myelogenous leukemia (AML), the presence of minimal residual disease (MRD) by targeted next-generation sequencing (NGS) during complete remission (CR) predicts relapse and survival, particularly after exclusion of pre-leukemic mutations. MRD assessment is not routinely performed for AML prior to transplant, partly because consensus regarding assay methodology, appropriate timing, interpretation of results, and therapeutic value prior to SCT is lacking. We therefore sought to describe the rates of mutational clearance and correlate these with relapse rates post-transplant.MethodsWe conducted a retrospective review of sequential AML or myelodysplastic syndrome (MDS) patients undergoing allogeneic hematopoietic cell transplant (alloHCT) at our institution between 2014 and 2017. There were 119 patients with AML/MDS who were treated with either myeloablative or reduced intensity conditioning regimens. Of the 119 patients transplanted, 60 had both pre- and post-treatment NGS results and were included in the analysis. 56 patients had somatic mutations on initial NGS and were therefore eligible for mutational clearance analysis. Twelve patients were in active disease and excluded from further analyses. The remaining patients (n=44) represent the core dataset.Blood and/or marrow specimens were analyzed via a clinical NGS panel targeting 68 leukemia-associated genes. Median coverage (across 88 samples) was 2817 reads. Mutations were considered persistent if present at variant allele frequencies (VAF) ≥ 1% for single nucleotide variants (SNV) or ≥ 2 copies for insertions and deletions (indels). Validated laboratory reporting practice at our institution reports VAF > 4% for SNVs and ≥ 1% for indels with a minimum of 250 total reads. We therefore defined three levels of mutational clearance on the basis of the VAF of residual mutations: VAF for SNV <1% (and/or indels ≤1 copy), between 1-4% (and/or indels <1% and ≥ 2 copies), and >4% (and/or indels > 1%). Patients with ≥ 1 mutation meeting these thresholds were designated NGS(-), NGS-low and NGS(+), respectively. The median follow-up was 332 days.ResultsOn review of NGS data, 120 mutations were present in initial sequencing, with 64 mutations persistent in pre-transplant samples from 26 patients. The most commonly mutated genes from initial samples were FLT3 (18), ASXL1 (11), TET2 (10), NPM1 (9), RUNX1 (8), SRSF2 (8), and DNMT3A (7) (Figure 1A). Mutational clearance varied widely, with the putative pre-leukemic genes DNMT3A, TET2, and ASXL1 (DTA) demonstrating low rates of mutational clearance (Figure 1A). Mutations persisting below the validated reporting threshold were present in 20 patients, including 10 patients otherwise negative by NGS.There were 16 patients categorized as NGS(+), 10 NGS-low, and 18 NGS(-), with relapse rates of 31%, 22%, and 30%, respectively. No difference in relapse risk was observed between NGS(-) and NGS-low subgroups (p = 0.72), and no RFS benefit was observed for patients without persistent mutations > 4% relative to the NGS(+) subgroup (p = 0.56, Figure 1B). Recent work has shown a survival benefit in AML patients in CR without persistent mutations that is enhanced when DTA genes were excluded from the analysis (Jongen-Lavrencic, NEJM 2018). In our cohort, after exclusion of DTA mutations, 6 patients were reclassified by mutational clearance status, and 2 were excluded from the analysis as they had only DTA mutations in pre-treatment samples. Similar to the more comprehensive cohort, no RFS benefit based on NGS status was observed in the post-transplant period (p = 0.42, Figure 1C).ConclusionsThere were similar outcomes regardless of molecular MRD findings by NGS for patients with advanced myeloid malignancies who were in morphologic CR prior to alloHCT. These results contrast with those in the published literature that address a more uniform patient population of clinical trial participants, not all of whom went on to transplant. Further detailed analyses from larger more homogeneous populations will be useful to determine the prognostic significance of MRD by NGS prior to allogeneic HCT. [Display omitted] DisclosuresFrey:Servier Consultancy: Consultancy; Novartis: Consultancy. Perl:Novartis: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Actinium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; NewLink Genetics: Membership on an entity's Board of Directors or advisory committees; Arog: Consultancy; Pfizer: Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy; Daiichi Sankyo: Consultancy. Stadtmauer:Takeda: Consultancy; Celgene: Consultancy; AbbVie, Inc: Research Funding; Amgen: Consultancy; Janssen: Consultancy. Porter:Genentech: Other: Spouse employment; Kite Pharma: Other: Advisory board; Novartis: Other: Advisory board, Patents & Royalties, Research Funding. Gill:Extellia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Carisma Therapeutics: Equity Ownership; Novartis: Research Funding.

