Patient-specific Markers Research Articles

The Comparison of Endovascular and Open Surgical Treatment for Ruptured Abdominal Aortic Aneurysm in Terms of Safety and Efficacy on the Basis of a Single-Center 30-Year Experience.

Ruptured abdominal aortic aneurysm (rAAA) is a critical condition with a high mortality rate. Over the years, endovascular aortic repair (EVAR) has evolved as a viable treatment option in addition to open repair (OR). The primary objective of this study was to compare the safety and efficacy of EVAR and OR for the treatment of rAAA based on a comprehensive analysis of our single-centre 30-year experience. Patients treated for rAAA at the Department of Vascular and Endovascular Surgery, University Hospital Düsseldorf, Germany from 1 January 1993 to 31 December 2022 were included. Relevant information was retrieved from archived medical records. Patient survival and surgery-related complications were analysed. None of the patient-specific markers, emergency department-associated parameters, and co-morbidities were associated with patient survival. The 30-day and in-hospital mortality was higher in the OR group vs. in the EVAR group (50% vs. 8.7% and 57.1% vs. 13%, respectively). OR was associated with more frequent occurrence of more severe complications when compared to EVAR. Overall patient survival was 56 ± 5% at 12 months post-surgery (52 ± 6% for OR vs. 73 ± 11% for EVAR, respectively) (p < 0.05). Patients ≥70 years of age showed poorer survival in the OR group, with a 12-month survival of 42 ± 7% vs. 70 ± 10% for patients <70 years of age (p < 0.05). In the EVAR group, this age-related survival advantage was not found (12-month survival: ≥70 years: 67 ± 14%, <70 years: 86 ± 13%). Gender-specific survival was similar regardless of the applied method of care. OR was associated with more severe complications in our study. EVAR initially outperformed OR for rAAA regarding patient survival while re-interventions following EVAR negatively affect survival in the long-term. Elderly patients should be treated with EVAR. Gender does not seem to have a significant impact on survival.

Targeted locus amplification to develop robust patient-specific assays for liquid biopsies in pediatric solid tumors.

Liquid biopsies combine minimally invasive sample collection with sensitive detection of residual disease. Pediatric malignancies harbor tumor-driving copy number alterations or fusion genes, rather than recurrent point mutations. These regions contain tumor-specific DNA breakpoint sequences. We investigated the feasibility to use these breakpoints to design patient-specific markers to detect tumor-derived cell-free DNA (cfDNA) in plasma from patients with pediatric solid tumors. Regions of interest (ROI) were identified through standard clinical diagnostic pipelines, using SNP array for CNAs, and FISH or RT-qPCR for fusion genes. Using targeted locus amplification (TLA) on tumor organoids grown from tumor material or targeted locus capture (TLC) on FFPE material, ROI-specific primers and probes were designed, which were used to design droplet digital PCR (ddPCR) assays. cfDNA from patient plasma at diagnosis and during therapy was analyzed. TLA was performed on material from 2 rhabdomyosarcoma, 1 Ewing sarcoma and 3 neuroblastoma. FFPE-TLC was performed on 8 neuroblastoma tumors. For all patients, at least one patient-specific ddPCR was successfully designed and in all diagnostic plasma samples the patient-specific markers were detected. In the rhabdomyosarcoma and Ewing sarcoma patients, all samples after start of therapy were negative. In neuroblastoma patients, presence of patient-specific markers in cfDNA tracked tumor burden, decreasing during induction therapy, disappearing at complete remission and re-appearing at relapse. We demonstrate the feasibility to determine tumor-specific breakpoints using TLA/TLC in different pediatric solid tumors and use these for analysis of cfDNA from plasma. Considering the high prevalence of CNAs and fusion genes in pediatric solid tumors, this approach holds great promise and deserves further study in a larger cohort with standardized plasma sampling protocols.

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.

A Personalized Longitudinal Strategy in Low-Grade Glioma Patients: Predicting Oncological and Neural Interindividual Variability and Its Changes over Years to Think One Step Ahead.

