Molecular Monitoring Of Residual Disease Research Articles

Feasibility of tumor-informed circulating tumor DNA (ctDNA) for molecular residual disease (MRD) assessment in pediatric patients with solid tumors.

10058 Background: Circulating Tumor DNA (ctDNA) is a noninvasive biomarker which has been validated as a prognostic tool in multiple adult cancers. There is evolving evidence that ctDNA may be prognostic in pediatric solid tumors, but experience with commercially available assays in pediatric patients is scarce. Signatera is a ctDNA assay which utilizes whole exome sequencing of tumor and whole blood to identify 16 tumor-specific, clonal, somatic variants. These variants are then monitored in plasma as a molecular residual disease (MRD) assessment. Methods: From November 2021 through August 2023, 30 patients were enrolled on a single center study evaluating the feasibility and utility of Signatera MRD monitoring in pediatric patients with solid tumors. Patients 6 months to 25 years of age with any malignant extracranial solid tumor including lymphoma were eligible. Peripheral blood has been collected every 3 weeks to 3 months while on therapy and every 3 to 6 months while off therapy for up to 2 years. A minimum of 22mL of blood was required for the first draw, and 16mL for subsequent draws. Timing of blood draws was aligned with standard of care blood draws when possible. Results of ctDNA MRD were compared with imaging obtained for disease response and surveillance as standard of care. Results: The cohort consisted of 6 cases of neuroblastoma, 6 osteosarcomas, 4 Wilms tumors, 4 carcinomas, 3 soft tissue sarcomas, 2 Ewing sarcomas, 2 germ cell tumors, 2 lymphomas, and one hepatoblastoma. Of 30 patients enrolled, a tumor-informed ctDNA assay was successfully created for 27 (90%). Of 25 patients who had radiographic evidence of disease at any time of monitoring, 23 (92%) had simultaneous detectable ctDNA. Seven patients have had disease recurrence, of whom all seven had detectable ctDNA at or preceding time of recurrence. Conclusions: Signatera tumor-informed ctDNA assay is a feasible noninvasive tool which may be utilized in parallel with standard of care imaging to monitor for disease recurrence in pediatric patients with extracranial solid tumors. Additional analysis of a larger patient cohort is needed to identify tumor types and clinical scenarios for which its application is most useful.

Longitudinal circulating tumor DNA monitoring for detection of molecular residual disease in patients with penile squamous cell carcinoma.

11 Background: Penile squamous cell carcinoma (SCC) is an extremely rare, aggressive disease, representing 0.4-0.6% of all malignancies in males in the U.S. and Europe. Early diagnosis is imperative, as lymphatic spread is associated with a dismal prognosis. The estimated 5-year survival rate in patients with pelvic lymph node involvement is 0%. Moreover, there are limited treatment options that carry significant toxicity risks. Accurate biomarkers to evaluate treatment response and guide treatment to improve outcomes are needed. Molecular residual disease monitoring with circulating tumor DNA (ctDNA) may be helpful in these scenarios. Methods: This is a retrospective analysis of a cohort of 7 patients with advanced HPV-unassociated penile SCC treated at Rush University Medical Center who monitored closely for molecular residual disease (MRD). Signatera (Natera Inc.), a personalized tumor-informed ctDNA assay, was utilized for detection and quantification of ctDNA in patients’ plasma samples, measured at regular intervals. Progression of disease was based on radiographic imaging. Results: In our cohort, personalized, tumor-informed ctDNA assays were designed for 100% (7/7) of the patients. ctDNA was detectable in at least one time point in all of the patients. One patient underwent partial penectomy and lymph node (LN) dissection with 1/11 LN involved. He is being monitored without adjuvant treatment and has sustained ctDNA clearance. Another patient underwent surgery, and had positive post-operative ctDNA. He was treated with adjuvant TIP (paclitaxel, ifosfamide, and cisplatin) with subsequent ctDNA clearance. In 3 of the patients, ctDNA was checked at regular intervals and rising ctDNA prompted imaging studies that showed progression. These patients proceeded to second line treatment options. The remaining 2 patients are non-surgical candidate with LN positivity, who will begin treatment with TIP and monitoring of ctDNA after each cycle of chemotherapy. Twenty-eight percent (2/7) of patients remain alive with sustained ctDNA clearance. Twenty-eight percent (2/7) of patients remain alive with ctDNA positivity, planned to initiate treatment and monitoring of ctDNA at regular intervals. Forty-three percent (3/7) of patients who had persistent ctDNA positivity are now deceased. Conclusions: Our data suggests that a personalized, tumor-informed ctDNA assay is not only useful for MRD monitoring, but also highly predictive of treatment response and imaging-based progression in patients with penile SCC. In our sample, MRD status correlated with both radiographic progression and clinical outcomes. Prospective studies are needed to better understand how ctDNA can be used to guide neoadjuvant and adjuvant approaches, including treatment intensification or de-escalation, in patients with penile SCC.

