Validation of QClamp as Next Generation Liquid Biopsy Technique for Colorectal Cancer He James Zhu M.D. Ph.D
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1 Validation of QClamp as Next Generation Liquid Biopsy Technique for Colorectal Cancer He James Zhu M.D. Ph.D Department of Radiation Oncology University of Florida College of Medicine
2 Outline Objective Background Protocol Overview Selection Criteria Tissue Process Future of Clinical Applications Potential for International Collaborations
3 Background Conventional methods to monitor tumor response and normal tissue toxicity during and after chemotherapy and radiotherapy (CRT) Liquid biopsy for cancer mutation profiling QClamp as a new liquid biopsy technique Colorectal cancer (CRC) as a model tumor for validation of new cancer gene mutation detection methods
4 Evaluation of Clinical Response to Radiotherapy All patients ask the same question after the first week of radiation: How is my tumor doing after this much chemotherapy and radiation doses dumped on me? Conventional methods: post-treatment imaging and biomarkers (e.g. CEA, CA19-9 etc). These take months to be useful, and are typically not very sensitive or specific They can not improve the radiation treatment since the treatment decisions have to be made long before the images and markers can help improve the treatment.
5 Liquid Biopsy Generally means any specific tumor marker that can be used to detect and possibly quantify tumor mass. A good liquid biopsy must be: Tumor specific Stable in body fluid/specimen with sufficiently long half life Adequate for reliable quantification Example: PSA for prostate cancer New approaches for tumor liquid biopsy: Circulating tumor cells Cell-free circulating tumor DNA (ctdna)
6 ctdna Capturing and quantifying circulating tumor cells are technically complex and time consuming with suboptimal results New approach: Cell free tumor DNA (ctdna) can be tumor specific and personalizable based on individual mutation profiles. To be useful ctdna for liquid biopsy needs to overcome key challenges: Ratio of wild-type:tumor DNA in blood is overwhelming (100-10,000:1 or greater) >95% of oncogenic mutations are point mutations (hard to detect and quantify) Mutations can occur in cells that do not have cancer (e.g. BCL2 in lymphocytes cause false positives) Example- Cologuard: detecting colorectal cancer (CRC) specific DNA in stool for CRC screening Requires central lab testing (in Wisconsin) because it includes epigenetics
7 QClamp New technique to amplify then quantify tumor specific mutations Sequence-specific wild-type template xenonucleic acid (XNA) clamp prevents wild type DNA from PCR amplification Extremely accurate for simple point mutation
8 QClamp Highly Sensitive & Specific Detection of a point mutant EGFR in background of wild-type. There were 100,000 wells. In the clamping control, no DNA was added. 0.1% well had a mix of 1000:1 wild-type:mutant with about 2 pieces of DNA per well. The 0.01% was a 10,000:1 ratio. In the 0% control, no mutant DNA was added. Therefore, quantification of ~1:100,000 mutant:wild-type was possible. no false positives were detected (CC)
9 QClamp Highly Sensitive & Specific ColoScape is the assay developed for CRC screening using Qclamp technique
10 QClamp Easily Personalizable Existing panel of XNA clamps accounting for majority of the exons that harbor cancer driver mutations cover 95% of CRC and lung cancers If a patient s panel of mutations is known, XNA clamps can be multiplexed within minutes to detect and quantify all ctdna strains that contain different point mutations. If a patient s tumor has a unusual mutation not accounted for in present XNA clamp library, new clamps specific to this mutation can be made within a week at a reasonable cost Quantification of ctdna can be completed the same day that the specimen is submitted
11 QClamp Test Widely Depolyable ctdna quantification using QClamp requires only regular PCR for most tests and at best, digital PCR for tests requiring high quantitative accuracy for subclinical disease (for example screening) QClamp can be used on many types of samples: plasma, stool, urine or formalin-fixed paraffin-embeded (FFPE) samples Next generation sequencing (NGS) of individual s tumor can be done locally without need for central lab processing; if no NGS is available Qclamp multiplex should still offer substantial sensitivity and specificity
12 CRC as Model Tumor Type to Validate QClamp Technique CRC is one of most common and deadly cancers worldwide CRC mutation profiles have been well characterized with 95% of tumor somatic mutations already mapped out at our institution CRC usually presents with substantial tumor size, resulting in high ctdna CRC response to treatment can be well assessed on both imaging and direct visualization (colonoscopy, sigmoidoscopy or DRE) CRC treatment involving neoadjuvant chemoradiation and surgery has been standardized in the U.S. Tumor will be removed after completion of radiation, making it available to compare with QClamp based analysis
13 QClamp Verification Protocol Overview: NG PCR vs. QClamp
14 Potential Protocol Tailored for Chinese Patients Patients with early stage lung or colon cancer; use broad mutation multiplex instead of requirement for NGS. Diacarta has a CLIA certified lab in China It would be preferable to be able to send tissue to the USA if possible for analysis
15 Selection Criteria (China) Patient with existing DNA and FFPE from colon or rectal or lung cancer Age > 18 Clinically stage II or stage III (T1-4N1-2) colon or rectal cancer; or stage I or II lung cancer No prior GI malignancies