Diffuse low-grade glioma (LGG) is a rare cerebral cancer, mostly involving young adults with an active life at diagnosis. If left untreated, LGG widely invades the brain and becomes malignant, generating neurological worsening and ultimately death. Early and repeat treatments for this incurable tumor, including maximal connectome-based surgical resection(s) in awake patients, enable postponement of malignant transformation while preserving quality of life owing to constant neural network reconfiguration. Due to considerable interindividual variability in terms of LGG course and consecutive cerebral reorganization, a multistage longitudinal strategy should be tailored accordingly in each patient. It is crucial to predict how the glioma will progress (changes in growth rate and pattern of migration, genetic mutation, etc.) and how the brain will adapt (changes in patterns of spatiotemporal redistribution, possible functional consequences such as epilepsy or cognitive decline, etc.). The goal is to anticipate therapeutic management, remaining one step ahead in order to select the optimal (re-)treatment(s) (some of them possibly kept in reserve), at the appropriate time(s) in the evolution of this chronic disease, before malignization and clinical worsening. Here, predictive tumoral and non-tumoral factors, and their ever-changing interactions, are reviewed to guide individual decisions in advance based on patient-specific markers, for the treatment of LGG.

Use of Plain Radiography of Uninjured Wrists as Patient-Specific Markers of Successful Reduction of Unilateral Distal Radius Fractures.

The standard of care of distal radius fractures requiring operative intervention involves restoration of anatomical alignment radiologically by comparing preoperative films and intraoperative fluoroscopy with established values based on population norms. The objective of this study is to evaluate the use of plain radiographs obtained from the uninjured wrist of patients who present with unilateral displaced distal radius fractures as a measure of successful achievement of anatomical realignment. A retrospective review was performed on 133 consecutive patients who presented from August 2020 to August 2021 with a diagnosis of unilateral distal radius fracture as confirmed on 3-view plain radiography. Patients who had bilateral radiographs and underwent open reduction and internal fixation were included. The primary outcome measure was comparison of radial inclination, radial height, tilt, and ulnar variance measured by 3 observers on preoperative, 1-week postoperative, and uninjured contralateral wrist films. Twenty-one patients were included for analysis. Comparison of postoperative radiologic parameters with the contralateral uninjured extremity revealed a mean radial inclination difference of 3.8°, radial height difference of 2.0 mm, volar tilt difference of 6.3°, and ulnar variance difference of 0.9 mm. The average postreduction radial height was found to deviate from contralateral radial height significantly more than from the historic radial height parameter (2.0 vs 0.6 mm, P < .001). Attempts at achieving distal radius fracture reduction to within historical normal limits may result in an increased deviation from patient-specific anatomical parameters, especially with respect to radial height.

Radiographic Bone Loss and Its Relation to Patient-Specific Risk Factors, LDL Cholesterol, and Vitamin D: A Cross-Sectional Study.

The influence of patient-specific factors such as medical conditions, low-density lipoprotein cholesterol (LDL-C) or levels of 25-hydroxyvitamin D (25OHD) on periodontal diseases is frequently discussed in the literature. Therefore, the aim of this retrospective cross-sectional study was to evaluate potential associations between radiographic bone loss (RBL) and patient-specific risk factors, particularly LDL-C and 25OHD levels. Patients from a dental practice, who received full-mouth cone beam CTs (CBCTs) and blood-sampling in the course of implant treatment planning, were included in this study. RBL was determined at six sites per tooth from CBCT data. LDL-C and 25OHD levels were measured from venous blood samples. Other patient-specific risk factors were assessed based on anamnesis and dental charts. Statistical analysis was performed applying non-parametric procedures (Mann–Whitney U tests, error rates method). Data from 163 patients could be included in the analysis. RBL was significantly higher in male patients, older age groups, smokers, patients with high DMFT (decayed/missing/filled teeth) score, lower number of teeth, and high LDL-C levels (≥160 mg/dL). Furthermore, patients with high 25OHD levels (≥40 ng/mL) exhibited significantly less RBL. In summary, RBL was found to be associated with known patient-specific markers, particularly with age and high LDL-C levels.

Hemodynamic Assessment in Bicuspid Aortic Valve Disease and Aortic Dilation: New Insights From Voxel-By-Voxel Analysis of Reverse Flow, Stasis, and Energetics.