Abstract 6694: Evaluation of a multi-omics approach to molecular residual disease detection

Abstract Introduction: Advancements in DNA sequence detection technology and assay design have enabled molecular residual disease (MRD) assays to detect cancer earlier and with greater sensitivity. In Tumor-Informed (TI) strategies, mutations from the tumor are used to define a set of patient-specific markers that are tracked in the blood as circulating tumor DNA (ctDNA). Whereas tumor-naïve (TN) approaches do not require prior genomic information from the tumor and instead employ a fixed panel of marker(s). Methylated DNA Markers (MDMs) can be used to distinguish cancer from non-cancer and can be found in plasma. We have developed a proprietary TN MRD platform for colorectal cancer (CRC) capable of measuring as few as 1-5 strands of MDM target. Additionally, we are actively developing a TI MRD assay platform. Here we describe the evaluation of both TI and TN approaches for the detection of circulating tumor DNA (ctDNA) in blood across all stages of colorectal cancer and explore the complementarity of these two approaches in the context of MRD monitoring. Procedures: A 20-sample cohort of paired tumor/normal and pre-surgery plasma specimens from stage I (6/20), stage II (7/20), stage III (6/20), and stage IV (1/20) CRC patients and 20 demographically matched non-cancer controls were acquired for our study. All cancer plasma samples were collected prior to tumor resection. Neat and diluted plasma samples were tested for the presence of ctDNA containing short variants specifically derived from the corresponding primary tumor and for a pre-defined set of MDMs and protein cancer biomarkers. Tumor-specific short variants were characterized by whole exome sequencing of the primary tumors. MDMs were detected from bisulfite converted cfDNA by Target Enrichment Long probe Quantitative Amplified Signal (TELQAS) for 35 MDMs. A cancer detection classifier for the TN assay was trained and validated using a separate cohort of non-cancer and CRC plasma samples and employed for this study. Protein was detected by high throughput ELISA.Results: Tumor sequencing of the 20 CRC samples identified between 122 and 7146 (median = 230) somatic mutations per tumor sample. The combination of features from TI and TN assays achieved 95% accuracy, demonstrating an improvement over either assay in isolation. Indeed, 5 “positive” calls were made for cancer samples for which only one of the two methods met pre-defined RUO thresholds for ctDNA detection. Comparison of median variant allele fraction from the TI assay with median percent methylation of CRC markers for these CRC samples had a Pearson’s correlation of 0.967. Conclusion: TI and TN assays show high concordance in cancer detection, but also complement each other. Additionally, ctDNA quantifications produced from TI and TN assays are linearly correlated. The combination of data from both assays can be used to further differentiate cancer samples from non-cancer samples. Citation Format: Alanna Chan, Martin You, Oliver Limbo, Billyana Tsevtanova, Gerald Dodson, Brenda Curiel, Karma Farhat, Samantha Scott, Archana Ramesh, Jacquelyn Hennek, Viacheslav Katerov, Michael Kaiser, Hatim Allawi, Brady P. Culver, Michael Reshatoff, Jorge Garces, Gina Costa. Evaluation of a multi-omics approach to molecular residual disease detection. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6694.

Integrative analysis of multi-omics data for liquid biopsy.

