Monitor Tumor Burden Research Articles

Discovery of methylated circulating DNA biomarkers for comprehensive non-invasive monitoring of treatment response in metastatic colorectal cancer

ObjectiveMutations in cell-free circulating DNA (cfDNA) have been studied for tracking disease relapse in colorectal cancer (CRC). This approach requires personalised assay design due to the lack of universally mutated...

Circulating tumor DNA shows variable clonal response of breast cancer during neoadjuvant chemotherapy.

Circulating tumor DNA (ctDNA) correlates with tumor burden and provides early detection of treatment response and tumor genetic alterations in breast cancer (BC). In this study, we aimed to identify genetic alterations during the process of tumor clonal evolution and examine if ctDNA level well indicated clinical response to neoadjuvant chemotherapy (NAC) and BC recurrence.We performed targeted ultra-deep sequencing of plasma DNAs, matched germline DNAs and tumor DNAs from locally advanced BC patients. Serial plasma DNAs were collected at diagnosis, after the 1st cycle of NAC and after curative surgery. For the target enrichment, we designed RNA baits covering a total of ∼202kb regions of the human genome including a total of 82 cancer-related genes.For ctDNA, 15 serial samples were collected and 87% of plasma SNVs were detected in 13 BC samples that had somatic alterations in tumor tissues. The TP53 mutation was most commonly detected in primary tumor tissues and plasma followed by BRCA1 and BRCA2. At BC diagnosis, the amount of plasma SNVs did not correlate with clinical stage at diagnosis. With respect to the therapeutic effects of NAC, we found two samples in which ctDNA disappeared after the 1st NAC cycle achieved a pathologic complete response (pCR). In addition, the amount of ctDNA correlated with residual cancer volume detected by breast MRI.This targeted ultra-deep sequencing for ctDNA analysis would be useful for monitoring tumor burden and drug resistance. Most of all, we suggest that ctDNA could be the earliest predictor of NAC response.

Tumor DNA: an emerging biomarker in head and neck cancer.

Head and neck cancer (HNC) includes a diverse range of malignancies arising commonly from mucosal epithelia of the upper aerodigestive tract. Head and neck squamous cell carcinoma (HNSCC), the most common form of HNC, develops in the oral cavity, pharynx, and larynx and is associated with tobacco exposure, alcohol abuse, and infection with oncogenic viruses. Despite global advances in cancer care, HNSCC often presents with advanced disease and is associated with poor 5-year survival of ~50%. Genotyping tumor tissue to guide clinical decision-making is becoming commonplace in modern oncology, but in the management of HNSCC, tissue biopsies with cytopathology or histopathology remain the mainstay for diagnosis. Furthermore, conventional biopsies are temporally and spatially limited, often providing a brief snapshot of a single region of a heterogeneous tumor. In the absence of a useful biomarker, both primary and recurrent HNSCCs are diagnosed with conventional imaging and clinical examination. As a result, many patients are diagnosed with advanced disease. Tumor DNA is an emerging biomarker in HNSCC. DNA fragments are constantly being shed from tumors and metastatic lesions, and can therefore be detected in blood and other bodily fluids. Utilizing next-generation sequencing techniques, these tumor DNA can be characterized and quantified. This can serve as a minimally invasive liquid biopsy allowing for specific tumor profiling, dynamic tumor burden monitoring, and active surveillance for disease recurrences. In HNSCC, analysis of tumor DNA has the potential to enhance tumor profiling, aid in determining patient prognosis, and guide treatment decisions.

Abstract 3834: Tracking CAD-ALK gene translocation in urine and plasma of a colorectal cancer patient treated with ALK blockade