Objectives: Clinical management decisions surrounding ascending aorta (AAo) dilation in bicuspid aortic valve (BAV) disease benefit from personalized predictive tools. 4D-flow MRI may provide patient-specific markers reflective of BAV-associated aortopathy. This study aims to explore novel 4D-flow MRI parametric voxel-by-voxel forward flow, reverse flow, kinetic energy and stasis in BAV disease. We hypothesize that novel parametric voxel-by-voxel markers will be associated with aortic dilation and referral for surgery and can enhance our understanding of BAV hemodynamics beyond standard metrics. Methods: A total of 96 subjects (73 BAV patients, 23 healthy controls) underwent MRI scan. Healthy controls had no known cardiovascular disease. Patients were clinically referred for AAo dilation assessment. Indexed diameters were obtained by dividing the aortic diameter by the patient’s body surface area. Patients were followed for the occurrence of aortic surgery. 4D-flow analysis was performed by a single observer in five regions: left ventricular outflow tract (LVOT), AAo, arch, proximal descending aorta (PDAo), and distal descending aorta (DDAo). In each region peak velocity, kinetic energy (KE), forward flow (FF), reverse flow (RF), and stasis were measured on a voxel-by-voxel basis. T-tests (or non-parametric equivalent) compared flow parameters between cohorts. Univariate and multivariate analyses explored associations between diameter and parametric voxel-by-voxel parameters. Results: Compared to controls, BAV patients showed reduced stasis (p < 0.01) and increased RF and FF (p < 0.01) throughout the aorta, and KE remained similar. In the AAo, indexed diameter correlated with age (R = 0.326, p = 0.01), FF (R = −0.648, p < 0.001), RF (R = −0.441, p < 0.001), and stasis (R = −0.288, p < 0.05). In multivariate analysis, FF showed a significant inverse association with AAo indexed diameter, independent of age. During a median 179 ± 180 days of follow-up, 23 patients (32%) required aortic surgery. Compared to patients not requiring surgery, they showed increased KE and peak velocity in the proximal aorta (p < 0.01), accompanied by increased RF and reduced stasis throughout the entire aorta (p < 0.01). Conclusion: Novel voxel-by-voxel reverse flow and stasis were altered in BAV patients and are associated with aortic dilation and surgical treatment.

Analytical Quality Controls for ddPCR Detection of Minimal Residual Disease in Acute Lymphoblastic Leukemia.

Droplet digital PCR (ddPCR) is a promising technique for absolute quantification of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), but there is no comprehensive quality assurance program to enable its application in clinical laboratories. Current guidelines for real-time quantitative PCR (qPCR) assays targeting immunoglobulin/T-cell receptor (Ig/TCR) gene rearrangements needed adaptation for ddPCR to cover droplet generation, intraassay variation, and interassay variation in the absence of standard curves. Six qPCR MRD assays for Ig/TCR gene rearrangements and a standard albumin control gene assay were migrated to a ddPCR platform and used to test 82 remission samples from 6 patients with ALL. Three analytical quality controls (QC) were developed and evaluated for ddPCR MRD detection. Analytical QC for droplet number generation (DN-QC), for albumin ddPCR assay performance (Alb-QC) and for patient-specific marker assay performance (PS-QC) were established with pass/fail limits and corresponding QC rules. Compared to established qPCRs, the ddPCR assays had comparable sensitivity and quantitative range. Overall, there was close agreement (91%) of MRD results between qPCR and ddPCR (κ = 0.86, P < 0.0001) and stronger concordance in 32 quantifiable samples (R2 = 0.97, P < 0.0001). The use of this newly developed quality control system for ddPCR MRD testing avoids the need to repeat standard curves and provides reliable results comparable to standardized qPCR methods for MRD detection in ALL.

A scoring system for predicting individual treatment effects of statins in type 2 diabetes patients on haemodialysis.

Haemodialysis patients have high cardiovascular disease risk. Although statins reduce this risk in chronic kidney disease, randomised trials in haemodialysis patients show no benefit. Post-hoc analyses of the German Diabetes Dialysis (4D) study identified patient-specific markers associated with heterogeneous treatment effects. We combined these markers to develop a score for predicting individual effects of statins in these patients. We used data from the 4D study, enrolling 1255 haemodialysis patients with type 2 diabetes mellitus, randomised to atorvastatin or placebo and followed for a composite cardiovascular endpoint. We calculated two scores: score 1 based on all 23 predictive markers and score 2 based on 17 clinically accessible markers. Groups stratified by score 1 showed differential treatment effects: for score <26 (458 patients; 36%), the hazard ratio (95% confidence interval) was 1.54 (1.16-2.03), suggesting harm; for 26-31 (331 patients; 26%), it was 1.03 (0.72-1.48), suggesting a neutral effect; and for >31 (466 patients; 38%), it was 0.43 (0.30-0.60), suggesting a benefit. Statins also significantly reduced all-cause mortality in the benefit group. Stratification by score 2 yielded similar results but a smaller group gaining benefit (360 patients). Statin effects in haemodialysis patients can be predicted by markers associated with plausible relevant mechanisms including cholesterol metabolism, atherosclerosis, protein energy wasting, or competing risks. In clinical practice, the score could aid in risk stratification, not only to select patients who benefit from statins but also to identify those whom treatment could harm.