The innovation of liquid biopsy holds great potential to revolutionise cancer management through early diagnosis and timely treatment of cancer. Integrative analysis of different tumour-derived omics data (such as genomics, epigenetics, fragmentomics, and proteomics) from body fluids for cancer detection and monitoring could outperform the analysis of single modality data alone. In this review, we focussed on the discussion of early cancer detection and molecular residual disease surveillance based on multi-omics data of blood. We summarised diverse types of tumour-derived components, current popular platforms for profiling cancer-associated signals, machine learning approaches for joint analysis of liquid biopsy data, as well as multi-omics-based early detection of cancers, molecular residual disease monitoring, and treatment response surveillance. We also discussed the challenges and future directions of multi-omics-based liquid biopsy. With the development of both experimental protocols and computational methods dedicated to liquid biopsy, the implementation of multi-omics strategies into the clinical workflow will likely benefit the clinical management of cancers including decision-making guidance and patient outcome improvement.

Recommendations for laboratory testing of UK patients with acute myeloid leukaemia.

Recommendations for laboratory testing of UK patients with acute myeloid leukaemia.

Circulating tumour DNA dynamics for assessment of molecular residual disease and for intercepting resistance in breast cancer.

Longitudinal evaluation of circulating tumour DNA (ctDNA) represents a promising tool for monitoring tumour evolution. In patients with breast cancer, ctDNA dynamics for the assessment of molecular residual disease (MRD) and resistances may, respectively, help clinicians in treatment modulation of adjuvant treatments, and in anticipating resistance to ongoing treatments and switch treatments before clinical progression, to improve disease control. Anyway, the introduction of this dynamic biomarker into clinical practice requires the demonstration of analytical validity, clinical validity and clinical utility. In early breast cancer setting, several observational studies demonstrated the clinical validity of MRD monitoring through ctDNA in identifying patients at a higher risk of relapse, but many clinical trials evaluating the clinical utility are still ongoing, and few data resulted in inconclusive results.Instead, ctDNA dynamics for intercepting resistance have not been fully evaluated in terms of clinical validity, because monitoring schedules of most observational studies are not intensive. The only trial assessing their clinical utility (PADA-1) demonstrated a benefit in terms of progression-free survival, portraying a new landscape for clinical trials in this space. Rigorous clinical trials with adequate assays and patient-relevant endpoints are paramount to demonstrate the clinical utility of ctDNA dynamics and eventually increase clinical outcomes.

Detection of molecular residual disease (MRD) for cancer management: Lessons from longitudinal profiling of NSCLC patients undergoing chemotherapy or targeted therapy.

e15020 Background: Identification of cancer patients who are at ongoing risk of relapse remains of high priority to direct effective adjuvant therapy. Plasma circulating cell-free DNA (cfDNA) analyses offer an unprecedented approach to identify patients with molecular residual disease (MRD) earlier than clinical or radiological evidence, detecting drug resistance, progression and relapse with relatively high sensitivity and specificity. Here we used longitudinal surveillance to profile cfDNA mutational landscape undergoing chemotherapy or EGFR-TKI. We demonstrated how cutting-edge NGS and digital qPCR technologies are defining new subgroups of patients with MRD for individually tailored treatment strategy. Methods: Plasma samples were collected from 8 subjects with lung adenocarcinoma, 5 patients were treated with EGFR-TKI and 3 cases by chemotherapy. Overall, 92 longitudinally collected specimens (duration of 31-143 weeks) were analyzed with a 520-gene ultra-deep NGS platform as well as a digital qPCR assay. Standard-of-care tests of CEA, CA19-9 biomarkers and CT radiography were also performed in parallel. Results: The number of somatic mutations detected ranging from 0 to 52 in a sample and correlated strongly with TMB (tumor mutational burden). The concordance of actionable EGFR mutations (L858R, T790M, Ex19Del) detected by NGS and digital qPCR assays was 100% (n = 49/49). NGS assay detected additional driver mutations in ∼20 non-EGFR genes. Majority of EGFR mutations showed directional decline in allelic fraction (AF) after treatment with EGFR-TKI. Patient progressed following EGFR-TKI were registered to undergo chemotherapy based on real-time mutational profiles and did show good responses and PFS. Longitudinal ultra-deep NGS analyses can help detect and stratify MRD, including non-druggable genetic information that could guide best clinical decision-making. Conclusions: Longitudinal ctDNA surveillance of patients treated with EGFR-TKI by a highly sensitive multi-gene NGS panel enables MRD monitoring and can potentially guide a selection of optimal adjuvant therapies earlier, and provide new surrogate endpoints for early registration of these therapies.