Abstract A metastatic colorectal cancer (mCRC) patient carrying CAD-ALK translocation achieved partial response to an experimental ALK inhibitor and then progressed after 5 months. We studied whether urine cell-free, trans-renal DNA (tr-DNA) could be used to monitor tumor burden and patient’s response. A NGS panel was developed to interrogate 52 common cancer gene rearrangements and 14 frequently mutated genes in cancer patients. A TP53 p.R248W mutation and the CAD-ALK genomic breakpoint (rearrangement) were identified in the tumor tissue and matched plasma circulating tumor DNA (ctDNA) Urine samples were longitudinally obtained from the patient during ALK inhibitor treatment in parallel with blood. To detect the CAD-ALK translocation in urine tr-DNA we designed ultra-short (51 bp amplicon) primer pairs spanning the genomic breakpoint as a unique tumor marker. This approach allowed the non-invasive monitoring of the gene fusion in urine with amounts paralleling tumor burden. Of note, CAD-ALK gene fusion was apparent in urine tr-DNA before radiological confirmation of disease progression. The same strategy was applied to plasma ctDNA and the results were compared. To detect point mutations in urine tr-DNA, we exploited a peptide nucleic acids (PNA)-CLAMP PCR coupled with droplet digital PCR (ddPCR) analysis to specifically suppress amplification of wild-type DNA fragments. Custom PNA probes were designed for TP53 codon 248, and a ddPCR assay was optimized to detect the TP53 p.R248W mutation, which was then identified in all urine tr-DNA samples, with absolute copies correlating with tumor burden throughout ALK inhibitor treatment. In conclusion, we find that urine tr-DNA can be exploited to non-invasively monitor tumor burden by detecting tumor-specific gene fusions as well as point mutations. Citation Format: Giulia Siravegna, Andrea Sartore-Bianchi, Benedetta Mussolin, Laura Palmieri, Federica Tosi, Andrea Cassingena, Luca Novara, Michela Buscarino, Giorgio Corti, Giovanni Crisafulli, Alice Bartolini, Mark Erlander, Federica Di Nicolantonio, Salvatore Siena, Alberto Bardelli. Tracking CAD-ALK gene translocation in urine and plasma of a colorectal cancer patient treated with ALK blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3834. doi:10.1158/1538-7445.AM2017-3834

Abstract 4741: Biomarker significance of plasma and tumor miR-21, miR-221, and miR-106a in pediatric osteosarcoma

Abstract Osteosarcoma is the most common malignant bone tumor in children and young adults. Despite the use of surgery and multi-agent chemotherapy, osteosarcoma patients who respond poorly towards chemotherapy or develop relapses have a dismal outcome. Identification of biomarkers for active disease may help to monitor tumor burden, detect early relapses, and predict prognosis in these patients, so that they can be treated as early as possible to improve their survival. In this study, we examined if circulating microRNAs (miRNAs) can be used as biomarkers in osteosarcoma patients. We performed genome-wide miRNA profiling on a discovery cohort of OS plasma samples (n=32) collected from the Texas Children’s Hospital. A total of 56 miRNAs were upregulated and 164 miRNAs were downregulated in osteosarcoma samples when compared to normal plasma samples (n=8). miRNA-21, miR-221 and miRNA-106a were selected for further validation based on their known biological importance. We showed that all three circulating miRNAs were expressed significantly higher in OS samples (n=29) than normal samples (n=17) in an independent cohort obtained from the Children’s Oncology Group. Furthermore, we demonstrated that miR-21 was expressed significantly higher in osteosarcoma tumors (n=89) compared with normal bone controls. More importantly, lower expressions of miR-21 and miR-221, but not miR-106a, significantly correlated with a poor outcome. In conclusion, our results indicate that miR-21, miR-221 and miR-106a are elevated in the circulation of osteosarcoma patients, whereas tumor expressions of miR-21 and miR-221 are prognostically significant. Further investigation of the miRNAs may lead to a better prognostic method and potential miRNA therapeutics for osteosarcoma. Citation Format: Manjula Nakka, Wendy Allen-Rhoades, Yiting Li, Aaron Kelly, Jianhe Shen, Aaron Taylor, Donald A. Barkauskas, Jason Yustein, Irene Andrulis, Jay Wunder, Richard Gorlick, Paul Meltzer, Ching Lau, Tsz-Kwong Man. Biomarker significance of plasma and tumor miR-21, miR-221, and miR-106a in pediatric osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4741. doi:10.1158/1538-7445.AM2017-4741

Abstract 4867: Exome- and whole genome sequencing for clinical evaluation and precision medicine in neuroblastoma