Reduced Intensity Conditioning (RIC) Regimens Hematopoietic Cell Transplantation (HCT) for Acute Myeloid Leukemia (AML): A Comparison of Fludarabine/Busulfan (FB) and Fludarabine/Melphalan (FM) Based Regimens from the CIBMTR

Background: There is limited data comparing the outcomes of the two most common (FM or FB) RIC regimens in AML. Previous smaller retrospective studies suggest increased non-relapse mortality (NRM) but decreased relapse with FM based RIC with a variable effect on overall survival as compared to FB.Methods: AML patients aged ≥18 years who had their 1st allo HCT using FM or FB (iv Bu only) RIC between 2001-2015 who had data reported to CIBMTR were studied. Patients with cord blood or identical twin donors, TBI as a part of conditioning, GVHD prophylaxis with T cell depletion/CD34 selection or post HCT Cyclophosphamide were excluded. Primary endpoint was overall survival (OS), and secondary endpoints included non-relapse mortality (NRM), relapse, leukemia free survival (LFS) and acute and chronic GVHD. Cox proportional hazards multivariable regression was used to compare the two RIC regimens while adjusting for significant patient, disease and transplant related factors. Major subgroup analysis included patients in CR pre-HCT.Results: 622 patients received FB and 791 had FM RIC. Median age of the FM group was 59 years, range (18 to 76) and median age of FB group was 61 years, range (18- 77). Compared to FB RIC regimen, FM group had more patients with active disease (36% vs 18%), with mismatched donors (24% vs 15%), with bone marrow grafts (19% vs 9%), HCT before 2005 (29% vs 12%) and without ATG/alemtuzumab (58 % vs 48%); but fewer patients in CR1 (46% vs 62%) and matched sibling donor grafts (21% vs 30%). There was significantly increased NRM in the FM vs FB within 3 mo post HCT (hazard ratio (HR)=3.85 (95% confidence intervals [CI] 2.46-6.03), but not beyond 3 mo FM vs FB , HR= 1.14 (95% CI 0.88-1.47). There was significantly decreased relapse for FM vs FB, (HR=0.65 (0.55-0.76), p<0.001) and a marginally superior LFS for FM vs FB, (HR=0.89 (95% CI 0.78-1.00), p=0.05). There was a lower OS FM vs FB within 3 mo post HCT (HR=1.82 (95% CI 1.36-2.45), p<0.001) but similar OS for FM vs FB beyond 3 mo post HCT (HR=0.87 (95% CI 0.76-1.00), p=0.05). Analysis restricted to only patients in CR pre-HCT showed similar results: equivalent survival, higher early NRM and lower relapse with FM.Conclusion: The current study of this large CIBMTR AML transplant cohort has showed that in these two most common RIC regimens for allogeneic HCT, long-term survival with FM was similar to FB. While the FM regimen showed higher early NRM, it was associated with lower risks of relapse. Further analysis to evaluate FB and FM regimens in relationship to comorbidities, comprehensive geriatric assessments, and presence of minimal residual disease is warranted to better define the relative efficacy of these two RIC regimens in AML. [Display omitted] DisclosuresWeisdorf:Pharmacyclics: Consultancy; Seattle Genetics: Consultancy; SL Behring: Consultancy; FATE: Consultancy; Equillium: Consultancy.