Coexisting opportunities and challenges: In which scenarios can minimal/measurable residual disease play a role in advanced non-small cell lung cancer?

Curative therapy was not previously available for patients with advanced non-small cell lung cancer (NSCLC); thus, the concept of minimal/measurable (or molecular) residual disease (MRD) was not applicable to these patients. However, advances in targeted and immunotherapy have revolutionized the treatment landscape for patients with advanced NSCLC, with emerging evidence of long-term survival and even the hope of complete remission (CR) by imaging examination. The latest research shows that patients with oligometastatic lung cancer can benefit from local treatment. After removing the lesions, the choice of follow-up therapy and monitoring of the lesions could remain uncertain. MRD plays a role in identifying early-stage NSCLC patients with high risks of recurrence and determining adjuvant therapy after radical treatment. In recent years, evidence has been accumulating regarding the use of circulating cell-free tumor DNA (ctDNA) to assess MRD in solid tumors. This study discussed the possible applications of ctDNA-based MRD monitoring in advanced NSCLC and described the current challenges and unresolved problems in the application of MRD in advanced NSCLC.

Molecular Residual Disease Monitoring Provides Insufficient Lead-Time to Prevent Morphologic Relapse in the Majority of Patients with Core-Binding Factor AML

IntroductionCore-binding factor (CBF) acute myeloid leukemias (AML) are characterized by the chromosomal abnormalities t(8;21) or inv(16)/t(16;16) and their fusion proteins RUNX1/RUNX1T1 and CBFB-MYH11, respectively. Despite their relatively favorable prognosis, it has been reported that up to 45% of patients with CBF-AML relapse. Current guidelines recommend monitoring the peripheral blood (PB) or bone marrow (BM) for molecular evidence of residual disease (RD) every 3 months for 2 years following remission. By monitoring patients for rising molecular transcripts, those at risk of impending relapse can be identified and treated prior to the emergence of overt disease. However, the relapse kinetics of CBF-AML are poorly-understood, and it is unknown if serial RD monitoring can detect molecular relapses with sufficient lead-time to intervene and prevent morphologic relapse.MethodsAfter local REB approval, we identified patients with CBF-AML treated at the Princess Margaret Cancer Centre in Toronto, Canada between 2000 and 2017. Patients underwent induction and consolidation chemotherapy according to standard protocols followed by RD monitoring with polymerase chain reaction (qPCR) of RUNX1/RUNX1T1 or CBFB-MYH11 transcripts in a CAP/CLIA certified lab every 3 months for a median of 1.2 years (range 0-5.3). RD assessment was performed on BM aspirates, but PB could be tested in patients who could not tolerate repeat BM aspirates. Morphologic relapse was defined as emergence of >5% blasts in the PB or BM, and molecular relapse as a positive transcript PCR (if previous PCR measurements were undetectable) or an increase by 1 log (if previous PCR measurements were detectable) on 2 successive samples without morphologic relapse. Rapid relapse was defined as <100 days from molecular to morphologic relapse (i.e. timeframe insufficient to administer pre-emptive treatments such as allotransplant to prevent morphologic relapse). We extracted relevant clinical and cytogenetic data and performed unpaired t-tests and Fisher's exact tests as appropriate using GraphPad statistical software and version 9.4 of the SAS System for Windows (SAS Institute, Cary, NC).ResultsWe included 114 patients with CBF-AML. Median age was 46.5 years (range 18-79). t(8;21) was present in 59% and inv(16)/t(16;16) in 41% of patients. All patients achieved remission with 7+3 induction chemotherapy. Over a median follow-up time of 3.7 years (range 0.2-14.3), RD measurements were performed a mean of 5 times per patient with mean sampling interval of 103±54 days. Remission was maintained in 71 (62%) patients but 43 (38%) developed morphological (n=34) or isolated molecular relapse (n=9), with median time to relapse of 4.4 months (range 1.4-31.4). Patients with relapsed disease were significantly less likely to have achieved ≥3 log reduction in RUNX1/RUNX1T1 or CBFB-MYH11 BM transcripts at the end of consolidation chemotherapy compared to patients who remained in remission (75% vs 90%, p=0.046). Of the 43 patients who relapsed, the majority (74.4%, n=32) had rapid relapse kinetics. Of these 43 patients, 25 received reinduction chemotherapy with achievement of CR2, 6 had refractory disease or death, 1 was lost to follow-up, and 17 went on to receive allotransplantation. RD monitoring enabled timely detection of impending relapse and permitted intervention prior to morphologic relapse in only 11 patients (25.6%). Among these 11 patients with slower relapse kinetics, 6 received reinduction chemotherapy with achievement of CR2, 2 received reinduction chemotherapy with refractory disease, and 8 went on to receive allotransplantation. Median overall survival was 20 months (range 3-128) for patients with rapid relapse kinetics and 28 months (range 14-166) for patients with slow relapse kinetics (p=0.02). Clinical features, BM vs PB RD measurements, additional molecular mutations, and cytogenetic abnormalities did not distinguish patients with rapid vs slow relapse kinetics.ConclusionsCurrent guidelines recommend molecular RD monitoring every 3 months for CBF-AML. However, in the majority of patients who relapsed at our institution, RD monitoring every 3 months provided insufficient lead-time to identify molecular relapses prior to morphologic relapse. Further research is warranted to identify the patients with CBF at the highest risk of relapse and the best strategies to monitor these patients over time. DisclosuresGupta:Novartis: Consultancy, Honoraria, Research Funding; Incyte: Research Funding. Maze:Novartis: Consultancy, Honoraria. Schuh:Celgene: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Jazz: Consultancy; Amgen Inc.: Consultancy; Shire: Consultancy; Teva: Consultancy; Otsuka: Consultancy. Yee:Celgene, Novartis, Otsuka: Membership on an entity's Board of Directors or advisory committees; Agensys, Astex, GSK, Onconova, Genentech/Roche: Research Funding. Schimmer:Otsuka Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; Medivir AB: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Chronic Myeloid Leukemia: Molecular Monitoring of Residual Disease by Genomic DNA Compared to Conventional mRNA Analysis in Follow-Ups up to 8 Years