Abstract Background. Despite major progress in treatment of pediatric cancer, aggressive neuroblastoma (NB) still constitutes a major clinical problem. Currently, the event free survival of high-risk NB is about 40% implicating that 60% either don’t go into full remission or falls into relapse. Most relapses have an acquired drug resistance meaning that conventional treatment options may not work. Therefore, further studies of this group are of great importance. Massive parallel sequencing has now become a valuable tool in both cancer research and in clinical evaluation as it provides valuable information regarding patient specific alterations that might be used in targeted therapy. Material and Methods. DNA from 54 patients in total have been subjects for massive parallel sequencing; 16 tumor/normal pairs and 27 single tumors have been analyzed using exome sequencing, 8 tumor/normal pairs with whole genome sequencing and three using both exome and whole genome sequencing. Exome sequences were mapped using BWA with GATK realignment followed by variant calling through SNPeff and copy number analysis trough ControlFreec. Whole genome data were analyzed using the CLC Genomic Workbench with annotation and filtering done in Ingenuity variant analysis software. Results. On average, 14 somatic protein changing single nucleotide variants were detected per patient (range 2-71) with recurrent alterations mainly detected mainly in ALK. Predicted deleterious germ line alterations in well-established cancer genes were detected in three patients. Structural variants included genes such as ATRX and TERT, as described by others previously, but also novel homozygous focal deletions at chromosome 19. Shortest regions of overlapping deletions of this chr19 region include seven gens whereof CIC (Capicua) might be the most interesting in a cancer context. Copy number analysis of both exome- and whole genome sequencing gives genomic profiles that are comparable to the genome profiles generated from the SNP-microarrays that current are in clinical use. Specific breakpoints for structural variants could be detected in all sample subjects for whole genome sequencing as well as for all for all MYCN amplified neuroblastomas that subjects for exome sequencing. Conclusions. Exome- and whole genome sequencing can be used for clinical evaluation of NB providing information regarding both constitutional and tumor specific alterations. The information can be used for clinical decision of patient specific therapeutic options such as ALK- or CDK4/6 inhibitors, but can also in rare cases give information regarding which treatments not to use. Furthermore, whole genome sequencing also gives the possibility to identify tumor specific structural alterations such as translocation breakpoints that can be used to monitor tumor burden in liquid biopsies. Citation Format: Angela Martinez Monleon, Susanne Reinsbach, Niloufar Javanmardi, Anna Djos, Rose-Marie Sjoberg, Per Kogner, Tommy Martinsson, Susanne Fransson. Exome- and whole genome sequencing for clinical evaluation and precision medicine in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4867. doi:10.1158/1538-7445.AM2017-4867

Abstract 5700: CircSarc: Disease monitoring by liquid biopsies in sarcomas

Abstract The CircSarc study aims to provide new insights into the clinical utility of liquid biopsies in soft tissue sarcomas. At present, mutational profiles of solid tumours are obtained from tissue biopsies or surgical specimens. Recent advances in technology now allows to use blood plasma as a “liquid biopsy”, examining circulating tumour DNA (ctDNA) shed by the tumour cells into peripheral blood. ctDNA in plasma carries tumour-specific alterations that can be used to monitor minimal residual disease, response to therapy, tumour burden and evolution throughout the course of the disease. In CircSarc, we have enrolled 30 high-grade soft tissue sarcoma patients, with localised disease, that are being followed throughout the course of their disease. Plasma from each patient is collected longitudinally; before and after surgery, at each routine control and before and after each treatment cycle. Patient’s tumour and germline DNA were exome sequenced to identify tumour-specific mutations, and ctDNA is being sequenced using a comprehensive 900 cancer gene panel. The level of ctDNA in plasma, represented by the tumour-specific biomarkers, are then monitored throughout the course of the treatment, and acts as an indicator of tumour burden. In addition, we are analysing plasma samples from 70 Gastro Intestinal Stroma Tumour samples using targeted resequencing with Anchored Multiplex PCR. The levels of KIT and PDGFRA mutations in ctDNA are being correlated with clinical and pathological features. The established targeted resequencing protocols for ctDNA provide different levels of complexity and sensitivity. In the first screening, we have successfully detected somatic mutations in plasma at time of surgery in 70% of the samples analysed. Mutations present in the tumour at an allele frequency large than 20% can be robustly identified in plasma. In addition, analysis of plasma samples identified novel mutations not detected in the primary tumour, possibly reflecting intra-tumour heterogeneity. These mutations are being followed throughout the course of the disease to monitor tumour evolution and resistance. In a proof-of-concept study, we have shown that that levels of tumour-specific mutations in liquid biopsies correlated to clinical manifestation of metastatic disease in aggressive sarcoma, and have the potential to detect disease progression at an early stage. Our work provides new insights into the clinical significance of ctDNA in sarcomas. By repeated sampling of liquid biopsies, somatic mutations identified in ctDNA can be used as unique non-invasive tumour-specific biomarkers for monitoring tumour burden and disease evolution throughout the course of the disease. Citation Format: Heidi M. Namløs, Seyed Hossein Moosavi, Bodil Bjerkehagen, Olga Zaikova, Synnøve Granlien, Stine Næss, Nina Louise Jebsen, Skyler Mishkin, Eva W. Stratford, Else Munthe, Kirsten S. Hall, Lars B. Aasheim, Eivind Hovig, Brian Kudlow, Ola Myklebost, Kjetil Boye, Leonardo A. Meza-Zepeda. CircSarc: Disease monitoring by liquid biopsies in sarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5700. doi:10.1158/1538-7445.AM2017-5700