Design of a liver cancer-specific selector for the analysis of circulating tumor DNA.

Circulating tumor DNA (ctDNA) has been frequently investigated to monitor tumor dynamics and measure tumor burden. This non-invasive method concerning ctDNA has been recognized as a promising biomarker. Recently, next generation sequencing has been used in ctDNA detection by researchers. However, those reports have been limited by modest sensitivity, and only a minority of patients with cancer were applicable. Additionally, a limited number of cases of liver cancer have been analyzed. A more precise method is required to be established to evaluate ctDNA noninvasively. In the present study, a novel method to design a liver cancer-associated chip region (spanning 211 kb, containing 159 genes) was performed with high specificity using International Cancer Genome Consortium datasets. Following evaluation with datasets from The Cancer Genome Atlas and data from 3 patients with liver cancer, the selected regions were demonstrated to be beneficial to locate specific somatic mutations associated with liver cancer therapy and to monitor cancer dynamics in the plasma samples of the patients. In addition to establishing performance benchmarks supporting direct clinical use, the chip designed and the high-resolution sequencing analyses pipeline would allow the development a set of patient specific markers that could monitor the process of cancer with high accuracy and low cost. Furthermore, the present study is essential to understanding the dynamics and providing insight into the basic mechanisms of liver cancer.

CLINICAL PROGNOSTIC MARKERS FOR PREDICTING VASOVAGAL SYNCOPE REMISSION

CLINICAL PROGNOSTIC MARKERS FOR PREDICTING VASOVAGAL SYNCOPE REMISSION

Prospective Molecular Monitoring of Minimal Residual Disease after Non-Myeloablative Allografting in Newly Diagnosed Multiple Myeloma

