Clinical trial NCT02775695
Conditions: Resectable Pancreatic Cancer
Keywords: pancreatic cancer Resectable Pancreatic Cancer Doxycycline Metakaryote cells Eukaryotic cells Cancer Stem Cells Targeted Cancer Stem Cell Therapy Multi-metakaryocide therapy Doxycycline Hyclate Tetracycline
Sponsor: Medical College of Wisconsin
Start date: 2017-04-03; Completion date: 2022-05-26
Enrollment: 12 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This is a window-of-opportunity study that examines the efficacy of doxycycline, and FDA-approved oral antibiotic, on metakaryotic (cancer stem cells) in resectable pancreatic cancer following eight weeks of treatment.

Detailed description:
BACKGROUND AND RATIONALE:

Pancreatic tumors have two distinct cell populations -- eukaryotic tumor cells and metakaryotic cells. The first cell type divides quickly but must stop at a certain point. Metakaryotic cells, also called cancer stem cells, divide less frequently but have an unlimited number of cell divisions. Chemotherapy works well on eukaryotic cells. Metakaryotic cells are resistant to chemotherapy and radiation, so they are more difficult to eliminate.

Massachusetts Institute of Technology basic science researchers working with the Medical College of Wisconsin pancreatic cancer group demonstrated in the laboratory that doxycycline can kill both eukaryotic and metakaryotic cells.

This study's goal is to discover if the metakaryocidal drug doxycycline kills any significant fraction of the metakaryotic cells found in treated pancreatic tumors. Targeting metakaryotic cells may decrease cancer relapse and metastases. The development of antimetakaryotics is vital for pancreatic cancer patients, who are at risk for disease recurrence and cancer-related death.

STUDY OBJECTIVES:

Primary Objectives:

To assess the efficacy of doxycycline on inducing metakaryotic cell death in primary pancreatic tumors from patients with resectable pancreatic cancer.

Secondary Objectives:

* To determine the plasma drug concentrations of the study drug at baseline and at days 1, 3, 5, 8, 15, 22, 29, and at restaging and at the time surgery.
* To assess the histopathologic treatment response of the primary tumors which have undergone neoadjuvant gemcitabine based chemoradiation and concurrent doxycycline therapy.
* To enumerate the number of observed dead/dying metakaryotes per 1 gram of resected pancreatic tissue.

STUDY PROCEDURES:

Patients will take 100 mg doxycycline twice daily for a period of eight weeks (56 days). Following standard-of-care (not study trial-related) chemotherapy, patients will receive radiation therapy. Patients will receive doxycycline beginning on the first day of radiation therapy. Following this, patients will undergo surgery four to five weeks after completion of chemoradiation. Doxycycline will be discontinued five to seven days prior to surgery.

This study involves pharmacokinetic studies, which means that patients will have blood draws several times so that serum levels may be evaluated.

Arms: arm 1: Patients will receive oral doxycycline and trough serum concentrations for pharmacokinetic studies will be obtained.
Interventions: intervention 1: Doxycycline
Intervention descriptions: intervention 1: Treatment with doxycycline hyclate will be administered as an oral agent on an outpatient basis. Patients will receive doxycycline 100 mg twice daily for a period of 8 weeks (56 days). Upon registration (baseline) and at the first, third, and fifth days of doxycycline therapy, patients will be seen to obtain trough serum concentrations for pharmacokinetic studies. Additional serum levels will be checked on days 8, 15, 22 and 29.

Protocol documents:
Prot_SAP_000.pdf: 
Clinical Trials.gov number:  NCT02775695 
Protocol Title:  Efficacy of doxycycline on metakaryote cell death in patients with 
resectable pancreatic cancer 
 
Document date:  1/12/18 
 


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PRO 28944 IIT Tsai v1/12/18 
Efficacy of doxycycline on metakaryote cell death in patients with resectable 
pancreatic cancer 
 
Principal Investigator: 
Susan Tsai, MD, MHS 
 
Assistant Professor   
 
Division of Surgical Oncology 
 
Froedtert Hospital and the Medical College of Wisconsin 
 
9200 W. Wisconsin Ave. 
 
Milwaukee, WI 53226 
 
Tel: 414 955 7646 
 
stsai@mcw.edu 
 
Protocol Team: 
Kathleen K. Christians, MD, FACS,  
Beth Erickson, M.D. 
Douglas B. Evans, MD 
Ben George, MD 
William Hall, MD 
Bryan Hunt, M.D. 
Matt Riese, M.D., Ph.D. 
Paul Ritch MD 
Medical College of Wisconsin 
9200 W Wisconsin Ave. 
Milwaukee, WI  53226 
 
 
William G. Thilly, Sc.D. 
 