Abstract 1687 Background:Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder resulting from the t(9;22)(q34;q11) balanced reciprocal translocation within a pluripotent stem cell (SC). The resulting Philadelphia (Ph) chromosome produces BCR-ABL1 fusion gene coding for a deregulated Abl tyrosine-kinase with constitutive and tumorigenic activity. The first line therapy of CML is imatinib mesylate, which targets Bcr-Abl protein, inhibiting proliferation pathways. Complete cytogenetic response can be achieved in 95% of patients treated in the early chronic phase (CP)1. Molecular monitoring of minimal residual disease is crucial to detect poor responses to imatinib and optimizing treatment with second generation tyrosine-kinase inhibitors or allogeneic stem cell transplantation. Residual leukemia is assessed by a quantitative manner evaluating levels of BCR-ABL1 transcripts by real-time reverse transcriptase PCR (qRT-PCR). Although qRT-PCR detects mRNA levels in a very sensitive manner, the negative result is difficult to interpret, because undetectable levels of chimeric transcript can reflect either an effective elimination of leukemia cells, or the presence of a quiescent leukemia SC transcriptionally silent. Methods:We developed a novel highly sensitive method to identify quiescent leukemic cells through quantitative real-time PCR (Q-PCR) targeting the genomic breakpoint sequence1. In CML each patient shows a unique genomic fusion sequence1, that need to be characterized in order to design a specific genomic assay. We selected 14 patients with CML diagnosed in the early CP. We identified junctions sequences by long-range PCR2. We carried out Q-PCR assay using common primer forward and probe in BCR, and 2 different primers reverse, in ABL or BCR, used as control1. The percentage of leukemic cells (LCs) was calculated using the derivation of the δCt formula1: LC= [100*(2/2δCt+1)]/n], where δCt is the difference between amplification cycles of BCR-ABL1 and BCR reactions, and n is the number of experimental replicates. We tested the sensitivity and the efficiency of the method on K562 cell line. According to the human C value, K562 were diluted in normal commercial genomic DNA until 10−4 dilutions. Eight CML patients in early CP were the object of this study. A patient specific Q-PCR assay was performed on DNA obtained at diagnosis and subsequently applied to monitor minimal residual disease during Imatinib treatment for up to 8 years, for a total of 61 samples. In parallel the same peripheral blood samples were tested by standard qRT-PCR, and the percentage of residual disease (international scale) measured by mRNA was compared with DNA analysis. Results:Positive levels of mRNA were obtained in 79% of samples analyzed by qRT-PCR,while we detected Ph-positive cells in 92% of samples. In all positive samples for chimeric transcript we measured positive levels of corresponding genomic DNA, confirming the sensitivity of the Q-PCR method. In 13% of samples, with undetectable levels of mRNA, we observed the persistence of quiescent leukemic cells, transcriptionally silent like shown by patient 2 in figure 1. This could probably indicate the presence of pluripotent LSCs or progenitor cells, that does not respond to imatinib treatment. Finally undetectable levels of mRNA were confirmed by a correspondent DNA negativity in 8,2% of the samples. This datum should be investigated further in order to establish if the disease was been eradicated. Patients negative by mRNA detection in several consecutive follow-ups could be candidates to stop imatinib therapy. The development of a DNA base technique could be a powerful tool to evaluate the effective presence/absence of leukemic cells. Patient 8 resulted negative at 70 months monitored by RNA and DNA technique could be a candidate to stop the therapy (figure 2). Conclusion:Although the initial characterization of genomic breakpoint sequence is still time consuming, it may provide a patient-specific DNA biomarker that can be used to detect the presence of quiescent leukemic cells otherwise undetectable using a conventional qRT-PCR. The DNA genomic Q-PCR could be a very sensitive and direct technique to detect minimal residual disease in CML patients treated with tyrosine-kinase inhibitors and allogeneic transplantation.We thank AIL Varese and Bergamo. Disclosures:No relevant conflicts of interest to declare.