Background Evaluation of minimal residual disease (MRD) by molecular methods is used as a surrogate of potential eradication of myeloma cells. Prolonged high-rates of molecular remission were reported after myeloablative allografting. However, recent studies on consolidation therapy after an autograft reported 18% of PCR negativity by qualitative nested PCR and a molecular response of 63% by quantitative RQ-PCR (Ferrero et al. Leukemia 2014). The aim of our study was that of evaluating depth of response by molecular methods induced by graft-vs.-myeloma following tandem autologous/non-myeloablative allografting in newly diagnosed myeloma.Patients and Methods. Twenty-six patients enrolled in prospective clinical trials (ClinicalTrial.gov Identifier: NCT-00702247, and NCT01264315) with a diagnostic bone marrow (BM) specimen suitable for immunoglobulin heavy-chain gene rearrangement sequencing were evaluated for MRD by qualitative nested PCR and quantitative RQ-PCR. A patient specific marker was generated for 19/26 (73%). Sensitivity of nested PCR was 3 neoplastic rearrangements in 106 normal cells while each RQ-PCR showed a standard curve correlation coefficient of at least 0.95 with a slope of 3.0 to 3.9 with minimum sensitivity of 10-5 (according to Euro-MRD criteria). Transplant plan consisted of a standard autograft (melphalan 200 mg/m2) followed by 200 cGy TBI and an allograft. Post-transplant donor lymphocyte infusions or maintenance/consolidation with new drugs were not allowed until clinical relapse. BM samples were collected at diagnosis, after the autograft, at month 1, 3, 6 after the allograft and then every 6 months or as clinically indicated. MRD negativity by nested-PCR or by RQ-PCR was defined as al least two consecutive samples scored negative.Results. At a median follow-up of 11.9 years (6.1-15.2) from diagnosis and 11.0 years (5.0-14.2) from the allograft, median overall survival (OS) for the entire patient cohort was not reached and median event-free survival (EFS) was 4 years from the allograft. Overall transplant-related mortality was 11.5% at 1 year and 15.4% at 5 years. Relapse was 3.8% at 1 year, 30.8% at 3 years and 34.6% at 5 years. Cumulative incidence of acute II-IV GVHD was 26.9%. At follow up, only 2 patients were on immunosuppression for limited chronic GVHD. In the 19/26 (73%) patients with a molecular marker, the rate of nested-PCR negativity remained low at month 3 (3/19, 16%) after the allograft and gradually increased at month 6 and 12 up to 44% (8/18) and 47% (7/15) respectively. By RQ-PCR analysis, the overall tumor reduction of the whole treatment was 13.80 ln and progressively increased through the autograft, the allograft and the post-transplant period (p<0.001). After the autograft a median reduction of 4.59 ln and one month after the allograft a further decrease of 4.83 ln were observed. At month 3, MRD levels were similar to those at month 1 whereas a further decrease of 4.61 ln was observed at month 6 suggesting progressive graft-vs.-myeloma effect. At 2 years, the overall molecular response by RQ-PCR was 63%. Overall, 8 relapses occurred. Five/8 in the 11 patients who never showed a molecular response; 1 in a patient negative by RQ-PCR but positive by nested PCR. In those who reached nested PCR negativity, 1 showed molecular relapse two years before clinical relapse and 1 relapsed 8 months after the last PCR-negative sample. At median follow up, median OS and EFS were not reached in patients who achieved nested PCR negativity whereas median OS and EFS were 3.3 years and 1.5 years in the remaining patients (p 0.023 and p 0.009). Similarly, median OS and EFS were not reached in all patients who achieved a molecular response by RQ-PCR as compared with those who did not (p 0.046 and p 0.025).Conclusions.Graft-vs-myeloma after non-myeloablative allografting reported prolonged rates of qualitative (nested PCR) molecular remissions similar to those after myeloablative allografting and higher than the recently reported combination of autografting and consolidation with anti-myeloma agents. Quantitative RQ-PCR analysis showed a deeper tumor reduction after non-myeloablative allografting than consolidation with new drugs. In the light of our results, it may be ethical to evaluate the combination of graft-vs-myeloma with newer anti-myeloma agents on larger series of young high risk and early relapsed patients where life expectancy is poor also in the era of new drugs. DisclosuresNo relevant conflicts of interest to declare.

Abstract 5461: The role of gene body cytosine modifications in MGMT expression and sensitivity to temozolomide

Abstract The goal of precision medicine is to use patient-specific markers to design individualized treatment strategies. Promoter methylation of O6-methylguanine-DNA methyltransferase (MGMT), which encodes for the DNA repair protein MGMT, has been shown to predict glioblastoma patient survival after treatment with temozolomide. Unfortunately, there is no approved therapeutic alternative for patients whose tumors have an unmethylated MGMT promoter and are unlikely to respond to temozolomide treatment. Promoter methylation status of MGMT is used to predict expression levels of MGMT, despite some observed discordance between promoter methylation and protein levels. We hypothesized that incorporation of MGMT gene body cytosine modification levels in models of temozolomide response may lead to better prediction of MGMT expression levels and, more importantly, improved methods of prediction for glioblastoma patient response to temozolomide. Using 91 human glioblastoma samples from The Cancer Genome Atlas, we observed significant variation in MGMT expression levels in patients with an unmethylated promoter, with higher levels of gene body cytosine modification correlating with higher gene expression levels. Furthermore, inducing hypomethylation across the MGMT gene body with decitabine resulted in decreased levels of MGMT gene expression in lymphoblastoid and glioblastoma cell lines. These data indicate an important functional role for gene body cytosine modifications in maintaining gene expression levels. We reasoned that the decrease in MGMT expression induced by decitabine might render resistant glioblastoma cell lines more sensitive to temozolomide. Indeed, MGMT-expressing glioblastoma cell lines pre-treated with decitabine became significantly more sensitive to temozolomide-induced cytotoxicity. Overall, our results demonstrate a functional role for gene body cytosine modification in regulating gene expression of MGMT and suggest that patients with an unmethylated MGMT promoter could benefit from pre-treatment with decitabine to increase tumor sensitivity to temozolomide. Citation Format: Erika L. Moen, Amy L. Stark, Wei Zhang, M. Eileen Dolan, Lucy A. Godley. The role of gene body cytosine modifications in MGMT expression and sensitivity to temozolomide. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5461. doi:10.1158/1538-7445.AM2014-5461

Monitoring residual disease in the ph-negative myeloproliferative neoplasms post-allogeneic stem cell transplantation: more mutations and more methodologies.