77 Massachusetts Ave 
 
Cambridge, MA 02139 
 
Tel: 617 253 6221 
 
thilly@mit.edu 
 
 
Elena V. Gostjeva, Ph.D. 
 
77 Massachusetts Ave 
 
Cambridge, MA 02139 
 
Tel: 617 253 02139 
 
gostjeva@mit.edu 
 
 
William R. Krick 
 
State Laboratory of Hygiene 
 
2601 Agriculture Dr 
 
Madison WI 53718 
 
Tel: 608 224 6251 
 
william.krick@slh.wisc.edu 
 
 
 
 
 


 
 
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PRO 28944  1/12/18 
Statistician: 
Spencer Huang, Ph.D. 
 
Division of Biostatistics 
 
UW Milwaukee, School of Public Health 
 
1240 N 10th St 
 
Milwaukee, WI 53201 
 
Tel: 414 227 5006 
 
huangcc@uwm.edu 
 
 
Sponsor: 
Batterman Foundation Award 
 
 
 
Study Coordinator: 
 
Cancer Center Clinical Trials staff 
Froedtert Hospital and the Medical College of Wisconsin 
9200 W. Wisconsin Ave. 
Milwaukee, WI 53226 
Tel: 414-805-8900 
  
 
Pharmacists 
David Herrman, PharmD 
Kate Lewis, PharmD 
Angela Urmanski, Pharm D.  
Froedtert Hospital  
Inpatient Pharmacy  
9200 W Wisconsin Ave  
Milwaukee, WI 53226 


 
 
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PRO 28944  1/12/18 
CLINICAL STUDY PROTOCOL  
Efficacy of doxycycline on metakaryote cell death in patients with resectable pancreatic 
cancer 
 
 
Indication: 
Resectable Pancreatic Cancer  
Phase: 
Phase II 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Protocol Histoy 
Original 
 
           Original / Version #1 /09/30/16 
 
 
           Version #2 Amendment v042817 
 
 
           Version #3 Amendment v1/12/18    
 
 
 
 
 
 
 
 
 
This is an investigator-initiated study.  The principal investigator Susan Tsai, MD, MHS (who 
may also be referred to as the sponsor-investigator), is conducting the study and acting as the 
sponsor.  Therefore, the legal/ethical obligations of the principal investigator include both those 
of a sponsor and those of an investigator. 


 
 
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PRO 28944  1/12/18 
PROTOCOL SUMMARY  


 
 
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PRO 28944  1/12/18 
Study Title:  Efficacy of doxycycline on metakaryote cell death  in patients with resectable 
pancreatic cancer 
Phase:  Phase II 
Number of Patients:   Expected enrollment of 12 patients to for an evaluable primary endpoint in 10 
patients. 
Study Objectives  
Primary Objectives: 
To assess the efficacy of doxycycline on inducing metakaryotic cell death in primary pancreatic 
tumors from patients with resectable pancreatic cancer. 
Secondary Objectives: 
• 
To determine the plasma drug concentrations of the study drug at baseline and at days 1, 3, 
5, 8, 15, 22, 29, and at restaging and at the time surgery. 
• 
To assess the histopathologic treatment response of the primary tumors which have 
undergone neoadjuvant gemcitabine based chemoradiation and concurrent doxycycline 
therapy. 
• 
To enumerate the number of observed dead/dying metakaryotes per 1 gram of resected 
pancreatic tissue. 
Overview of Study Design:   
This is a window of opportunity trial which will test the efficacy of doxycycline on metakaryotic cell 
death after 8-10 weeks of treatment. 


