Increasingly, clinicians and scientists now support the hypothesis that the majority of patients with pancreatic cancer (PC) will have systemic disease at the time of diagnosis, even in the absence of radiographic evidence of distant metastases.[1-3] Radiographic underestimation of metastatic disease is a major impediment in the management of PC. Among patients with localized PC who undergo surgical resection, disease recurrence occurs in up to 60% of patients within 6.9 months of surgery [4] and median survival is only 24 months, suggesting that radiographically occult metastatic disease is present in many patients at the time of surgery.(5) Furthermore, the delivery of postoperative (adjuvant) therapy for micrometastatic disease is unpredictable due to unanticipated perioperative morbidity and can only be achieved in 50% of patients.[5, 6] Therefore, inaccurate staging has significant consequences, and immediate surgery for presumed localized disease may temporarily or permanently delay access to systemic therapy for patients at high risk for metastatic disease. Furthermore radiographic imaging also underestimates treatment response and may not necessarily correlate with resectability.[7, 8] As the paradigm begins to shift from postoperative (adjuvant) to preoperative (neoadjuvant) therapy, objective and quantitative methods to assess treatment response and overall extent of disease will be critical to optimize patient selection and oncologic outcomes.

Use of tumor-specific cell free DNA as a treatment response biomarker

In recent years, the field of oncology has recognized the potentially revolutionary application of cell-free DNA. Cell-free DNA is a naturally occurring component of plasma that originates from cellular death when cellular DNA is released into free circulation. The utility of cell-free DNA monitoring has seen the greatest success in the detection of fetal DNA in the maternal blood, including point mutations and aneuploidy, and has become part of the standard of care in prenatal assessment in high-risk patients.[9, 10] In oncology, detection of cell-free tumor DNA (ctDNA) may be particularly relevant, as the pathogenesis of many cancers is the result of acquired genetic mutations. Therefore ctDNA may have exquisite biologic specificity as a biomarker. Especially in PC, monitoring of ctDNA represents a unique opportunity, in that over 90% of PCs have a KRAS oncogene point mutation and therefore a ubiquitous genetic target should exist . [11, 12] We hypothesize that monitoring of KRAS mutations detected in plasma ctDNA may be a clinically useful biomarker.

The use of ctDNA as a biomarker has largely been studied in patients with metastatic lung cancer as means to both monitor tumor burden and to detect molecular resistance to targeted therapies.[13, 14] The utility of ctDNA has been more limited among patients with earlier stage disease, as current technologies have insufficient sensitivity to quantitate extremely low mutant allelic frequencies. In PC, approximately 59-75% of patients with metastatic disease have ctDNA detectable by PCR-based single gene methods.[15, 16] However, detection of ctDNA is challenging in earlier stage disease. In the largest experience of ctDNA monitoring in patients with PC reported by Takai et al, only 18% and 8% of locally advanced and resectable patients with PC had detectable mutant KRAS, respectively.[15] The inability to detect KRAS mutations from patients with localized PC may be limited by the sensitivity of the assay. Our laboratory has demonstrated the limit of quantification to be 0.5% using next generation sequencing technology (Figure 1). Alternative techniques which provide greater sensitivity are needed to detect rare mutant allelic frequencies at very low levels (<0.001%). Our co-investigator, Dr. Aoy Mitchell, is developing a novel assay to detect rare mutations at or below the current level of detection without using next generation sequencing. [17] This assay utilizes a preferential amplification of the mutant allele relative to the wide-type allele.

Karl Storz Proposal

We proposed to develop a highly sensitive assay to identify rare mutant allelic frequencies from plasma cell free DNA and correlate quantitative levels of KRAS mutations at two time points (prior to surgery and following surgery) with disease-free survival at 1 year. To date, we have isolated total cell free DNA from 90 patients. Total cell free DNA was lower in patients with localized PC as compared to metastatic disease (median 5.53 ng/mL vs 14.56 ng/mL, respectively, p = 0.15). Among patients with localized PC, median total cell free DNA was lower in patients that were CA19-9 non-producers as compared to CA19-9 producers (median 3.6 ng/mL vs 5.7 ng/mL, respectively, p = 0.06). We have optimized the next generation sequencing assay to quantitate mutant KRAS down to an allelic frequency of 0.5%. Of the metastatic patient samples which were tested, mutant KRAS could be detected in a range from 1.5-15.8% of the total cell free DNA.