Droplet digital PCR for detection and quantification of circulating tumor DNA in plasma of head and neck cancer patients

BackgroundDuring posttreatment surveillance of head and neck cancer patients, imaging is insufficiently accurate for the early detection of relapsing disease. Free circulating tumor DNA (ctDNA) may serve as a novel biomarker for monitoring tumor burden during posttreatment surveillance of these patients. In this exploratory study, we investigated whether low level ctDNA in plasma of head and neck cancer patients can be detected using Droplet Digital PCR (ddPCR).MethodsTP53 mutations were determined in surgically resected primary tumor samples from six patients with high stage (II-IV), moderate to poorly differentiated head and neck squamous cell carcinoma (HNSCC). Subsequently, mutation specific ddPCR assays were designed. Pretreatment plasma samples from these patients were examined on the presence of ctDNA by ddPCR using the mutation-specific assays. The ddPCR results were evaluated alongside clinicopathological data.ResultsIn all cases, plasma samples were found positive for targeted TP53 mutations in varying degrees (absolute quantification of 2.2–422 mutational copies/ml plasma). Mutations were detected in wild-type TP53 background templates of 7667–156,667 copies/ml plasma, yielding fractional abundances of down to 0.01%.ConclusionsOur results show that detection of tumor specific TP53 mutations in low level ctDNA from HNSCC patients using ddPCR is technically feasible and provide ground for future research on ctDNA quantification for the use of diagnostic biomarkers in the posttreatment surveillance of HNSCC patients.

Biomarker significance of plasma and tumor miR-21, miR-221, and miR-106a in osteosarcoma.

Osteosarcoma is the most common malignant bone tumor in children and young adults. Despite the use of surgery and multi-agent chemotherapy, osteosarcoma patients who have a poor response to chemotherapy or develop relapses have a dismal outcome. Identification of biomarkers for active disease may help to monitor tumor burden, detect early relapses, and predict prognosis in these patients. In this study, we examined whether circulating miRNAs can be used as biomarkers in osteosarcoma patients. We performed genome-wide miRNA profiling on a discovery cohort of osteosarcoma and control plasma samples. A total of 56 miRNAs were upregulated and 164 miRNAs were downregulated in osteosarcoma samples when compared to control plasma samples. miR-21, miR-221 and miR-106a were selected for further validation based on their known biological importance. We showed that all three circulating miRNAs were expressed significantly higher in osteosarcoma samples than normal samples in an independent cohort obtained from the Children’s Oncology Group. Furthermore, we demonstrated that miR-21 was expressed significantly higher in osteosarcoma tumors compared with normal bone controls. More importantly, lower expressions of miR-21 and miR-221, but not miR-106a, significantly correlated with a poor outcome. In conclusion, our results indicate that miR-21, miR-221 and miR-106a were elevated in the circulation of osteosarcoma patients, whereas tumor expressions of miR-21 and miR-221 are prognostically significant. Further investigation of these miRNAs may lead to a better prognostic method and potential miRNA therapeutics for osteosarcoma.

Future perspectives of circulating tumor DNA in colorectal cancer.

Tumor biopsy is currently the gold standard for diagnosis and in determining cell signaling pathways involved in the development of treatment resistance. However, there are major challenges with this technique, including the need for serial sampling to monitor treatment resistance, which is invasive and also has the potential for selection bias due to intra-tumoral and inter-tumoral heterogeneity. These challenges highlight the need for more effective methods for obtaining Tumor samples. Liquid biopsy analyzes genetic material or tumor cells shed into the blood from the primary tumor and metastatic sites and consequently provides a comprehensive, real-time picture of the tumor burden in an individual patient. Indeed, liquid biopsy has the potential to revolutionize cancer management. Here, we review recent studies on the potential clinical applications of liquid biopsy using circulating tumor DNA in colorectal cancer, including screening, diagnosis, detection of minimal residual disease after surgery, detection of recurrence, prognosis, predicting treatment response, monitoring tumor burden or response during treatment, and tracking resistance. We also discuss recent data demonstrating the utility of detecting KRAS-mutated circulating tumor DNA, both at diagnosis to determine an appropriate treatment strategy and during anti-epidermal growth factor receptor therapy to predict treatment resistance. The future integration of liquid biopsy into clinical practice is discussed, together with alternative approaches and key questions that need to be answered in future clinical studies before this technology can be implemented and used routinely.