 
 
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PRO 28944  1/12/18 
Study Population:   
Inclusion criteria: 
1. Patients must have histologically or cytologically confirmed pancreatic adenocarcinoma 
2. Patients will be receiving neoadjuvant therapy for resectable pancreatic cancer in 
anticipation of surgical resection. Resectable stage will be defined by radiographic criteria 
including; (a) no radiographic evidence of arterial abutment of the celiac, superior 
mesenteric, or hepatic arteries, and (b) < 50% tumor-induced narrowing of the superior 
mesenteric vein or portal vein. 
3. Patients who will receive neoadjuvant therapy (chemoradiation) are eligible.   
4. Age ≥18 years.  
5. ECOG performance status ≤2 (Karnofsky ≥60%). 
6. Life expectancy of greater than 6 months 
 
 
 
 
 
7. Patients must have normal organ and marrow function as defined below: 
a. leukocytes 
≥3,000/mcL 
b. absolute neutrophil count ≥1,500/mcL 
c. platelets 
≥100,000/mcL 
d. Creatinine clearance ≥60mL/ min or cretinine ≤1.5mg/dL 
e. Bilirubin ≤2mg/dL: If the subject’s serum bilirubin is greater than two at the 
time of enrollment but the patient has demonstrated a progressive decline and 
is anticipated to be ≤2mg/dL prior to start of therapy they are eligible, at the 
discretion of the trial investigators and their treatment assignment.  
8. Have no active or chronic infection with HIV, Hepatitis B or Hepatitis C 
9. Ability to understand and the willingness to sign a written informed consent document. 
Exclusion Criteria: 
1. Patients with more clinically advanced pancreatic cancer (borderline resectable, locally 
advanced, or metastatic) 
2. Uncontrolled intercurrent illness including, but not limited to, ongoing or active infection, 
symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, or 
psychiatric illness/social situations that would limit compliance with study requirements. 
3. Pregnant women are excluded from this study because the effects of metakaryocidal agents 
have the potential for teratogenic or abortifacient effects.   
4. Previous history of other malignancy (other than cured basal or squamous cell carcinoma of 
the skin or cured in-situ carcinoma of the cervix) within 2 years of study enrollment. 
5. Active or chronic HIV, hepatitis B or hepatitis C 
6. Patients who are receiving other investigational drugs or enrolled in other clinical trials 


 
 
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PRO 28944  1/12/18 
7. Inability to undergo scheduled blood acquisition per protocol. 
8. Drug specific exclusion including history of allergic reactions to tetracyclines. 
9. Prior treatment with doxycycline within a 7 day washout period prior to initiating treatment 
with alternate antimetakaryocidal medication. 
 
Duration of Study:  Accrual period: Approximately 12 months. 
 
 
 


 
 
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PRO 28944  1/12/18 
ABBREVIATIONS USED: 
CT: 
 
COMPUTED TOMOGRAPHY 
MRI:  
MAGNETIC RESONANCE IMAGING 
AE:  
 
ADVERSE EVENT 
AESIS:  
ADVERSE EVENTS OF SPECIAL INTEREST  
DLT:   
DOSE LIMITING TOXICITY  
FDA:   
FOOD AND DRUG ADMINISTRATION 
MTD:   
MAXIMUM TOLERATED DOSE  
PK:  
 
PHARMACOKINETIC 
SAE:   
SERIOUS ADVERSE EVENT 
 
 


 
 
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STUDY SCHEMA  
 
 


 
 
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PRO 28944  1/12/18 
TABLE OF CONTENTS 
STUDY SCHEMA ..........................................................................................................................9 
1.1 
Primary Objective ..................................................................................................12 
1.2 
Secondary Objective ..............................................................................................12 
2. 
BACKGROUND ...............................................................................................................12 
2.1 
Metakaryotic Biology ............................................................................................12 
2.2 
Amitotic division: replication and segregation of the metakaryotic nuclear 
genome via dsRNA/DNA intermediates ................................................................13 
2.3 
Metakaryotes Are Present in Resected Pancreatic Cancers ...................................14 
2.4 
Determining metakaryocidal activity .....................................................................14 
2.5 
Identification of metakaryocidal agents .................................................................16 
2.6 
Doxycycline Hyclate ..............................................................................................19 
3. 
PATIENT SELECTION ....................................................................................................23 
3.1 
Eligibility Criteria ..................................................................................................23 
3.2 
Exclusion Criteria ..................................................................................................24 
3.3 
Inclusion of Women and Minorities ......................................................................24 
4. 
REGISTRATION PROCEDURES ...................................................................................24 
4.1 
General Guidelines.................................................................................................24 
4.2 
Registration Process ...............................................................................................25 
5. 
STUDY DESIGN...............................................................................................................25 
5.1 
Treatment Plan .......................................................................................................25 
5.2 
Surgery ...................................................................................................................26 
5.3 
Doxycycline pharmacokinetics ..............................................................................26 
5.4 
Pathologic Assessment...........................................................................................26 
5.5 
General Concomitant Medication and Supportive Care Guidelines ......................26 
5.6 
Duration of Therapy ...............................................................................................27 
5.7 
Duration of Follow Up ...........................................................................................27 
5.8 
Patient Discontinuation ..........................................................................................27 
6. 
DOSE DELAYS/DOSE MODIFICATIONS ....................................................................28 
7. 
ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS ............................29 
7.1 
Definitions..............................................................................................................29 
7.2 
Relationship ...........................................................................................................29 
7.3 
Reporting of Adverse Events .................................................................................30 