The next step of the project will utilize biospecimens prospectively collected from patients with localized PC who were enrolled in a clinical trial. We will utilize biospecimens collected from patients with resectable and borderline resectable PC who have been enrolled in an investigator-initiated clinical trial (NCI01726582). This trial utilizes immunohistochemical profiling from fine needle aspirate biopsies and surgical specimens to guide chemotherapeutic selection for neoadjuvant and adjuvant therapy, respectively. As a secondary endpoint of the trial, planned blood collection occurred at defined staging intervals. Blood was collected prior to the initiation of any therapy and following neoadjuvant therapy prior to surgery, after surgery, and with every restaging evaluation (Q3 month) until the date of disease progression (Figure 2). Restaging imaging prior to surgery (Figure 2: time point 1) consisted of a dual phase computed tomography (CT) of the abdomen and pelvis, abdominal magnetic resonance imaging, positron emission test, and laboratory tests, including CA19-9. Subsequent restaging evaluations consisted of an abdomen and pelvis CT and laboratory tests. To date 130 patients have been enrolled and are evaluable for the primary endpoint of the study; 81 (85%) of patients completed all neoadjuvant therapy and surgery and 14 (15%) had disease progression during neoadjuvant therapy. The goal of these studies is to develop a highly sensitive biomarker to improve the clinical management of patients with PC by allowing clinicians to identify patients at high risk for disease relapse within 1 year of surgery and to improve assessment of treatment response.

1. Rhim, A.D., et al., EMT and dissemination precede pancreatic tumor formation. Cell, 2012. 148(1-2): p. 349-61. 2. Sohal, D.P., et al., Pancreatic adenocarcinoma: treating a systemic disease with systemic therapy. J Natl Cancer Inst, 2014. 106(3): p. dju011. 3. Heestand, G.M., J.D. Murphy, and A.M. Lowy, Approach to patients with pancreatic cancer without detectable metastases. J Clin Oncol, 2015. 33(16): p. 1770-8. 4. Oettle, H., et al., Adjuvant chemotherapy with gemcitabine vs observation in patients undergoing curative-intent resection of pancreatic cancer: a randomized controlled trial. JAMA, 2007. 297(3): p. 267-77. 5. Mayo, S.C., et al., Management of patients with pancreatic adenocarcinoma: national trends in patient selection, operative management, and use of adjuvant therapy. J Am Coll Surg, 2012. 214(1): p. 33-45. 6. Wu, W., et al., The impact of postoperative complications on the administration of adjuvant therapy following pancreaticoduodenectomy for adenocarcinoma. Ann Surg Oncol, 2014. 21(9): p. 2873-81. 7. Katz, M.H., et al., Response of borderline resectable pancreatic cancer to neoadjuvant therapy is not reflected by radiographic indicators. Cancer, 2012. 118(23): p. 5749-56. 8. Ferrone, C.R., et al., Radiological and surgical implications of neoadjuvant treatment with FOLFIRINOX for locally advanced and borderline resectable pancreatic cancer. Ann Surg, 2015. 261(1): p. 12-7. 9. Ghanta, S., et al., Non-invasive prenatal detection of trisomy 21 using tandem single nucleotide polymorphisms. PLoS One, 2010. 5(10): p. e13184. 10. Meijerink, H., et al., Heroin use is associated with suppressed pro-inflammatory cytokine response after LPS exposure in HIV-infected individuals. PLoS One, 2015. 10(4): p. e0122822. 11. Hruban, R.H., et al., K-ras oncogene activation in adenocarcinoma of the human pancreas. A study of 82 carcinomas using a combination of mutant-enriched polymerase chain reaction analysis and allele-specific oligonucleotide hybridization. Am J Pathol, 1993. 143(2): p. 545-54. 12. Witkiewicz, A.K., et al., Whole-exome sequencing of pancreatic cancer defines genetic diversity and therapeutic targets. Nat Commun, 2015. 6: p. 6744. 13. Diehl, F., et al., Circulating mutant DNA to assess tumor dynamics. Nat Med, 2008. 14(9): p. 985-90. 14. Forshew, T., et al., Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA. Sci Transl Med, 2012. 4(136): p. 136ra68. 15. Takai, E., et al., Clinical utility of circulating tumor DNA for molecular assessment in pancreatic cancer. Sci Rep, 2015. 5: p. 18425. 16. Bettegowda, C., et al., Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med, 2014. 6(224): p. 224ra24. 17. Cha, R.S., et al., Mismatch amplification mutation assay (MAMA): application to the c-H-ras gene. PCR Methods Appl, 1992. 2(1): p. 14-20.


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