Tumor burden monitoring using cell-free tumor DNA could be limited by tumor heterogeneity in advanced breast cancer and should be evaluated together with radiographic imaging

BackgroundAccurate measurement of tumor burden in breast cancer disease is essential to improve the clinical management of patients. In this study, we evaluate whether the fluctuations in the fraction of PIK3CA mutant allele correlates with tumor response according to RECIST criteria and tumor markers quantification.MethodsEighty six plasma samples were analyzed by digital PCR using Rare Mutation Assays for E542K, E545K and H1047R. Mutant cfDNA and tumor markers CA15-3 and CEA were compared with radiographic imaging.ResultsThe agreement between PIK3CA mutation status in FFPE samples and circulating tumor DNA (ctDNA) was moderate (K = 0.591; 95% IC = 0.371–0.811). Restricting the analysis to the metastatic patients, we found a good agreement between PIK3CA mutation status assessed in liquid and solid biopsy (K = 0.798 95%; IC = 0.586–1). ctDNA showed serial changes with fluctuations correlating with tumor markers 15.3 and CEA in 7 out of 8 cases with Pearson correlation coefficients ranging from 0.99 to 0.46 and from 0.99 to 0.38 respectively. Similarly, fluctuations in the fraction of PIK3CA mutant allele always correlated with changes in lesion size seen on images, although in two cases it did not correlate with treatment responses as defined by RECIST criteria.Conclusiononcogenic mutation quantification in plasma samples can be useful to monitor treatment outcome. However, it might be limited by tumor heterogeneity in advanced disease and it should be evaluated together with radiographic imaging.

Comprehensive measurement of UVB-induced non-melanoma skin cancer burden in mice using photographic images as a substitute for the caliper method.

The vernier caliper has been used as a gold standard to measure the length, width and height of skin tumors to calculate their total area and volume. It is a simple method for collecting data on a few tumors at a time, but becomes tedious, time-consuming and stressful for the animals and the operator when used for measuring multiple tumors in a large number of animals in protocols such as UVB-induced non-melanoma skin cancer (NMSC) in SKH-1 mice. Here, we show that photographic images of these mice taken within a few minutes under optimized conditions can be subjected to computerized analyses to determine tumor volume and area as accurately and precisely as the caliper method. Unlike the caliper method, the photographic method also records the incidence and multiplicity of tumors, thus permitting comprehensive measurement of tumor burden in the animal. The simplicity and ease of this method will permit more frequent monitoring of tumor burden in long protocols, resulting in the creation of additional data about dynamic changes in progression of cancer or the efficacy of therapeutic intervention. The photographic method can broadly substitute the caliper method for quantifying other skin pathologies.

Circulating tumor DNA: A potential biomarker from solid tumors' monitor to anticancer therapies

Circulating tumor DNA (ctDNA) in the peripheral blood is a liquid biopsy that contains representative tumor information including gene mutations. ctDNA is a promising new avenue for real-time monitoring of tumor progression. As a noninvasive biomarker and potential surrogate for the entire tumor genome, it has been applied to the detection of driver gene mutations and epigenetic alteration as well as monitoring of tumor burden, acquired resistance, tumor heterogeneity, and early diagnosis. Since precise therapy is a strategy that optimal therapy is decided based on simultaneous tumor genome information, ctDNA may help perform dynamic genetic surveillance. Dynamic marker surveillance may provide critical information to identify disease progression and guide therapeutic options. This review provides an overview on related articles about ctDNA, with a focus on monitoring response of solid tumors to anticancer therapies.

Dynamic Changes in MDS Clonal Architecture Following Allogeneic Stem Cell Transplant