 
 
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7.4 
Study Risks ............................................................................................................30 
7.5 
Risk Discussion ......................................................................................................30 
7.6 
Study Benefits ........................................................................................................30 
8. 
STUDY CALENDAR .......................................................................................................30 
9. 
DRUG PROCUREMENT .................................................................................................32 
9.1 
Drug Dispensing ....................................................................................................32 
10. 
MEASUREMENT OF EFFECT........................................................................................32 
10.1 
Definitions..............................................................................................................32 
10.2 
Methods for Evaluation of Measurable Disease ....................................................32 
10.3 
Response Criteria ...................................................................................................33 
10.4 
Specimen Collection ..............................................................................................33 
11. 
DATA REPORTING / REGULATORY REQUIREMENTS ...........................................34 
11.1 
Data Reporting .......................................................................................................34 
11.2 
Data Monitoring .....................................................................................................34 
11.3 
Collaborative Agreements Language .....................................................................34 
11.4 
Ethics Consideration ..............................................................................................35 
11.5 
Publication Policy ..................................................................................................35 
12. 
STATISTICAL CONSIDERATIONS...............................................................................35 
12.1 
Study Design/Endpoints.........................................................................................35 
12.2 
Sample Size/Accrual Rate ......................................................................................36 
13. 
REFERENCES ..................................................................................................................38 
 
 


 
 
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PRO 28944  1/12/18 
OBJECTIVES 
1.1 Primary Objective 
To assess the efficacy of doxycycline on inducing metakaryotic cell death in primary pancreatic 
tumors from patients with resectable pancreatic cancer using cytometric analysis. 
1.2 Secondary Objective 
1. To determine the plasma drug concentrations of the study drug at baseline and at days 1, 
3, 5, 8, 15, 22, 29, and at restaging and at the time surgery. 
2. To assess the histopathologic treatment response of the primary tumors which have 
undergone neoadjuvant gemcitabine based chemoradiation and concurrent doxycycline 
therapy. 
3. To enumerate the number of observed dead/dying metakaryotes per 1 gram of resected 
pancreatic tissue. 
 
2. BACKGROUND 
2.1 Metakaryotic Biology 
Pancreatic cancer is a lethal disease which is distinguished by tumors which metastasize and 
cannot be cured by present day chemotherapy or radiotherapy. Even in the earliest stages of the 
disease, patients who undergo “curative” resection inevitably succumb to tumor recurrence and 
metastases. It is hypothesized that a small fraction of all cells in a human tumor are responsible 
for tumor regrowth after treatment. Such cells are called "cancer stem cells". Tumors are 
hypothesized to be composed of two distinct cell populations:  eukaryotic tumor cells which 
often rapidly divide but have a finite number of cellular divisions, and a stem-cell population 
(possibly metakaryotic cells) which divide less frequently and may have an unlimited number of 
cellular divisions.  Eukaryotic cells can be exquisitely sensitive to chemotherapeutic agents and 
radiotherapy which capitalize on the interruption of cellular mitotic division.  In contrast, stem 
cells have previously been described as being relatively chemoresistant and radioresistant, and 
therefore are difficult to eradicate. Cell antigens such as CD44 and CD133 have been associated 
with cancer stem cells, but have variable specificity as a marker; not all cells that were labeled as 
CD44+/CD133+ behaved as stem cells.  
Using an alternative approach, our colleagues at MIT have identified and reported a novel 
amitotic cell type, the metakaryote, using 
clear easily reproduced cellular 
morphological criteria.  Before 2003, 
cancer stem cells could not be identified 
by microscopic examination. At the 
Massachusetts Institute of Technology, 
Dr. Elena Gostjeva in conjunction with 
Professor William Thilly, identified cells 
which had unusual large, hollow, bell-
shaped nuclei on one end of the cell, 
which they termed “metakaryotic cells. 1-3 
In particular, these cells are remarkable 
because unlike eukaryotic cells, when 
Figure 1: Metakaryotic division. (a) Symmetric division 
similar to two paper cups separating (b) asymmetric division, 
yielding a bell-shaped nuclei and a close nuclei 
 