Molecular Monitoring of Residual Disease in Chronic Myeloid Leukemia by Genomic DNA Compared with Conventional mRNA Analysis

Translocation t(9;22), which produces the BCR-ABL gene, is pathognomonic of chronic myeloid leukemia. For clinical purposes, the amount of chimeric transcript is considered proportional to the leukemic clone; thus, mRNA is commonly used for molecular monitoring of patients. However, there is no consensus regarding the degree of increase in mRNA that should cause concern or whether the absence of transcript indicates a "cure." In this study, we analyzed 57 samples from 10 chronic myeloid leukemia patients undergoing imatinib treatment. For each sample, we compared BCR-ABL mRNA levels with the actual proportion of leukemic cells, which were measured through a novel genomic approach based on the quantitative amplification of DNA breakpoints. The two approaches gave similar patterns of residual disease, and the majority of patients were still positive after an average treatment period of 2 years. Nevertheless, in one of two patients with confirmed undetectable levels of chimeric transcript, DNA still revealed the persistence of leukemic cells at 42 months. These findings appear to justify the clinical practice of maintaining imatinib treatment indefinitely. However, the absence of leukemic DNA (observed in 1 of 10 patients) could be used to identify possible candidates for drug discontinuation. In conclusion, DNA analysis proved to be a reliable index of residual disease with potential applications in the field of clinical diagnostics and research.

Molecular Monitoring of Residual Disease (MRD) during Induction and Intensification Phases in Low Risk Adult B Cell ALL Treated According to the MRC UKALL12 Protocol.