Monitoring residual disease in the ph-negative myeloproliferative neoplasms post-allogeneic stem cell transplantation: more mutations and more methodologies.

Monitoring of residual disease by next-generation deep-sequencing of RUNX1 mutations can identify acute myeloid leukemia patients with resistant disease

We studied the utility and clinical relevance of RUNX1 (runt-related transcription factor 1) mutations and their application as residual disease detection markers using next-generation deep-sequencing. Mutation screening was prospectively performed in 814 acute myeloid leukemia patients. At diagnosis, 211/814 (25.9%) patients harbored mutations with a median clone size of 39% (range: 2-96%). Furthermore, in 57 patients paired samples from diagnosis and relapse were analyzed. In 47/57 (82.5%) cases the same alterations detected at diagnosis were present at relapse, whereas in 1/57 (1.8%) cases the mutation from the diagnostic sample was no longer detectable. Discrepancies were observed in 9/57 (15.8%) cases, also including the occurrence of novel RUNX1 mutations not restricted to those regions affected at diagnosis. Moreover, in 103 patients the prognostic impact of residual levels of RUNX1 mutations during complete remission was studied. Separation of patients according to median residual mutation burden into 'good responders' and 'poor responders' (median: 3.61%; range: 0.03-48.0%) resulted in significant differences of both event-free (median 21.0 vs. 5.7 months, P<0.001) and overall survival (OS; median 56.9 vs. 32.0 months, P=0.002). In conclusion, deep-sequencing revealed that RUNX1 mutations qualify as patient-specific markers for individualized disease monitoring. The measurement of mutation load may refine the assignment into distinct risk categories and treatment strategies.

The Landscape of RUNX1 Mutations in Acute Myeloid Leukemia: Investigations On Stability of Mutations At Relapse and Utility As a Marker for Minimal Residual Disease

AbstractAbstract 657 Introduction:RUNX1 mutations constitute disease-defining aberrations in acute myeloid leukemia (AML) and were demonstrated to be particularly frequent in secondary and de novo AML with normal karyotype or non-complex alterations and to confer an unfavorable prognosis. Monitoring minimal residual disease (MRD) in AML has been shown to provide prognostic information and is increasingly used for treatment decisions. A variety of molecular markers has been identified suitable for MRD assessment, yet there still is a lack of such markers in a significant number of patients. The use of RUNX1 mutations may bridge a gap. Aims:1.Evaluate the stability of RUNX1 mutations between diagnosis and relapse.2.Test the utility and clinical relevance of RUNX1 mutations as MRD marker. Patients and Methods:RUNX1 mutation screening was prospectively performed in 814 patients with AML at diagnosis (645 de novo, 109 s-AML, and 60 t-AML). The median age of the patients was 69.6 years (range: 1 – 93 years), including 375 female and 439 male patients, respectively. 50.5% (411/814) of cases presented with a normal karyotype, 38.8% (316/814) with non-complex cytogenetic alterations, 9.6% (78/814) with a complex aberrant karyotype, and 1.1% (9/814) with prognostically favorable cytogenetics. Mutation analysis was performed using a sensitive next-generation amplicon deep-sequencing assay (454 Life Sciences, Branford, CT). Moreover, in a subset of 44 AML patients and additional 59 retrospectively analyzed cases the prognostic impact of MRD levels of RUNX1 mutations was studied at a second time point after completion of intensive induction therapy (median sampling interval: 128 days after diagnosis; range 60 – 180 days). In these follow-up samples the RUNX1 mutations already detected at diagnosis were investigated with a higher coverage (835-fold median coverage) as compared to the diagnostic assessment (759-fold median coverage) resulting in a sensitivity level of 1%. Furthermore, in 57 patients paired samples from diagnosis and relapse were analyzed to assess the stability of RUNX1 mutations. Results:211/814 patients (25.9%) were detected to carry RUNX1 mutations. The median clone size was 39% and revealed a significant heterogeneity ranging from 2% to 96%. 73.9% (156/211) of mutated patients carried one mutation only, whereas 26.1% (55/211) harbored two (n=46) or more (n=9) mutations. In detail, the 211 patients harbored a total number of 275 alterations in RUNX1: 42.5% (117/275) frame-shift mutations, 34.9% (96/275) missense, 14.2% (39/275) nonsense, 4.4% (12/275) exon-skipping/splicing, and 4.0% (11/275) in-frame insertion/deletion alterations, respectively. Regarding MRD assessment, patients were separated according to the median MRD level (3.92%; range 0.03% - 48.00%) into “good responders” (n=78) with MRD levels below 3.92% and “poor responders” (n=25) with MRD levels above 3.92%. This resulted in significant differences in both event-free survival (median 21.4 vs 5.7 months, p<0.001) and overall survival (73.3% vs 66.1% at 2 years, p=0.016). Moreover, in 57 cases the stability of individual RUNX1 mutations was studied at the time of relapse. In 46/57 (80.7%) cases the same alterations detected at diagnosis were present at relapse, whilst in 2/57 (3.5%) cases the RUNX1 mutation from the diagnostic sample was no longer detectable at relapse. Importantly, in 7/57 (12.3%) patients novel RUNX1 mutations were detected in regions different from those affected at diagnosis. Conclusion:Next-generation deep-sequencing accurately detects and quantifies RUNX1 mutations in AML with high sensitivity. RUNX1 mutations qualify as patient-specific markers for individualized disease monitoring. Thus, the measurement of mutation load by next-generation sequencing may contribute to refine the assignment into distinct risk categories in AML. Analysis of RUNX1 mutations should be considered for the complete coding region at relapse to detect new RUNX1 mutations. Disclosures:Kohlmann:MLL Munich Leukemia Laboratory: Employment; Roche Diagnostics: Honoraria. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership.