Background: MDS is a genetically complex, oligoclonal disease consisting of a founding clone and typically one or more subclones derived from the founding clone. Previously we have shown that in MDS patients treated with chemotherapy, a minor subclone present at diagnosis can expand during disease progression from MDS to secondary AML, highlighting the clinical implications of clonal heterogeneity. Whether a similar pattern of clonal evolution occurs in MDS patients that relapse following allogeneic hematopoietic stem cell transplant (alloHSCT) is not known.Methods: We identified 9 MDS patients who progressed after receiving either a myeloablative (n=3) or reduced-intensity (n=6) alloHSCT (median time to progression 309 days, range 98-881). We performed enhanced exome sequencing (EES) to define the clonal architecture of 23 tumor samples at the following clinical landmarks (with matched skin as a source of normal DNA): diagnosis, <2 months pre-alloHSCT (where available), and relapse post-alloHSCT. Somatic mutations were validated in the 23 discovery samples and genotyped in 35 additional serial banked samples at various time-points post-alloHSCT, including day 30 and 100, using capture probes targeting all putative single-nucleotide variants (SNV) and short insertions and deletions (INDELs) identified by EES. The variant allele fraction (VAF) of all validated somatic mutations was determined. Ultra-deep, error-corrected sequencing (i.e., barcoded sequencing) was performed on 49 tumor samples to provide increased sensitivity to detect low-level mutations post-alloHSCT. Copy number alterations were identified using exome and SNP array data.Results: Averaged sequencing coverage depth was 246x for tumors subjected to EES; 537x for validation sequencing, and 24,150x total and 5,180x unique for the ultra-deep sequencing. In all cases, we observed that mutations found in the diagnostic founding clone were always detected at relapse. However, using SNVs, INDELs, and copy number analysis, we show that the dominant clone at relapse was often derived from a population that was subclonal at presentation. We observed the following, non-mutually exclusive patterns of clonal evolution at relapse: i) A subclone expanded or emerged and became the dominant clone at relapse as compared to presentation (n=6). In 2 of these cases, the subclone contained mutations that were not detected at presentation even via ultra-deep sequencing. ii) A subclone was cleared with alloHSCT (defined as VAF<1% by EES, n=4), confirmed by ultra-deep sequencing when available (n=2). iii) The founding/dominant clone at diagnosis was also the dominant clone at relapse (n=3). However, in 2 of these 3 cases, changes in clonal architecture were observed with evidence of rising or contracting subclones. Although our sample size is relatively small, the intensity of the alloHSCT conditioning regimen did not impact the relapse pattern. No acquired abnormalities were detected in the MHC locus, and no mutations in a particular gene family or cellular pathway were consistently observed in rising or contracting subclones.Finally, we used ultra-deep sequencing to determine if mutations (i.e., tumor cells) could be detected at day 30 post-alloHSCT in 7 of the 8 patients with no evidence of disease, who had available data. Mutations were detected in 6 of 7 patients. The average detectable mutation VAF per patient was 0.37% (ranged from 0.04% to 0.93%)(i.e., 1 mutant cell in 135).Conclusion: Complex clonal dynamics and clonal evolution are observed at relapse post-alloHSCT for MDS. Although minor subclones rise and may become the dominant clone at relapse, mutations present in the dominant (i.e., founding) clone of the diagnostic MDS sample pre-alloHSCT are always detected at relapse. This is similar to the pattern of clonal evolution previously observed for MDS progression to secondary AML following chemotherapy. These observations have implications for targeted therapy and tumor burden monitoring. Ultra-deep sequencing can detect persistent or emerging mutations at early time-points post-alloHSCT that are associated with subsequent relapse. The predictive value of detecting persistent mutations early after post-alloHSCT merits testing in future studies. DisclosuresJacoby:Quintiles: Consultancy; Sunesis: Research Funding; Celgene: Speakers Bureau. DiPersio:Incyte Corporation: Research Funding.

Abstract A140: Check point inhibitor modulation of tumor microenvironment at orthotopic and metastatic sites using bioluminescent syngeneic cell line models in immune competent mice

Abstract Introduction: Syngeneic models are widely used to model the impact of cancer immunotherapy on tumor growth and tumor invading leucocytes (TILs), and the majority of such work is typically carried out in the subcutaneous setting. However, orthotopic models are known to better model cancer in patients as they form a single focal disease area as in the patient situation, facilitate metastatic spread via intra- and extra-thoracic lymph nodes and, in the case of syngeneic models, strain-specific tumor microenvironment interactions (immune and stromal components). Bioluminescent imaging (BLI) increases the usefulness of such models, as it allows for non-invasive longitudinal monitoring of tumor burden, allowing for optimal randomisation and reduction of false positives. It also allows continuous feedback allowing one to optimise treatment regimen mid-study. Herein we describe the generation of several bioluminescent variants of syngeneic cell lines commonly used for immunotherapy studies and assess the impact of orthotopic growth on response to immune check point therapy. Experimental Procedures: Bioluminescent cell line variants of syngeneic cell lines were established by lentiviral transduction for: 4T1 (breast), B16-F10 (melanoma), MBT-2 (bladder) H22 (Hepatoma), LL/2 (lung), Pan02 (Pancreatic) and RM-1 (prostate). Following establishment of stable cultures, DNA profiling and in vitro cytotoxicity assays was carried out to ensure there was no significant changes in DNA, cell doubling time or response to SoC agents following transduction. Subcutaneous growth of wild-type and bioluminescent variants was compared to assess any impact of luciferase expression on tumor growth, inflammation and TILs. Orthotopic models were established for most cell lines, and a metastatic model for B16-F10, BLI was carried out to assess real-time tumor growth and tumor burden at end stage (Spectrum CT; PerkinElmer). Immune checkpoint therapy was also assessed and TIL infiltration by FACs analysis and IHC. Results: Stable transduced bioluminescent cell lines were established; cell doubling time, morphology and growth in vitro was found to be consistent with their wild-type counterparts. Bioluminescent 4T1 cells exhibited growth consistent to that previously reported and readily metastasised to the lungs from both the orthotopic and subcutaneous sites; B16-F10 cells readily metastasise to bone following intracardiac administration. Subcutaneous response to immunotherapy did not appear to be affected. Modulation of immune cell infiltration will be reported and correlated to response. Conclusions: The growth and response to immunotherapy does not appear to be significantly impacted in the bioluminescent cell line models tested; as such they are a useful tool for further assessing the impact of complex orthotopic, spontaneous and experimental metastasis modelling in immune competent mice. Citation Format: Andrew McKenzie, Nektaria Papadopoulou, Simon Jiang, Jane Wrigley, Kelly Jones, Russell Garland, Neil Williams, Rajendra Kumari. Check point inhibitor modulation of tumor microenvironment at orthotopic and metastatic sites using bioluminescent syngeneic cell line models in immune competent mice [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A140.