Introduction and aims. Molecular monitoring of minimal residual disease (MRD) using Immunoglobulin (IG) and T cell receptor (TCR) targets has provided an independent and prognostically significant parameter of outcome in adult and childhood acute lymphoblastic leukaemia (ALL). The aim of our study was to evaluate the impact of molecular tests carried out during the first 20 to 24 weeks of chemotherapy for MRD in a standard risk group of adult B cell ALL patients.Patients and Methodology. We evaluated MRD tests in 63 patients with B cell ALL (37M/26F). Median age of our cohort was 24 yrs (range: 15.5–54.6 yrs) while median WBC was 9.9 (range: 1.1–553 × 109l). All were negative for the t(9;22) or t(4;11) translocation and received standard induction chemotherapy or auto stem cell transplant (A-SCT) only. All patients had at least one molecular marker which was tested by quantitative or semi-quantitative PCR with sensitivity ≥1E4 and all tests were carried out at time of morphological remission. End-points were relapse or continuous clinical remission (CCR) with follow up ≥12 months. Time point for molecular evaluation were post induction phase 1, TP1 (up to 1.8 mo), post induction phase 2 (1.8–3.5 mo), post intensification (3.5–5.7 mo). Median follow up was 92 months for patients in CCR (range 12–141 mo) and 15.5 months for patients who relapsed (range: 2.4–49 mo).Results. Thirty-five patients in complete morphological remission were tested at TP1. Relapse free survival (RFS) tests showed a statistically significant correlation (p=0.02) between MRD positive tests and relapse (n=19 pts) and MRD negative tests (n=16 pts) and relapse free survival. In addition, pts with resistant disease at TP1 (n=10; not included above) and MRD positive pts were indistinguishable as far as relapse FS and both faired extremely poorly (Figure A).Thirty patients were analysed at TP2. RFS confirmed the significant association between MRD positivity and poor outcome (n=16 pts) and MRD negativity and CCR (n=14 pts) (p=0.03) (Figure B).MRD data were also available for 43 patients at TP3. Association of MRD positive tests (n=15 pts) and MRD negative tests (n=28 pts) with poor and good outcome, respectively (p=0.0006) (Figure C) was strongest at this time point. Outcome correlated with level of MRD at all time points, with poorer outcome in patients with MRD >1E3.Conclusions. Molecular monitoring during induction and intensification for standard risk B cell ALL patients treated with UKALL12 protocol provides a prognostically significant parameter for the management of adult ALL in otherwise morphological remission and may in the future be used for patients' stratification. [Display omitted]

Molecular Monitoring of Residual Disease in Patients with Mantle Cell Lymphoma and CLL after Allogeneic Bone Marrow Transplantation Post Reduced Intensity Conditioning.

To control circulating lymphoma cells after allogeneic transplantation in order to induce or manipulate graft-versus-lymphoma (GvL) reactivity in case of relapse, molecular monitoring of circulating lymphoma cells was carried out by quantitative real-time PCR for clone-specific IgH(CDR3) sequences on six patients (CLL n=3; mantle cell lymphoma (MCL) n=2; variant hairy cell leukaemia (vHCL) n=1). All patients were considered as high risk for recurrence or relapse because of age, refractory disease, failure after autografting or poor performance status. They had at least one or more high-risk features that precluded the application of a standard myeloablative conditioning regimen. Therefore, they were treated with allogeneic blood stem cell transplantation (BSCT) from unrelated donors after toxicity-reduced conditioning with treosulfan and fludarabine. Molecular relapse was defined as an increase of circulating lymphoma cells by more than three orders of magnitude in the absence of symptoms or signs of clinical relapse.In the three patients with CLL and one MCL patient leukemic cells disappeared from peripheral blood and bone marrow within the first six months after transplant as demonstrated by quantitative PCR. Only short periods of acute GvHD were observed and successfully treated. In 2/6 patients (one with vHCL and one with MCL) a molecular relapse was detected one month (MCL) and 6 months (vHCL) after transplant. A 3-log increase of circulating lymphoma cells was observed within about 20 days (MCL) and 3 months (vHCL) starting from <1 lymphoma cell/500,000 cells to about 1,000 lymphoma cells/100,000 cells. Rapid reduction and withdrawal of immunosuppressive therapy combined with the infusion of the monoclonal antibody rituximab led to the disappearance of circulating lymphoma cells in both patients. It has to be emphasized that in both patients with documented molecular relapse rituximab alone or in combination with chemotherapy has not led to a stable CR or molecular remission before allogeneic BSCT. The disappearance of recurrent lymphoma cells in both patients correlated well with the development of acute GvHD. This fact demonstrates the curing potency of GvL. Serial molecular monitoring at short-term intervals provided the additional important information that further therapeutic interventions, c.f. infusion of donor lymphocytes, were not necessary. All patients monitored are still in continuous complete clinical and molecular remission for almost three years.Clinical relapse of malignant B cell lymphoma after allogeneic BSCT can be successfully treated by immune therapeutic intervention, e.g. reduction and rapid withdrawal of immunosuppression and infusions of peripheral blood lymphocytes from the original blood stem cell donor by establishing graft versus lymphoma (GvL) reactions. In addition, B-cell specific monoclonal antibodies seem to be very helpful for treating relapses. These therapeutic interventions may be even more effective when introduced before clinical relapse, possibly already at molecular relapse detected by quantitative real time PCR. Therefore, molecular monitoring by quantitative real-time PCR carried out at ≤ 3 months intervals may further improve transplant results and the long-term outcome of patients treated within clinical studies.