Immunophenotypic Assessment of Minimal Residual Disease in Younger Acute Myeloid Leukemia Patients Is Highly Predictive of Outcome

Introduction: Mainstay of chemotherapy in acute myeloid leukemia (AML) is induction with a goal to achieve morphological complete remission (CR) as evident by less than 5% blasts. Post remission strategies then focus on either consolidation chemotherapy or allogeneic bone marrow transplantation (aBMT). However, not all patients by this remission criterion achieve long term remission and a subset of patients' relapse. This relapse originates from further expansion chemoresistant clones. Detection of minimal residual disease (MRD) following chemotherapy, early in the course of treatment, is highly predictive of outcome and offers a window of opportunity to intensify treatment and prevent relapse as seen in ALL. Here, we describe assessment of immunophenotypic MRD using a 10-colour two tube assay and a combination of difference from normal and leukemia associated immunophenotype (LAIP) approach in patients of adult AML.Methods: We accrued 310 patients of adult (>18 years) AML, other than with t(15;17), over a 66-month period (1st July 2012- 31st December 2017) after obtaining informed consent. All patients received standard induction chemotherapy. MRD testing was done post induction and after first consolidation with high dose cytarabine. Patients accrued from 1st July 2012 to 28th February 2015 (85 patients) were processed using a three tube 8 colour MRD assay. Subsequently samples of 225 patients were processed using a two tube 10 colour MRD assay. Identical panel was used for diagnostic sample, post induction and post consolidation. 500,000 events were acquired per tube with the 3-tube assay and 1.6 million events per tube obtained per tube with the 2 tube, 10 colour assay. Analysis of MRD was done using Kaluza 1.3 by a combination of difference from normal approach that focused on the development of Myeloid progenitors to mature cells and LAIP approaches. Cytogenetic studies by conventional karyotyping and FISH was done as per NCCN version 2 2014 recommendations. Patients were classified into favorable, intermediate and poor cytogenetic risk. The presence of FLT3-ITD, NPM1 and CEBPA mutations were detected by a fragment length analysis-based assay. Overall survival (OS) was calculated from date of diagnosis to time of last follow up or death. Relapse free survival (RFS) was calculated from date of CR till time to relapse or death or last follow up if in CR. Results of the MRD assays, cytogenetic and molecular risk groups were analyzed for their impact on OS and RFS using log rank test.Results: The median age of entire cohort was 33 years (M : F = 1.6 : 1). Based on cytogenetics, 35.1% were classified as favorable risk whereas 52.5% and 12.2% were intermediate and poor risk respectively. FLT3-ITD, NPM1 and CEBPA mutations were detected in 21.9%, 29.0% and 7.7% of patients respectively. Post induction MRD was assessed in 298 patients of which 114 (38.3%) had detectable residual disease (range 0.004-22.6%, median:0.9%). Post consolidation MRD was assessed in 186 patients of which 49 (26.4%) were MRD positive (range 0.002-19.8%, median: 0.37%).The 3 year OS, RFS and median RFS of the entire cohort was 59.0%, 44.8% and 18.7 months respectively. Poor risk cytogenetics as expected was predictive of inferior OS (median OS = 14.6 months vs not reached, p=0.05) as well as RFS (median RFS = 9.3 months vs 18.6 months, p=0.003 respectively). FLT3, NPM1 and CEBPA mutations were not informative as predictors of outcome.The presence of MRD at post induction time point predicted an inferior OS [(p=0.08), HR:1.53; 95% CI (0.93-2.51)] & RFS [(p=0.0002), HR:2.14; 95% CI (1.4-3.3)] as compared to the MRD negative group. Similarly, patients harboring MRD at the end of consolidation had an inferior OS [(p=0.04), HR:1.83; 95% CI (0.94-3.6)] & RFS [(p=0.02), HR:1.79; 95% CI (1.04-3.09)] as compared to the MRD negative group. On multivariate analysis post induction FCM MRD emerged as the most important independent prognostic factor predictive of inferior outcome for RFS [(p=0.01); HR:2.14; 95% CI (1.17 - 3.4)] followed by poor cytogenetic risk [(p=0.05); HR:1.8; 95% CI (1.0 to 3.26)].Conclusion: Our data demonstrates that MRD by flow cytometry at end of induction as well as consolidation are important prognostic factors together with known factors such as high risk cytogenetics. AML MRD is a very useful guide for post remission strategies in AML and should be incorporated into routine treatment algorithms. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Whole Exome Sequencing of Residual Disease in Multiple Myeloma: Searching for Novel Therapeutic Targets