Abstract 504: Circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) are superior to CA 15-3 in predicting tumor burden, patients response to treatment and overall survival (OS) rates in metastatic breast cancer patients from Egypt

Abstract Background: Monitoring of tumor burden in patients with metastatic breast cancer (mBC) is crucial to determine response to treatment. In addition to radiological procedures and serological biomarkers, circulating, tumor cells (CTCs) are now used to assess the tumor burden and patients’ response to treatment. The circulating cell-free tumor DNA (ctDNA) harboring tumor-specific aberrations has not been properly assessed yet. Methods: 100 Egyptian patients with locally mBC were assessed for CA 15-3 levels, CTCs number by flowcytometry (FCM) confirmed by RT-PCR (CK and mammaglobin expression), and for ctDNA. The results of the three techniques were compared to the radiographic imaging of tumors, patients response to treatment and overall survival (OS) rates. Paraffin blocks for 40 tumor samples, obtained from the studied patients (20 non-responders and 20 responders) were used to detect p53 gene mutations exons 5-9. CA 15-3 levels and CTC numbers were measured at the same time intervals. Results: Somatic mutations of p53 were detected in 23/40 (57.5%) sequenced tumors. ctDNA showing the identified mutations in tumor samples, was detected in 78 cases (78%) with high dynamic range. CTCs&amp;gt;4/7.5ml blood were present in 57 out of the 78 (73.1%) ctDNA positive cases, and CA 15-3 was detected in 30 (38.5%) cases. Changes in ctDNA levels and CTCs&amp;gt;4 correlated significantly with the tumor burden (p = 0.034), patients response to treatment (p&amp;lt;0.01), and lower OS rates (p = 0.034&amp;p = 0.01; respectively). CTCs number and ctDNA levels showed higher correlation with changes in tumor burden, compared to CA 15-3 (p&amp;lt;0.001 versus p = 0.046). However, ctDNA provided the earliest measure of treatment response in most of the patients (53%). Conclusions: ctDNA is an informative, highly sensitive and specific biomarker that could be used to monitor tumor burden in mBC. Together with enumeration of CTCs they can predict tumor response and OS in mBC patients with high accuracy. Citation Format: Abdel-Rahman N. Zekri, Abeer A. Bahnassy. Circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) are superior to CA 15-3 in predicting tumor burden, patients response to treatment and overall survival (OS) rates in metastatic breast cancer patients from Egypt. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 504.

Abstract 1443: IL-18 is associated with the onset and progression of gastric cancer