16 Tissue Process Purified DNA or FFPE These needs to be prepared in a CLIA appropriate manner such as might be used for patient diagnosis Plasma Specimens EDTA anti-coagulant (at least 5 ml, 10 ml prefered) Collected and placed on ice Plasma collected within an hour and frozen at -80 C (needs to be well labeled, best if aliquoted into 1 ml tubes)
17 USA: Clinical Applications in Process Quantification of tumor response and normal tissue toxicity during chemoradiation for rectal adenocarcinoma Quantification of normal tissue toxicity during proton radiotherapy for prostate cancer
18 Quantification of Tumor Response and Toxicities from Chemoradiation Using QClamp & QuantiDNA
19 QuantiDNA Technique Only human DNA can be captured in test wells using the ELISA like sandwished design Primate specific short interspersed element (SINE) Alu were abundant in human cells Consensus sequence to Alu locks human DNA to the bottom of test wells Another consensus sequence binds human DNA to 500X luminescence amplifer or QClamp can be applied to anchored human DNA to amplify only tumor DNA This approach can assure accurate quantification of human tumor DNA in stool samples where human DNA is not abundant
20 QClamp for Rectal CA Treatment Assessment ctdna quantity in plasma and stool during chemoradiation could be correlated with amount of tumor death in forms of tumor shrinkage or necrosis Amount of normal tissue damage by chemoradiation could be assessed by amount of non-tumor human DNA shed in plasma and stool and correlate with clinical symptoms Non-tumor DNA = total DNA (QuantiDNA) tumor DNA (QClamp) Screening ctdna immediately post-op + tumor response at time of surgery could predict risk of future recurrence or metastasis
21 QClamp for Rectal CA Treatment Assessment Screening ctdna immediately post-op + tumor response at time of surgery could predict risk of future recurrence or metastasis Complete reponse (CR) + no ctdna after surgery = no residual CR + ctdna present before surgery and persistent weeks after surgery = high chance of metastasis PR + ctdna present and persistent after surgery = high chance of microscopic residual tumor Rising ctdna over time could be early predictor of tumor recurrence. Consider systemic therapies (like PSA doubling time)
22 QuantiDNA for Pelvic Proton Therapy Toxicity Assessment Proton therapy for prostate cancer become attractive due to dosimetric advantage in reducing pelvic normal organ toxicity Clinical toxicity from proton therapy for intact prostate cancer has been published, but limited to post-pt assessment Conventional patient-report based toxicity assessment can only provide population statistics and can not be specific to individual patient s predisposition risks (e.g. treatment fields, autoimmune diseases etc.) Hypothesis: QuantiDNA technique can quantify amount of normal tissue damage during early weeks of PT vs XRT adjusted for individual patient s predisposition risks
23 Ongoing Qclamp Trial for Prostate Cancer Phase II diagnostic study, funded with accrual goal: 70 Primary objective: To determine if tissue damage measured using circulating DNA during treatment correlates with toxicity scoring. 2 nd Objectives: To determine if DNA release is lower with PT compared to conventional XRT To determine if DNA release is lower with smaller prostate cancer fields than larger fields
24 Selection Criteria Consent to participate in the study and a signed and dated an institutional review board (IRB)-approved consent form conforming to federal and institutional guidelines. 18 years of age. Patients must have an Eastern Cooperative Oncology Group (An ECOG) performance status of 0 or 1. A diagnosis of adenocarcinoma of the prostate. Not yet begun definitive therapy with chemotherapy or radiation but may have hormones or surgery. Clinically stage I to III tumors No prior pelvic RT or invasive rectal or other pelvic malignancy No active inflammatory bowel disease Psychiatric or addictive disorders such that patient unable to compliant with treatment
25 Eligibility Radiation for non-metastatic prostate cancer (stage I to III); prostate intact, positive margins, and salvage for rising PSA after prostatectomy are all eligible. Hormone treatment allowed. Pre-Treatment Pre-radiation collection of plasma (20 ml). Chart review for pertinent information (other treatments and confounders). Baseline toxicity score. Treatment plan, including DVH for tissues at risk and integral dose. Week 1 Daily plasma (10 ml qd) x 5 beginning 24 ± 4 hr and 48 ± 4 hr after the initiation of radiation therapy. Chart review for tumor response and toxicity information (throughout study). Toxicity score. Weeks 2-8 Chart review for tumor response and toxicity information. Toxicity score. Both intact prostate and post-prostectomy RT are eligible Pelvic XRT using IMRT to 45-46Gy to all patients with high risk of pelvic lymph node involvement Total dose 78Gy or 78CGE for intact prostate RT Total dose Gy or CGE for salvage RT Hormone therapy allowed Follow-up at 6 Months and 1 Year Chart review for tumor response and toxicity information. Toxicity score.
26 Potential for International Collaborations Learn potential demographic differences in tumor genetics Exchange expertise in treating challenging malignancies Field-test new technology in diverse clinical practice and patient populations Establish care for Chinese patients at UF Proton Therapy Institute (UFPTI) Dr. Paul Okunieff (Chair) Dr. Nancy Mendenhall (Medical Director) Dr. Robert Amdur (Residency Director) Dr. Zuofeng Li (Chief Physicist) Stuart Klein, MBA (CEO)
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