Introduction:Multiple myeloma (MM) is a plasma cell dyscrasia causing damage of multiple organs with fatal consequences for patients. Despite the success of modern therapies eliminating a vast bulk of the aberrant cells, surviving residual clones eventually lead to the relapse of the disease. Accumulation of genomic alterations during the stage of minimal residual disease (MRD) likely contributes to a selective grow advantage and survival under the drug pressure. Identification of specific mutations in MM patients with MRD can provide unique opportunities to target the residual plasma cell clones. Here we present the first whole exome sequencing (WES) analysis of 22 MM samples of patients with MRD that identified 814 mutated genes with 4% of genes previously implicated in the pathogenesis of MM.Methods:Aberrant plasma cells (A-PCs) and peripheral blood (PB) were collected from patients after signing informed consent form. Presence of MRD was assessed with EuroFlow protocol and A-PCs were sorted out from bone marrow according to their pathological immunophenotype based on the expression of antigens CD38, CD45, CD19 and CD56. DNA from A-PCs was isolated and amplified by Repli-g Single cell kit (QIAGEN). Sequencing libraries were prepared using SureSelect Human All Exon V6 Kit (Agilent Technologies) and sequenced by Macrogen Inc. on Illumina HiSeq 4000 platform with average coverage 50x and 2x 100bp read length. Sequencing data were processed using the Bcbio framework following the standard workflow for tumor-matched-normal studies. Specifically, reads were mapped to the human reference genome GRCh37, successively marking duplicates using Picard. Germline mutations were identified using GATK HaplotypeCaller, whereas somatic mutations were identified using MuTect2 reporting as significant variants observed in at least 5 reads and minimum allele frequency of 10%. Variants in homopolymer regions longer than 5 nucleotides were filtered out. The final set of calls were further characterised by assessing their functional impact using snpEff and by annotating each variant using data from 1000 Genomes Phase 3, ExAC, and ClinVar. We then used OncodriveCLUST to identify putative oncogenic genes, and later compared these results with a literature curated list of MM driver genes (Weaver & Tariman, 2017).Results:Our dataset comprises 22 samples from 21 patients (one patient was sampled in two time points) with MM MRD, who received bortezomib-based regimen (age 41-71, average 59 years, 11/22 males, 10/22 females). 8 patients reached complete response, 9 patients had very good partial response and 4 patients had partial response. In our analysis, we detected 1,014 tumour somatic variants (8-287 per sample, median 36), most of them being missense mutations (676/1014), splice site mutations (145/1014) and frameshift insertions (134/1014). The variants affected a total of 814 genes, 97 genes were shared in at least two samples. The most frequently mutated genes were KIAA1211 (11/22), the immunoglobulin gene IGLV3-1 (8/22), apoptotic chromatin condensation inducer ACIN1 (7/22) and CCR4-associated factor 3 CNOT3 (7/22). We also identified 32 genes known to be mutated in MM in 64% of our samples (14/22). We found mutations shared by at least 2 samples in KRAS (4/22), DIS3 (3/22), TRAF3 (3/22), NRAS (2/22), ANK2 (2/22), BRAF (2/22) and RBM15 (2/22). Further analysis with OncodriveCLUST identified 18 putative oncogenic genes (FDR < 0.1), including KRAS, DIS3, ACIN1.Conclusion:We presented the first whole exome study of MM MRD, providing a characterisation of the mutations observed in A-PCs. We overcame problem with low amount of A-PCs in this disease stage by using whole genome amplification and a highly customised bioinformatic analysis pipeline. Our study suggests that A-PCs are characterised by new MM MRD specific set of mutated genes, along with the presence of mutations in well-known multiple myeloma cancer driver genes. This offers a great potential for design of novel precise treatments targeting MRD after standard MM therapies.Supported by Ministry of Health of the Czech Republic (17-30089A, CZ-DRO-FNOs/2016) and Ministry of Education of the Czech Republic (SGS18/PřF/2017-2018) DisclosuresKryukov:JSC BIOCAD: Employment. Maisnar:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Hajek:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Novartis: Research Funding.

Blinatumomab for Minimal Residual Disease (MRD) in Adults with B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL): Median Overall Survival (OS) Is Not Reached in Complete MRD Responders at a Median Follow-up of 53.1 Months

Blinatumomab for Minimal Residual Disease (MRD) in Adults with B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL): Median Overall Survival (OS) Is Not Reached in Complete MRD Responders at a Median Follow-up of 53.1 Months