Changes in Biomarkers From Blood Over Time in Patients With Pancreatic Adenocarcinoma
Changes in Biomarkers From Blood Over Time in Patients With Pancreatic Adenocarcinoma
Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer mortality in the
 United States. The high mortality for these tumors is primarily attributed to the late stage
 in which most patients are diagnosed, leading to a dismal 5-year survival of 6% for all
 stages of PDAC. Surgical resection offers the best chance for survival, but most patients
 only present with symptoms after the tumor has metastasized, and as a result are not
 operative candidates. This creates a need to both identify patients at an earlier stage while
 their cancer is still resectable, and predict the aggressiveness of the disease in order to
 better target treatment. In addition, even patients who receive curative surgery are at a
 high risk of developing recurrence of disease. Thus, there is also a need to detect
 recurrence early so appropriate treatment can be provided. As several adjuvant
 chemotherapeutic regimens are now available, it will be important to identify as soon as
 possible that the cancer has become refractory to a given therapy. This will allow one to
 progress to second or third line therapy more quickly while the tumor burden is smaller. This
 purpose of this study is to identify biomarkers in the blood of patients with PDAC and
 determine how they can change over time in relation to treatment to assess for any
 correlation with patient outcomes, response to treatment, recurrence of disease and overall
 survival. This study will be limited to patients who present to the Johns Hopkins Hospital
 between January 1, 2015 and December 31, 2018 with PDAC. Blood will be drawn from all
 consenting patients at the time of initial diagnosis and after treatment. Patients will
 undergo treatment for their cancer based on personal preference, standard guidelines and
 discussion with medical, radiation, and surgical oncologists. Patients who undergo surgical
 resection will also have an additional blood sample collected after resection, and patients
 who undergo chemotherapy and/or radiation will have an additional blood sample draw at the
 end of this treatment. A patient could have blood collected at multiple intervals, i.e. a
 pre-treatment sample, sample post-neoadjuvant chemotherapy/radiation, sample post-surgery,
 and sample post-adjuvant chemotherapy/radiation. In patients, who have undergone curative
 resection of PDAC blood samples will be collected till they develop clinical recurrence of
 disease. For the first 2 years following surgery samples will be collected every 3-4 months.
 Beyond that the investigators will collect samples every 6 months for the next two years. For
 all patients found to be alive and disease free beyond 4 years after surgery samples will be
 collected once every year. These patients will be followed to determine disease-free and
 overall survival. With this study, the investigators aim to assess the potential utility of
 blood biomarkers over time for pancreatic tumors which will help both with early detection of
 disease and also recurrence of disease after surgery. Biomarkers identified would have the
 potential to create a new method for early diagnosis of patients with PDAC, predict overall
 survival, response to treatment, or risk of metastatic spread, and predict recurrence of
 disease, all of which has the potential to drastically improve outcomes for this deadly
 disease.
This is a single-institutional, prospective study at the Johns Hopkins Hospital which will be
 conducted from January 1, 2015 to December 31, 2018. All patients who present to the Johns
 Hopkins Hospital with a pancreatic adenocarcinoma (PDAC) for evaluation of treatment by
 chemotherapy, radiation therapy, and/or surgery will be approached to participate in this
 study, but only those who consent to the study will be included. All patients who consent to
 participate in this trial will have blood drawn (approximately 50 ml or 3-4 tablespoons) at
 the time of diagnosis but before any treatment had been administered. Further blood will be
 drawn after any treatment. For example, those patients that undergo surgical resection will
 also have blood drawn after resection prior to discharge. Those patients that undergo
 treatment with chemotherapy, radiation therapy, and or biologic/vaccine therapy will have
 blood drawn once after this treatment is completed. If a patient undergoes chemotherapy and
 then surgical resection, he or she will have blood drawn after chemotherapy but before
 surgical resection, and then again after surgical resection, all in addition to the
 pre-treatment blood sample (see schema included in IRB). For the first 2 years following
 surgery samples will be collected every 3-4 months. Beyond that the investigators will
 collect samples every 6 months for the next two years. For all patients found to be alive and
 disease free beyond 4 years after surgery samples will be collected once every year. This
 blood will be stored in the investigators' research facility and as such will be labeled with
 a de-identified code. If a patient refuses further blood draws at any time during the study
 or is loss to follow-up, the samples that have been collected will still be utilized.

 The blood will be processed on the day of collection, within 6 hours of being drawn. Twenty
 (20) ml of blood, collected in two purple topped tubes, will be used for isolation of
 circulating tumor cells (CTCs) through the Isolation by Size of Epithelial Tumor Cells assay
 (ISET), which separates tumor cells by size onto a membrane which can be used for further
 experiments. These 20 ml of blood will be mixed with ISET buffer per standard protocol and
 filtered through the ISET machine onto membranes. These membranes will be stored at -20
 degrees C under a de-identified number, and remain at these conditions for several years with
 full preservation of the cells. Further experiments will be done on these membranes, which
 include immunofluorescent staining, laser capture microscopy, and genetic analysis. The
 remaining 30 ml of blood, collected in red and purple topped tubes, will be processed by a
 standard protocol within 6 hours of collection. Blood will be double spun to remove white
 blood cells, and plasma and will be frozen. Driver gene mutations characterized in the
 resected tumors will be monitored by digital droplet Polymerase Chain Reaction (ddPCR),
 including the Kirsten rat sarcoma viral oncogene homolog (k-ras), p53, p16/cdkn2a and the
 Deleted in Pancreatic Cancer-4 gene (DPC4/SMAD4) to identify the presence of ctDNA and
 whether levels are rising.

 Data will be collected, de-identified and analyzed as a single dataset at the Johns Hopkins
 Hospital. Data will be obtained from patient electronic and paper clinical charts from the
 John Hopkins Hospital starting on 1/1/2015, with only data in existence as of 1/1/2015
 collected. More specifically, radiology, chemotherapy, operative and pathology reports,
 discharge summaries, and clinic notes will be reviewed and data from these sources collected.
 For patients lost to follow-up, the Social Security Death Index (but will not need to collect
 social security numbers) will be searched for evidence of death. Collected patient data will
 be entered into a dedicated database. Data will only be collected in order to correlate
 outcomes and tumor characteristics with the biomarkers identified during this study.
 Following data accrual and prior to analysis, all personal identifiers will be deleted and no
 identifiable information will be present at the time of statistical analysis. The subject
 data generated will not appear in the subject's medical record and will be used for research
 purposes only. The data will be recorded in a password-protected, secure database within the
 Principal Investigator's office and will be accessible only by the key personnel. After all
 data for a particular subject is collected, the subject's identity will be deleted to assure
 confidentiality.

 The data that are collected in this study will not be used for the clinical management of any
 patient on study.

 Inclusion/Exclusion Criteria

 The investigators will include all patients who present to the Johns Hopkins Hospital for
 treatment with a diagnosis of a pancreatic adenocarcinoma. Subjects will only be included if
 they are over the age of 18 year old and plan to undergo their treatment at the Johns Hopkins
 Hospital. All patients with non-primary pancreas tumors, pancreatic neuroendocrine tumors, or
 benign pancreatic masses will be excluded. Patients will be excluded if they have already
 undergone treatment of any kind at another institution.

 The investigators aim to include data from at least 200 patients over the three years of this
 study. The primary outcome will be the identify biomarkers in the blood from patients with
 pancreatic adenocarcinoma and the changes in these biomarkers over time in relation to
 treatment. These biomarkers will include specific antibodies, circulating tumor cells (CTCs)
 and circulating tumor DNA (ctDNA). Secondary outcomes will include the relationship of
 pre-treatment biomarker levels or changes in biomarker levels to disease-free and overall
 survival, risk of metastases, recurrence of disease and response to treatment. Patient,
 tumor, and surgical characteristics will be collected for all patients included in this
 study. Differences between characteristics will be statistically compared between patients.
 In addition, these characteristics will be analyzed as possible predictors of survival using
 univariate and multivariate analyses through the log-rank test and Cox-proportional hazard
 models. These characteristics will include:

 1. Patient and tumor clinical demographics including age, gender, race, primary tumor
 location, location of metastases, tumor grade, and Carbohydrate Antigen 19-9 (CA19-9)
 levels pre- and post-chemotherapy and pre- and post-resection.

 2. Chemotherapy variables including type, number of cycles, response, and timing
 (neoadjuvant and/or adjuvant).

 3. Operative variables including type of resection, number of lymph nodes resected,
 vascular proximity of tumor, histopathology, and post-operative mortality and
 complications.

 4. Long-term variables including presence and location of recurrence and status
 (alive/dead).

 5. Levels of specific biomarkers at the various time points of blood draws.

 6. Numbers of circulating tumor cells at each time point in addition to genetic mutations
 present in the cells.

 For all patients, overall survival will be calculated as the time between the date of the
 first pre-treatment blood draw to the date of the last known follow-up visit or death,
 whichever comes first. For patients who undergo surgical resection the investigators will
 calculate time to disease recurrence as measured from the day of surgery for pancreatic
 resection to the date of documented disease recurrence or death, whichever comes first. Time
 to disease recurrence and overall survival will be described with Kaplan-Meier curves and
 will be compared by log-rank test. The 1- and 5-year overall survival rate will be estimated
 with its 95% confidence interval. Statistical significance will be set for a p-value at or
 below 0.05.

 The primary risks to this patient will be associated with the blood collection, but are no
 more than minimal given collection with be by venipuncture and no more than 50 ml will be
 taken at a time. The general risks associated with venous blood draw include bruising or
 minor bleeding at the site of venous puncture, which would be managed with the application of
 pressure.

 There will be no direct benefit to patients in this study, as results generated will not be
 used for management of these patients. This study has the potential to benefit future
 patients by identifying methods for early diagnosis of patients with pancreatic cancer and
 predicting overall survival, response to treatment, or risk of metastatic spread, and
 predicting recurrence of disease, all of which has the potential advantage of improving
 outcomes for this deadly disease.

 There will be no compensation given to subjects in this study. Costs in this study will be
 related to the cost of blood draws during various scheduled clinic visits. These will be
 minimal.

 All patients who agree to participate in this study will be required to sign a consent form
 for the collection, storage, and use of their blood.
Inclusion Criteria:

 -  Patients who present to the Johns Hopkins Hospital for treatment with a diagnosis of
 pancreatic adenocarcinoma.

 Exclusion Criteria:

 -  Patients with non-primary pancreas tumors, pancreatic neuroendocrine tumors, or benign
 pancreatic masses

 -  Patients who have already undergone cancer treatment of any kind at another
 institution.