Molecular monitoring of residual disease using antigen receptor genes in childhood acute lymphoblastic leukaemia

Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements are assumed to be unique ‘fingerprint-like’ sequences for each acute lymphoblastic leukaemia (ALL). Various clonal Ig/TCR gene rearrangements can be identified at diagnosis in virtually all childhood ALL patients, representing molecular targets for detection of minimal residual disease (MRD) during follow-up analysis. The usage of at least two MRD-PCR targets per patient generally ensures high sensitivity (⩽1:104 normal cells) and prevents false-negative results owing to ongoing or secondary rearrangements.MRD monitoring in childhood ALL employing Ig/TCR gene rearrangements as PCR targets has significant prognostic value. This is particularly powerful for evaluation of early treatment response and consequently can be used for improved therapy stratification. Prolonged continuous MRD monitoring might be important for patients at intermediate or high risk of relapse. MRD monitoring in second complete remission identifies patients with excellent drug sensitivity and predicts outcome after stem cell transplantation.

Stem-cell transplantation for chronic lymphocytic leukemia: The 1999 perspective

The use of myeloablative intensive therapy followed by autologous or allogeneic stem-cell transplantation (SCT) for treatment of chronic lymphocytic leukemia (CLL) has largely increased over the last years. The present overview updates the available clinical results and discusses important aspects of SCT in patients with CLL including the type of SCT (autologous vs. allogeneic), myeloablative regimens, purging, the predictive value of molecular monitoring of residual disease, and prognostic factors for the outcome of transplant approaches. With appropriate supportive care, autologous SCT is safe and can induce long-lasting clinical and molecular remissions, which may improve the prognosis of patients with CLL. Feasibiliy and efficacy of autologous SCT appears to be best early during the course of the disease, but it is still unclear if autotransplantation can cure the disease even in this favorable subgroup. The role of purging is still unclear. The better disease control observed after allografting appears to be due to graft-versus-leukemia activity and may allow cure in at least a subset of poor-risk patients. Due to the extraordinarily high treatment-related mortality, however, the outcome after allogeneic SCT is still inferior to that after autologous SCT. Autologous transplantation is a valuable treatment option for younger patients with early or sensitive poor-risk CLL. Selected patients with advanced poor-risk disease and low probability of successful auto-SCT should be considered for allografting. However, it must be kept in mind that both autologous and allogeneic stem-cell transplantation are still experimental procedures and clinical trials further elucidating their value in the treatment of patients with CLL are warranted.

Molecular monitoring of residual disease in chronic myelogenous leukemia patients after therapy.

The degree of tumor load reduction after therapy, which is determined by the degrees of cytoreduction and cytogenetic response, is an important prognostic factor for patients with chronic myelogenous leukemia (CML). Conventional metaphase analysis is considered to be the "gold standard" for evaluating cytogenetic response. The frequency of cytogenetic analysis can be reduced considerably if patients are monitored by molecular methods, such as quantitative Southern blot, fluorescence in situ hybridization (FISH), quantitative western blot, or competitive reverse transcriptase polymerase chain reaction (RT-PCR). Molecular methods can be performed on peripheral blood specimens and are therefore less invasive than cytogenetic analyses of bone marrow metaphases. Furthermore these techniques are applicable to Ph-negative/BCR-AbL-positive cases. Results obtained by Southern blotting, western blotting, and FISH are readily quantifiable but their sensitivity is not generally superior to that of cytogenetic methods. RT-PCR is by far the most sensitive method. Quantitative RT-PCR analysis is the method of choice for monitoring patients after bone marrow transplantation (BMT). Using competitive PCR in patients after BMT, reappearance and/or rising levels of BCR-ABL transcripts can be detected prior to relapse. All complete cytogenetic responders to interferon-alpha are positive for BCR-ABL transcripts. The level of residual disease spans a range over found orders of magnitude. In both interferon-alpha-treated patients and patients after BMT a good correlation between BCR-ABL transcript numbers per microgram of RNA and cytogenetic results has been found. Variables in the competitive PCR assay may be controlled for by quantification of transcripts of the normal ABL gene as an internal standard. We suggest a stepwise strategy for diagnosis and follow-up of CML patients employing molecular methods.