Abstract INTRODUCTION: Gastric cancer (GC) is the fourth most prevalent, and the third most common cause of cancer-related death worldwide. The disease is generally asymptomatic, and consequently is often diagnosed at an advanced stage when metastasis is present, and limited treatment options are available. Chronic inflammation is recognized as an integral component in the development and progression of GC, and is associated with increased infiltration of immune cells into the tumor microenvironment. The production of pro-inflammatory cytokines by these cells may contribute to tumor progression through activation of pathways promoting tumor cell survival and proliferation. Previous work from our lab has shown that therapeutic inhibition of the inflammatory cytokine interleukin (IL)-11 is effective in ameliorating disease progression in gastrointestinal cancer, however, it is unclear what role other pro-inflammatory cytokines, such as IL-18, might have in disease progression. METHODS: To characterize the role of different pro-inflammatory cytokines in GC, we first analyzed microarray and qRT-PCR data of from human GC specimens and adjacent non-tumor tissue. Following the generation of a candidate list of cytokines deregulated in human GC, the role of individual cytokines in disease progression was examined using a validated mouse model of intestinal-type GC, referred to as gp130Y757F, by crossing into cytokine knock-out strains and monitoring tumor burden and the expression of genes and proteins classically associated with tumorigenesis. RESULTS: We found that the expression pro-inflammatory cytokines including IL-1β and IL-18 were significantly elevated in the tumors of human GC patients compared to non-tumor tissue. In gp130Y757F mice, genetic ablation of IL-18, but not of IL-1β significantly reduced gastric tumor burden, which was associated with a reduction in the number of intratumoral macrophages, but not lymphocytes. This observation correlated with decreased expression of pro-inflammatory (Ifng, Tgfb1), antimicrobial (Reg3b, Reg3g), and tissue remodeling (Mmp9) genes, and a reduction in apoptosis in the gastric lesions in these mice. CONCLUSION: Our results demonstrate that IL-18 has an important role in GC disease progression, and may serve as a potential therapeutic target. Citation Format: Paul Nguyen, Rita Busuttil, Lisa Mielke, Gabrielle Belz, Alex Boussioutas, Matthias Ernst, Tracy Putoczki. IL-18 is associated with the onset and progression of gastric cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1443.

Abstract 3138: Changes in tumor cell-free DNA copy number instability (CNI) predict therapeutic response in metastatic cancers

Abstract Background: Tumor cell-free DNA (cfDNA) has the potential to provide minimally invasive patient specific biomarkers to monitor tumor burden. Gains and losses of chromosomal regions - as a hallmark of cancer - have been detected in plasma as copy number aberrations (CNAs), and for several cancers a relation to tumor size has been reported. Longitudinal observations during anti-cancer therapy have been mostly anecdotal. We measured CNAs changes during treatment by computing a genomic copy number instability index (CNI) of cfDNA to evaluate its potential to predict cytotoxic chemotherapy (chemo) response. Methods: 24 patients (pts) with advanced esophageal cancer (EC; n = 2), colorectal cancer (CRC; n = 3), non-Hodgkin lymphoma (NHL; n = 3), pancreatic ductal adenocarcinomas (PDAC; n = 4), and non-small cell lung cancer (NSCLC; n = 12) were included and assessable for response. DNA was extracted from pre-treatment plasma samples at baseline and sequentially for up to six cycles of chemotherapy. Copy-numbers were called from shotgun sequencing (Illumina) after mapping and quality filtering reads were counted in 5.5MBp windows (sliding) yielding a read coverage of 24,000-fold per bin. The read counts were transformed into log2 ratios and converted into a score based on Gaussian transformations. Concentration of total cfDNA was determined by digital PCR. Treatment response by imaging was recorded by RECIST 1.1 or EORTC PET/CT criteria. For pts with baseline CNI&amp;gt;40 (representing the 99.99% level for non-cancer healthy controls), CNI change was considered predictive of response or stable disease when there was a reduction of CNI ≥50% relative to baseline. Disease progression was demonstrated when: 1) CNI was ≥50% over nadir and &amp;lt;80% reduction relative to baseline; OR, 2) CNI was ≥5 fold the nadir; OR, 3) CNI demonstrated an absolute increase by 1,000 above baseline CNI. For pts with baseline CNI≤40, CNI change was considered predictive of response or stable disease when CNI remained ≤40, and PD when CNI≥2X above the baseline CNI. CNI was an early predictor of response if these results were observed by ∼3-8 weeks prior to imaging or a major change in pt's clinical status. Results: CNI was measured in 124 samples. Median baseline cfDNA was 7,238 cp/mL (range 2,319-91,978) and CNI was 171 (range 26-10,170). In general, NHL and tumors with high lymph node disease burden had highest baseline cfDNA and CNI, whereas overall the CNI was not correlated to the cfDNA content. 22 of 24 pts (91.7%) had CNI changes that were predictive of treatment response. For at least 15 pts, CNI change predicted response ∼3-8 weeks prior to scan results demonstrating response or PD. Conclusions: CNI change may serve as predictor (potentially early predictor) of therapeutic response to standard chemo for the investigated cancer types. Highest baseline cfDNA and CNI appear to be present in lymph node predominant disease, suggesting more readily shed DNA into the circulation. Citation Format: Glen J. Weiss, Julia Beck, Donald P. Braun, Kirsten Bornemann-Kolatzki, Heather Barilla, Rhiannon Cubello, Ashish Sangal, Robert P. Whitehead, Madappa Kundranda, Vivek Khemka, Howard B. Urnovitz, Ekkehard Schutz. Changes in tumor cell-free DNA copy number instability (CNI) predict therapeutic response in metastatic cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3138.

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies.

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies.