Pilot-Feasibility Trial of EUS Guided Pancreas Fine Needle Core Biopsy for Whole Exome Sequencing and Genomic Profiling
Phase: NA
Conditions: Pancreatic Cancer
Sponsor: Mayo Clinic

Endoscopic Ultrasound Guided Core 
Biopsy Needle Versus Fine Needle 
Aspiration (FNA) In Tissue Sampling Of 
Pancreas Cancer For Whole Exome 
Sequencing And Genomic Profiling: A 
Prospective Randomized Controlled  
 Trial 
NCT# 02678442 
June 9, 2017 

Endoscopic ultrasound guided core biopsy needle versus fine needle aspiration (FNA) needle 
in tissue sampling of pancreas cancer for whole exome sequencing and genomic profiling: a 
prospective randomized controlled trial. 
CLINICAL STUDY PROTOCOL 

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Table of Contents 
STEERING COMMITTEE 
PROTOCOL SYNOPSIS 
LIST OF ABBREVIATIONS 
1.0 INTRODUCTION 
1.1 BACKGROUND 
1.2 RATIONALE FOR PERFORMING THE STUDY 
1.3 HYPOTHESIS 
1.4 BENEFIT/RISK ASPECTS 
2. STUDY OBJECTIVES
2.1 PRIMARY OBJECTIVE:
2.2 SECONDARY OBJECTIVES:
3. STUDY POPULATION
3.1 NUMBER OF PATIENTS:
3.2 INCLUSION CRITERIA
3.3 EXCLUSION CRITERIA:
3.4 PATIENT INFORMED CONSENT
3.5 REPLACEMENT POLICY
3.6 COMPENSATION AND FINANCIAL RESPONSIBILITY
3.7 PATIENT REMUNERATION
4. PRE-PROCEDURE EVALUATION
4.1 HISTORY AND PHYSICAL EXAMINATION
4.2 LABORATORY STUDIES
5. INTRA AND POST- PROCEDURE EVALUATION
5.1 EVALUATIONS DURING PROCEDURE

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5.2 POST-PROCEDURE EVALUATIONS 
6. SUBJECT WITHDRAWAL AND STUDY TERMINATION CRITERIA
6.1 SUBJECT WITHDRAWAL
6.2 CONFIDENTIALITY
7. ETHICS AND REGULATORY
7.1 INSTITUTIONAL REVIEW BOARD (IRB)
7.2 REGULATORY AUTHORITY AUTHORIZATION-APPROVAL-NOTIFICATION
7.3 ETHICAL CONDUCT OF THE STUDY
7.4 SUBJECT INFORMATION AND CONSENT
7.5 CONFIDENTIALITY OF SUBJECT DATA
7.6 STANDARDIZED NUMERICAL IDENTIFIER (SNI)
8. STUDY DESIGN:
8.1 PATIENT REGISTRATION AND DOCUMENTATION
8.2 STUDY PROTOCOL
9. STATISTICAL CONSIDERATIONS
9.1 STATISTICAL ANALYSIS ........................................................................................... 26 

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Steering Committee 
Principal Investigator: 
Michael B. Wallace MD, MPH 
Professor of Medicine  
Division of Gastroenterology and Hepatology 
Mayo Clinic  
wallace.michael@mayo.edu 
Coinvestigators: 
Massimo Raimondo, MD  
Division of Gastroenterology and Hepatology 
Mayo Clinic  
Raimondo.massimo@mayo.edu 
Timothy A. Woodward, M.D. 
Division of Gastroenterology and Hepatology 
Mayo Clinic  
Woodward.timothy@mayo.edu 
Victoria Gomez, M.D. 
Division of Gastroenterology and Hepatology 
Mayo Clinic  
gomez.victoria@mayo.edu 
Pujan Kandel, M.D. 
Research Fellow 
Division of Gastroenterology and Hepatology 
Mayo Clinic  

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Kandel.pujan@mayo.edu 
Biostatistician 
Julia Crook Ph.D. 
Biostatistics Unit 
Mayo Clinic 
Crook.julia@mayo.edu 
Pathologist:  
Aziza Nassar M.D. 
Department of Pathology 
Mayo Clinic 
Nassar.aziza@mayo.edu 

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Protocol Synopsis 
Title of Study 
Endoscopic ultrasound guided core biopsy needle versus fine 
needle aspiration (FNA) needle in tissue sampling of pancreas 
cancer for whole exome sequencing and genomic profiling: a 
prospective randomized controlled trial. 
Hypothesis 
Flexible core biopsy needles are more capable than fine needle 
aspiration in acquisition of tumor tissue that is important for 
pathological evaluation and genomic analysis.  This would 
specifically allow us to perform whole exome sequencing of a 
tumor tissue and could provide path for future individualized 
pancreas cancer treatment. Thus, we hypothesized that FNB is 
superior to FNA in acquiring tumor tissue from pancreas mass that 
is key for genomic profiling. 
Specific Aims 
 To compare FNB versus conventional FNA for whole exome 
sequencing and genomic profiling in tissue sampling of pancreas 
cancer. 
1. Per needle adequacy for un-amplified whole exome
sequencing
2. Per needle quantity of DNA.
3. Per needle adequacy for cytology
4. Per needle adequacy for histology
5. Adverse events
Study design 
Prospective, single blinded, randomized controlled trial of FNB 
versus FNA for whole exome sequencing and genomic profiling. 

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Sample size 
50cases 
Inclusion criteria 
 Male and female patients who are 18 years old or older and are
referred for the evaluation of pancreatic mass lesion
 International Normalized Ratio (INR) less than 1.5 and platelet
count of more than 50,000.
 Medically stable to undergo sedation for EUS.
 Signed informed consent
Exclusion criteria 
 Medical condition that preclude the patient from having a
therapeutic procedure regardless of the EUS finding
 Pregnant patients
Study design 
 This is a prospective, single blinded randomized controlled trial 
with a paired evaluation of FNB vs FNA for whole exome 
sequencing and genomic profiling. A minimum 2 passes (1 with 
each needle) will be performed from form pancreas stratified by 
the lesion location (pancreas head tumor vs pancreas body/tail). 
Based on the location and type of the abnormal lesion, if further 
investigation via FNA is deemed necessary by the participating 
endosonographer, conventional FNA will be alternated with FNB 
in the usual fashion for obtaining histological material.  The 
procedure will be performed with rapid onsite evaluation. Onsite 
cytopathologist will evaluate the adequacy and the degree of 
pathological changes. Based on the information provided by the 
cytopathologist, the endosonographer will repeat the FNA until 
enough histological material is obtained to confirm a diagnosis. 
Adverse effect of procedure will be assessed immediately after 
procedure and during the first 30 days with a follow-up phone call. 
Statistical 
analysis 
For descriptive analyses, continuous variables will be reported as a 
mean ± SD or median (interquartile range) and comparison 

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between two groups will be done by Paired t- test. Categorical 
variables will be reported as frequencies with percentages and 
compared using chi-square test or Fisher’s exact test. Two sided P 
values less than 0.05 will be considered statistical significance. 
Descriptive analysis will be performed in terms of DNA yield per 
sample, ability to complete WES, histology and cytology yield per 
sample. 
Trial duration 
Approximately 1 year 

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List of Abbreviations  
This list includes all abbreviations used in the document in alphabetic order. Abbreviated terms 
are spelled out and the abbreviation is indicated in parentheses at its first appearance in the text 
section.   
CBC 
Complete Blood Cell count 
EUS 
Endoscopic Ultrasound 
FNA 
Fine Needle Aspiration  
FNB 
Fine Needle Biopsy 
ROSE 
Rapid onsite evaluation 
GCP 
Good Clinical Practice 
GI 
Gastrointestinal 
INV 
Investigator 
INR 
International Normalized Ratio 
IRB 
Internal Revenue Board 
LN 
Lymph Node 
SNI 
Standardized Numerical Identifier 

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 1.0 INTRODUCTION 
1.1 Background  
Pancreas cancer is a highly fatal disease with a 5 year overall survival is of only 8%1. The 
shorter survival and the poor outcome may be due to late stage presentation, tendency toward 
early metastases and aggressive biologic behavior of pancreas cancer with diverse genetic 
alterations that drive the tumor to early dissemination. Endoscopic ultrasound (EUS) has been 
established as a one of the valuable tools for evaluation of pancreatico-billary disease. EUS- 
guided FNA is important for cytological and pathological evaluation of pancreatic cancer. It is 
minimally invasive, rapid and accurate for staging and diagnosis of pancreas cancer. The 
overall sensitivity of EUS-FNA is 85% (95% CI, 0.84-.86) and specificity of 98% (95% CI, 
0.97-0.99)2 for pancreatic tumor diagnosis. Over the past 20 years there have been significant 
developments done in the field of needle technology and sampling techniques to obtain the 
good quality of cytologic materials. However, over time it has been obvious that size of 
needle, use of stylet and suction techniques has small value in increasing the diagnostic yield 
of pancreas cancer3-5.   
There has been recent advancement in the field of FNB design including needle tip and use of 
more flexible shaft. However, multiple randomized trials and metaanalysis have failed to show 
the superiority of FNB vs FNA for pancreatic lesions6,7. Therefore difficulties still persist to 
diagnose pancreas cancer at early stage. The development of pancreas cancer is a multistep 
process that involves wide range of genetic alteration. However, the paradigm shift for early 
diagnosis of pancreas cancer has changed over recent years. Now a day multiple gene mutation 
and expression profiling of pancreas cancer has become a research priority.  A number of efforts 
have been made for molecular diagnosis of pancreas cancer at early stage that would facilitate to 
design a specific chemotherapy preoperatively. 
EUS- guided FNA is not only important for histopathologic characterization of pancreas lesion 
but has also opened the door for genomic analysis of pancreas cancer tissue. Several techniques 

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have been investigated for improving the pancreatic cancer diagnosis, mainly in on the genetic 
analyses8-10. Studies have demonstrated that the feasibility of genomic profiling, next generation 
sequencing and performing the multiple genetic tests from a small sample of pancreas tumor 
tissue obtained by endoscopic-ultrasound guided fine needle aspiration11. 
With the advances in the needle technology to increase the yield of core tissue in addition to 
cytology, a new needle has been developed termed “Shark Core needle”. The needle has a 
sharpened point in the shape of a fork-tip that promotes the collection of core sample by shearing 
a material form the target lesion during to-and fro-movement of the needle. Recently published 
studies have demonstrated its benefit in acquiring adequate core tissue compared to conventional 
FNA needle 12,13 for pathological evaluation. With this advancement, it will be possible to obtain 
more DNA from the tumor tissue and carry out comprehensive whole exome sequencing of 
pancreas cancer. The contribution will be significant because a large amount of DNA will 
facilitate to carry out genomic profiling and chemo sensitivity test of pancreas cancer 
preoperatively. This will be a significant breakthrough in the field of precision medicine. Though 
several studies have been conducted to characterize the expression profiles of pancreas cancer14 
using FNA specimens, the ability to characterize the global genetic mutational status form FNB 
specimens has not been proven yet. It has been understood that the low quantity of cells, 
obtained from the FNA specimens preclude the precise determination of a tumors genetic 
status15. None of the studies have reported the comparison between FNB needle “Shark Core” 
and Standard FNA needle for genomic profiling of pancreas cancer. Thus, to facilitate a 
development of personalized cancer treatment we sought to determine whether the new FNB 
needle “Shark Core” would be capable of performing widespread whole exome sequencing of 
pancreas tissue from FNB specimens. Therefore the main of this study is to compare the DNA 
yield and genomic profiling of pancreas cancer between FNB needle and standard FNA needle.  

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1.2 Rationale for performing the study 
Based on the above facts, there is still a need of prospective randomized trial to evaluate the 
efficacy of FNB in conducting the comprehensive whole exome sequencing of pancreas cancer. . 
We designed the trial to compare of FNB versus FNA for whole exome sequencing and genomic 
profiling of pancreas cancer. The rationale for choosing the 25g as the standard comparator is 
based on the recent systematic review of needle devices by Wani et al. which concluded “The 
use of a 25-gauge needle is associated with a higher diagnostic yield compared with a 22-gauge 
needle in patients undergoing EUS-FNA of pancreatic masses.”16.  
1.3 Hypothesis 
Hypothesis:  FNB is superior compared to FNA for whole exome sequencing and genomic 
profiling of tissue sampling of pancreas cancer. 
1.4 Benefit/Risk Aspects 
Procedures used in this study are standard medical practice for the evaluation of lesions within or 
adjacent to the GI tract. The shark core FNB is an FDA approved device for sampling of 
submucosal lesions, mediastinal masses, lymph nodes and intraperitoneal masses. FNB was 
introduced as a less invasive alternative to open surgical biopsy.  FNB has been proposed to 
increase the sampling yield and proposed to potentially require fewer needle passes compared to 
conventional FNA. This could potentially improve the tissue yield which is measured by the 
tumor DNA and will facilitate to perform whole exome sequencing of pancreas cancer. This will 
provide significant development for “personalized cancer therapy”. 

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2. STUDY OBJECTIVES
2.1 Primary Objective: 
To compare EUS guide fine needle biopsy versus fine needle aspiration for whole exome 
sequencing and genomic profiling in tissue sampling of pancreatic solid tumors. 
2.2 Secondary Objectives: 
 To compare the DNA yield of FNB versus FNA in tissue sampling of pancreas cancer
including sufficient DNA to complete un-amplified whole exome sequencing.
 To compare the number of needle passes necessary to obtain adequate tissue sample to make a
cytological/histological diagnosis when FNB is used as compared to the conventional FNA for
pancreas cancer
To compare the histology quality of the specimens obtained via FNB as compared to
conventional FNA.
To compare the yield of tissue sufficient for targeted (Foundation Medicine Assay) genomic
profiling of pancreas tumors.
To estimate the safety profile of FNB as compared to conventional FNA. (by historical
comparison)
 Per needle adequacy for cytology

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3. STUDY POPULATION
3.1 Number of Patients: 
50 
3.2 Inclusion criteria 

Male and female patients who are 18 years old or older and are referred for the evaluation
of pancreatic solid mass requiring FNA or FNB.

International normalized ratio (INR) less than 1.5 and platelet count of more than 50,000.

Medically stable to undergo sedation for EUS.

Signed informed consent.
3.3 Exclusion criteria: 

Any medical condition that preclude the patient from having a therapeutic procedure
regardless of the EUS finding.

Pregnant patients.
3.4 Patient Informed Consent 
The Institutional Review Board (IRB) of Mayo Clinic will approve a consent document with 
information about the nature and possible adverse events of the procedure. Patients that might be 

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included in the trial will be given sufficient time to study the written consent form and ask 
questions before signing the consent document. 
3.5 Replacement Policy 
Any patient who does not complete the study will be replaced.  In the case of any withdrawal or 
dropouts, existing data will be retained for evaluation at the end of trial.   
3.6 Compensation and Financial Responsibility 
Since this procedure is “standard of care”, the cost of procedure will be charged to the patient or 
their insurer. The cost of the FNB will be covered by a grant from Medtronic Corporation (grant 
application in process). 
3.7 Patient Remuneration 
No patient remuneration will be provided for this study. 

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4. PRE-PROCEDURE EVALUATION
4.1 History and Physical Examination (all standard of care) 
1) Inclusion/Exclusion Criteria
2) Medical History
3) Medication Review
4) Brief Physical Examination (heart, lungs, abdomen) with vital signs (BP, pulse), and weight
4.2 Laboratory Studies 
1) CBC and INR (within 30 days of enrollment which is standard of care)

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5. INTRA AND POST- PROCEDURE EVALUATION
5.1 Evaluations during Procedure 
Standard monitoring, including continuous cardiopulmonary evaluations, will be performed 
during the EUS exam. In case of immediate complications, including but not limited to, visceral 
perforation or extensive bleeding, the procedure will be terminated and appropriate standard care 
will be provided.  The precise time for each FNA procedure as defined by the first puncture 
attempt to the completion of expressing the cytologic material on the glass slide and formalin.  
5.2 Post-Procedure Evaluations 
All of the participants of the study will receive the standard post-EUS care until discharge. Any 
adverse effects of the procedure will be assessed immediately after procedure and during the first 
30 days with a follow-up phone call. 

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6. SUBJECT WITHDRAWAL AND STUDY TERMINATION CRITERIA
6.1 Subject Withdrawal 
Subjects will be withdrawn from the study for any of the following reasons: 
1) Voluntary withdrawal - Any patient may remove himself from the study at any time without
prejudice to his medical care.
2) Complication related Withdrawal - Any patient with a serious immediate complication due to
the EUS-FNA will be withdrawn.
6.2 Confidentiality 
Only the investigators and their office staff will have access to the data that identifies the patient 
by name.  Patients will be identified only by Standardized Numerical Identifier (SNI) and patient 
identity will remain confidential if material from the record is used for publication or for 
educational purposes.  
6.3 Interim Analysis 
No interim analysis is planned. Biannually the available data will be reviewed retrospectively 
and the rate of anticipated complications will be calculated. Serious adverse events including any 
life threatening events, inpatient hospitalization or prolongation of existing hospitalization or any 
unanticipated complication including any death as a result of the procedure will be reported to 
the Mayo Clinic IRB. 

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7. ETHICS AND REGULATORY
7.1 Institutional Review Board (IRB) 
Mayo Clinic Institutional Review Board Committee (IRB) will review the study protocol and 
any amendments.  The IRB will review the subject informed consent form, their updates (if any) 
and any written materials given to the subjects.   
7.2 Regulatory Authority Authorization-Approval-Notification 
The regulatory permission to perform the study will be obtained in accordance with applicable 
regulatory requirements.  All applicable ethical and regulatory approvals will be available before 
a subject is exposed to any study-related procedures, including screening tests for eligibility.   
7.3 Ethical Conduct of the Study 
This study will be conducted in accordance with the ethical principles that have their origins in 
the Declaration of Helsinki, in compliance with the approved protocol, Good Clinical Practice 
(GCP) and applicable regulatory requirements.   

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7.4 Subject Information and Consent 
The Investigator (or designee) will obtain a freely given written consent from each subject after 
appropriate explanation of the aims, methods, potential risks, and any other aspects of the study, 
which are relevant to the subject’s decision to participate.  The consent form must be signed and 
dated by the subject before conducting any study-related procedures, including screening tests 
for eligibility. 
The Investigator will inform the subjects that they are completely free to refuse to enter the study 
or to withdraw from it at any time, without any consequences to their further care and without a 
need to justify. The subject will receive a copy of his/her signed informed consent.   
Each subject will be informed that portions of his/her source records and source data related to 
the study may be reviewed and utilized in the study analysis. Data protection will be handled in 
compliance with Mayo Clinic bylaw. 
7.5 Confidentiality of Subject Data 
The Investigator will ensure that the confidentiality of the patients’ data will be preserved in a 
safe database.  The patients will not be identified by their names, but by SNI, which consists of 
their initials and number in the study. 
7.6 Standardized Numerical Identifier (SNI) 
Each subject will be assigned an identifier that will be used for identification purposes. SNI 
consists of the patient’s initials, a ten digit number that correlates with the date and time of the 
procedure and the initials of the participating investigator in the following format: 
 I 
I 
I 
M 
M 
Y 
H 
Y 
D 
D 
I 
I 
I 
H 
M 
M 

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Patient initial 
Date 
Time 
       Inv. Initial 

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8. STUDY DESIGN:
This is a single-center, prospective randomized controlled trial.  All male and female patients 18 
years old or older that are referred for evaluation of pancreatic solid mass can be considered for 
this trial.  
8.1 Patient Registration and Documentation 
Recruitment of patients will be initiated at the time of pre-procedure evaluation by one of the 
investigators. Any patient that has been referred to undergo an EUS exam for one of the above 
mentioned indications will be consulted regarding the ASPEN trial and his/her questions will be 
answered. If he/she is interested, the consent form will be provided to the patient and he/she will 
be asked to sign the consent form to participate in the study.  
Patient registration section of the “Registration and Data Collection” form will be completed 
prior to performing FNA by the investigator. At this point, each patient will be assigned with a 
Standardized Numerical Identifier (SNI) that will be used for identification and data entry 
purposes in the study database. This form is designed to collect the following information (See 
Appendix A): 
Participants name and date of birth and medical record number
Name of the investigator performing the EUS exam
SNI
Type of the cross-sectional study or endoscopic exam that warranted the EUS evaluation
Size, location and characteristic of the abnormal lesion

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After the EUS exam, the investigator will complete the post procedure information section of the 
“Registration and Data Collection” form that collects the following information (Appendix A): 
Size, location and characteristic of the lesion based on the EUS exam
Size and type of the needle that was used for conventional FNA (25-gauge needle). 25g
needle (brand of needle will be physicians choice) will be used as the standard needle for
this study. If an adequate sample is not obtained after a total of 2 passes, the endoscopist
may change to a different needle of their choice.
Immediate complications that were noticed during the procedure or during the recovery
period
8.2 Study Protocol 
1) Filling the “Pre-Procedure Patient Registration” section of the “Registration and Data
Collection” form
2) Review of the signed informed consent before the start of procedure.
3) All patients will undergo conscious sedation or Monitored Anesthesia Care (MAC) for the
duration of the procedure. Conscious section and/or MAC are the standard method of
sedation for all patients that have EUS at our institution, unless contraindicated.
4) Morphologic features of the lesion will be examined and documented in the procedure
report.  Based on the location and type of the lesion, the participating endosonographer
determines the necessity of performing an FNA. If FNA deemed necessary, the patient will
be included in the study.

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5) Randomization of needle order. The first needle to be used will be selected by
randomization using block randomization in envelopes (FNA first, FNB first) by tumor
location (1 block for head tumors, 1 block for body/tail tumors).
6) Conventional FNA will be alternated with FNB (22 gauge for  all sites) in the usual fashion
for obtaining histological material. A minimum of 2 passes (1 with each needle) will be
obtained from all lesions. Conventional FNA will be performed with a standard 25g needle
(our standard needle) in the usual fashion using back and forth passes for 30 seconds.
Negative pressure will be applied for both needle types using the “capillary” technique of
withdrawing the stylet slowly (5-10cm per second) during the to and fro movement of the
needle.  All FNA material will be expressed onto a glass slide. Visible core samples will be
removed and placed in formalin after an initial “touch prep” (light touch of tissue to slide).
The presence or absence and length (in mm) of grossly visible each core will be recorded by
the cytotechnologist/study personnel. Standard passes (alternating with each needle) will be
repeated in pairs until an adequate sample is obtained or the endoscopist feels that no further
sampling is warranted.  Then, a 2nd and 3rd set of passes (one with each needle) will be
performed to collect material for biobanking and DNA and RNA extraction. The sample
will be expressed into a sealed biospecimen container and flash frozen in liquid nitrogen,
then stored at -80C. If no material is obtained, the research pass will be repeated up to a
maximum of 2 passes for each needle.
7) After each pass of needle, the on-site cytotechnologist will evaluate the adequacy and the
degree of the pathological changes in the obtained material. Based on the information
provided by the cytotechnologist, the endosonographer will repeat the FNA until enough
material is obtained to confirm the clinical diagnosis. The initial determination of adequacy
will be made by the onsite cytotechnologist. The final determination of cytological
adequacy will be made by the study pathologist (AN) based on the criteria below.
8) Separate sets of slides and formalin jars will be produced in the GI suite from the tissue
obtained from each type of the needle. Each pass will be placed on separate slides and

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formalin jars with labelling (A, B). 9)    In order for the pathologist to remain blinded of the 
needle type that the tissue was obtained with, two separate slide sets will be generated and 
each set will be labeled as “Slide Set A or B”. Slides that are prepared from the FNB will be 
labeled as “Slide Set B” depending on the parity of the assigned SNI number. Slides that are 
prepared from the conventional FNA needle will be labeled as “Slide Set A” to complete 
each pair10) Pathology interpretation for clinical purposes will be done by the on-call 
pathologist of the day, using all material available. For study purposes a single study 
pathologist (AN) will interpret all of the slides. The pathologist will evaluate the slides for 
cell quantity (adequate vs. scanty/acellular), grade of dysplasia (atypical vs. malignant 
cells), and presence of core samples.  
11) Each slide set (A or B) will be evaluated and reported separately for research purposes and
accumulatively to generate the patient’s pathology report for medical records.
12) Quarterly, the available data will be reviewed retrospectively and the rate of anticipated
complications will be calculated.  Anticipated complications of EUS with FNA include, but
are not limited to post-procedure pancreatitis, bleeding, perforation, and infection. Serious
adverse events from FNA are rare but include life threatening events, inpatient
hospitalization, prolongation of existing hospitalization or any unanticipated complication
including death as a result of the procedure will be reported to Mayo Clinic IRB.
13) DNA extraction and quantification: One or two biopsy tissues are first digested, then DNA
extractions will be performed using the silica membrane-based column DNA extraction
method with the QIAamp DNA Mini Kit.  This column based extraction method provides
purified DNA which is free of protein, nucleases and other contaminants or inhibitors.  This
kit is used for fresh or frozen tissue, cells, and blood.  If larger tissue samples are obtained;
the Puregene DNA extraction will be used and is a salt precipitation method.  Tissues are
lysed and DNA is precipitated from the cells.  This method allows for purification of high
molecular weight DNA for downstream use.

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For DNA quantification, the Trinean DropSense96 spectrophotometer will be used which is 
a multichannel spectrophotometer for quick and precise UV/VIS spectral analysis of 
microliter droplets of DNA.   This method allows for the measurement of total and double 
stranded DNA as well as purity and quality ratios (A260/280 and A260/230). 
For whole exome sequencing, a minimum of 1.1 ug of DNA is needed (personal 
communication, Joshua Gorman; Department of Laboratory Medicine). Thus for this study 
purpose, 1.1 ug of DNA will be considered a “sufficient” sample.  Whole exome 
sequencing, using a minimum of 1.1 ug of DNA, sequencing will be performed using 
sequence type: Exome Sure Select v5 + UTR (71MB) at 50% on target (PE 101 base) on the 
Version 4 HiSeq instrument. 
In order to validate that WES can be performed a subset of 5 samples from each needle will 
be evaluated by whole exome sequencing using Illumina standard methods. Only samples 
with at least 5 micrograms of DNA will be evaluated. The WES will be performed in a de-
identified manner and will not be used clinically for the care of the patient.  
14. All the pathological data collection will done by following guidelines from the
Papanicolaou   Society of Cytopathology Guidelines 21, 22.
8.3 9. STATISTICAL CONSIDERATIONS 
9.1 Statistical Analysis 
This is paired sample study of FNB vs FNA for whole exome sequencing and genomic profiling. 
For descriptive analyses, continuous variables will be reported as a mean ± SD or median 
(interquartile range) and comparison between two groups will be done by Paired t- test. 
Categorical variables will be reported as frequencies with percentages and compared using chi-
square test or Fisher’s exact test. Two sided P values less than 0.05 will be considered statistical 

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significance. Descriptive analysis will be performed in terms of DNA yield per sample, histology 
and cytology yield per sample. Sample size calculation is done after interim analysis.Sample 
size calculation: 
From our preliminary data from 5 PDAC patients and using the Foundation Medicine adequacy 
criteria, 2 (40%) had an adequate sample of DNA with FNA, while all 5 (100%) patients had 
adequate DNA with FNB. Although this is a very small sample size it is consistent with the 
notion that FNB may be much superior to FNA. Based on this preliminary data, it seems 
reasonable to expect that the difference in proportions of adequate samples by the two methods 
might be 25% or more. Assuming we have 50 patients who all are sampled with both FNA and 
FNA, then with a McNemar’ s test we will have 80% or higher power to detect a difference in 
adequacy proportions between FNA and FNB (at the 5%   significance level) of ≥25% if ≤ 38% 
of patients have discordant results for FNA and FNB, or a difference of ≥ 20% if ≤ 25% are 
discordant. The following are just 3 of many possible examples of scenarios whereby we will 
have sufficient power with a sample size of 50 patients, all of which are consistent with the 
findings in the preliminary data: 
Example A (25% difference): 
FNA adequate? 
Yes 
No 
FNB 
adequate? 
Yes 
≥ 58.5 % 
≤ 31.5 % 
90 % 
No 
≤ 6.5 % 
≥ 3.5 % 
10 % 
65 % 
35 % 
Example B (25% difference): 
FNA adequate? 
Yes 
No 
FNB 
adequate? 
Yes 
≥ 48.5 % 
≤ 31.5 % 
80 % 
No 
≤ 6.5 % 
≥ 13.5 % 
20 % 
55 % 
45 % 

   (Ver.1.0) 
RC NUMBER:    
28
Example C (20% difference): 
FNA adequate? 
Yes 
No 
FNB 
adequate? 
Yes 
≥ 68% 
≤ 22% 
90 % 
No 
≤ 2% 
≥ 8% 
10 % 
70 % 
30 % 
References: 
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sciences. 2011;56(8):2409-2414.

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29
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