FAK, CD44v6, c-Met and EGFR in colorectal cancer parameters: tumour progression, metastasis, patient survival and receptor crosstalk

Research for reliable and patient-specific markers in colorectal cancer (CRC) is based on solid evidence that staging alone is not informative enough. Employing four cellular receptors, we embarked to identify aggressive tumour behaviour and impact of surrogate marker expression on patient prognosis. One-hundred eighty-three CRC patients were enrolled in our investigation that focused on an array of biological markers, namely epidermal growth factor receptor (EGFR), c-Met, focal adhesion kinase (FAK) and CD44v6. Tissue samples, clinicopathological data and patient's follow-up information were collected, and immunohistochemical assays evaluated the levels of the aforementioned molecules. All available data were correlated with tumour grade, stage, patient age, gender and survival. Expression of all receptors correlated closely with tumour stage (P < 0.01) exhibiting a connection with cancer's invasiveness and progress. Survival also proved to depend significantly on molecular expression (log-rank test for Kaplan-Meier; EGFR P = 0.030, c-Met P = 0.050, FAK P < 0.001, CD44v6 P < 0.001). Stage, FAK and CD44v6 emerged as independent predictors of survival in a stepwise regression analysis (FAK P = 0.001 Exp(B) = 2.517, 95% confidence interval (CI) = 1.704-5.831 and CD44v6 P = 0.005, Exp(B) = 2.299, 95% CI = 1.287-4.110). T-stage, nodal metastasis, all metastatic types (N/M) and size correlated with at least one of the receptors or their co-expression. Notably, increased staining for each receptor was followed by statistically significant expression elevation of at least one of the other markers. Our results suggest that the selected cellular receptors are suitable for use as biomarkers of survival and tumour progression in CRC. Furthermore, we provide additional evidence for receptor interaction, properly clarifying their importance, which could potentially lead to more effective anti-CRC regimens.

Decreased lactate concentration and glycolytic enzyme expression reflect inhibition of mTOR signal transduction pathway in B-cell lymphoma.

The application of kinase inhibitors in cancer treatment is growing rapidly. However, methods for monitoring the effectiveness of the inhibitors are still poorly developed and currently rely mainly on the tracking of changes in the tumor volume, a rather late and relatively insensitive marker of the therapeutic response. In contrast, MRS can detect changes in cell metabolism and has the potential to provide early and patient-specific markers of drug activity. Using human B-cell lymphoma models and MRS, we have demonstrated that the inhibition of the mTOR signaling pathway can be detected in malignant cells in vitro and noninvasively in vivo by the measurement of lactate levels. An mTOR inhibitor, rapamycin, suppressed lactic acid production in lymphoma cell line cultures and also diminished steady-state lactate levels in xenotransplants. The inhibition was time dependent and was first detectable 8 h after drug administration in cell cultures. In xenotransplants, 2 days of rapamycin treatment produced significant changes in lactic acid concentration in the tumor measured in vivo, which were followed by tumor growth arrest and tumor volume regression. The rapamycin-induced changes in lactate production were strongly correlated with the inhibition of expression of hexokinase II, the key enzyme in the glycolytic pathway. These studies suggest that MRS or (18) F-fluorodeoxyglucose positron emission tomography (FDG PET) detection of changes in glucose metabolism may represent effective noninvasive methods for the monitoring of mTOR targeting therapy in lymphomas and other malignancies. Furthermore, the measurement of glucose metabolic inhibition by MRS or FDG PET imaging may also prove to be effective in monitoring the efficacy of other kinase inhibitors given that the rapamycin-sensitive mTOR lies downstream of many oncogenic signaling pathways.

NCI First International Workshop on the Biology, Prevention, and Treatment of Relapse after Allogeneic Hematopoietic Stem Cell Transplantation: Report from the Committee on Disease-Specific Methods and Strategies for Monitoring Relapse Following Allogeneic Stem Cell Transplantation. Part II: Chronic Leukemias, Myeloproliferative Neoplasms, and Lymphoid Malignancies

Relapse has become the major cause of treatment failure after allogeneic hematopoietic stem cell transplantation. Outcome of patients with clinical relapse after transplantation generally remains poor, but intervention prior to florid relapse improves outcome for certain hematologic malignancies. To detect early relapse or minimal residual disease, sensitive methods such as molecular genetics, tumor-specific molecular primers, fluorescence in situ hybridization (FISH), and multiparameter flow cytometry (MFC) are commonly used after allogeneic stem cell transplantation to monitor patients, but not all of them are included in the commonly employed disease-specific response criteria. The highest sensitivity and specificity can be achieved by molecular monitoring of tumor- or patient-specific markers measured by polymerase chain reaction-based techniques, but not all diseases have such targets for monitoring. Similar high sensitivity can be achieved by determination of recipient-donor chimerism, but its specificity regarding detection of relapse is low and differs substantially among diseases. Here, we summarize the current knowledge about the utilization of such sensitive monitoring techniques in chronic leukemias, myeloproliferative neoplasms, and lymphoid malignancies based on tumor-specific markers and cell chimerism and how these methods might augment the standard definitions of posttransplant remission, persistence, progression, relapse, and the prediction of relapse. Critically important is the need for standardization of the different residual disease techniques and to assess the clinical relevance of minimal residual disease and chimerism surveillance in individual diseases, which in turn must be followed by studies to assess the potential impact of specific interventional strategies.

NCI First International Workshop on the Biology, Prevention, and Treatment of Relapse after Allogeneic Hematopoietic Stem Cell Transplantation: Report from the Committee on Disease-Specific Methods and Strategies for Monitoring Relapse following Allogeneic Stem Cell Transplantation. Part I: Methods, Acute Leukemias, and Myelodysplastic Syndromes

Relapse has become the major cause of treatment failure after allogeneic stem cell transplantation. Outcome of patients with clinical relapse after transplantation generally remains poor, but intervention prior to florid relapse improves outcome for certain hematologic malignancies. To detect early relapse or minimal residual disease, sensitive methods such as molecular genetics, tumor-specific molecular primers, fluorescein in situ hybridization, and multiparameter flow cytometry (MFC) are commonly used after allogeneic stem cell transplantation to monitor patients, but not all of them are included in the commonly employed disease-specific response criteria. The highest sensitivity and specificity can be achieved by molecular monitoring of tumor- or patient-specific markers measured by polymerase chain reaction-based techniques, but not all diseases have such targets for monitoring. Similar high sensitivity can be achieved by determination of donor chimerism, but its specificity regarding detection of relapse is low and differs substantially among diseases. Here, we summarize the current knowledge about the utilization of such sensitive monitoring techniques based on tumor-specific markers and donor cell chimerism and how these methods might augment the standard definitions of posttransplant remission, persistence, progression, relapse, and the prediction of relapse. Critically important is the need for standardization of the different residual disease techniques and to assess the clinical relevance of minimal residual disease and chimerism surveillance in individual diseases, which in turn, must be followed by studies to assess the potential impact of specific interventional strategies.