 
 
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PRO 28944  1/12/18 
metakaryotic cells divide the chromosomes do not condense and separate in the process of 
mitosis. Instead their nuclei divide by an amitotic process resembling the separation of two paper 
cups making two metakaryotic cells.  (Figure 1a) Other times they would give rise to a cell with 
a bell-shaped nucleus and one with a closed nucleus. (Figure 1b)  Some of the new cells with 
closed nuclei then divided many times by mitoses; others did not further divide. This ability of 
metakaryotic cells to both grow in number and divide asymmetrically to create eukaryotic cells 
marked them as "stem cells". Importantly, since metakaryotic cells do not require mitosis for 
division, these cells were predicted to be resistant to chemotherapy and radiotherapy. In passing, 
Dr. V. Koledova of the MIT group has found the CD33 and CD144 antigens embedded in the 
cytoplasmic wall of mononuclear metakaryotes but not associated with the tubular syncytial 
metakaryotes. Further she found many eukaryotic cells that expressed these antigens in colonies 
that did not contain metakaryotic stem cells. 
2.2 Amitotic division: replication and segregation of the metakaryotic nuclear genome via 
dsRNA/DNA intermediates 
Quantitative Feulgen-based DNA image cytometry demonstrated that bell shaped nuclei of 
metakaryotic cells double their DNA content during and after symmetric and asymmetric 
amitotic fissions rather than in the separate, pre-mitotic S-phase of eukaryotic cells[1,2,5, 8]. The 
MIT group has observed that metakaryotic cells undergo division by first transforming their 
dsDNA genomes into two pangenomic dsRNA/DNA intermediates.  The RNA/DNA 
intermediate is then segregated into sister nuclei in both symmetric and asymmetric amitotic 
divisions. Metakaryotic nuclei in amitosis stain strongly with an antibodies specific to 
dsRNA/DNA. In Figure 2a metakaryotic cells from human fetal spinal cord forming syncytia is 
shown illustrating this phenomenon. After segregation the dsRNA/DNA intermediate is 
retransformed to dsDNA form by combined action of RNAse H1 and a complex of other DNA 
polymerases, including DNA polymerases beta and zeta. Both DNA polymerases beta and zeta 
are expressed in large quantities (~300,000 copies per nucleus) in nuclei containing the 
dsRNA/DNA intermediate. Co-expression of these two DNA polymerases was discovered by 
Christopher Lawrence (U. Rochester) in S. cervesiae after ionizing irradiation; both polymerases 
are associated with high mutation rates. Mechanistically, it is believed that the two polymerases 
contribute to the highly error prone DNA repair process that by-passes otherwise lethal lesions in 
the DNA strand being copied.  
The phenomenon of using 
dsRNA/DNA replicative 
intermediates in conjunction 
with error prone DNA 
polymerases offers a 
mechanistic explanation of 
why the metakaryotic stem 
cells survive and demonstrate 
continued robust cell division 
after standard treatment 
regimens of radio- and chemo-
therapy. 
 
The research team at MIT has 
examined pancreatic, lung, and colon surgical specimens taken from patients who had undergone 
Figure 2:  Metakaryotic division via dsRNA/DNA intermediate. (a) 
Metakaryote in syncytium. (b) immunofluorescent staining for dsRNA/DNA 
(red) duplex, (c) counterstain DAPI (blue). 
 


 
 
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PRO 28944  1/12/18 
preoperative chemotherapy or radiotherapy and observed that in such specimens most of the 
eukaryotic cells but not the metakaryotic tumor cells were killed. Furthermore, chemo-resistant 
and radio-resistant metakaryotic stem cells were also found in established colon and pancreatic 
cell lines. The research team further identified multiple FDA- approved medications which 
specifically kill metakaryotic cells in vitro when given at high doses over a prolonged period of 
time.  Interestingly, such metakaryocidal doses of these drugs did not affect the growth of 
eukaryotic cells. One of these drugs is doxycycline, which has a tetracycline antibiotic with a 
long history of clinical use and a well described safety profile. Coincidentally, an independent 
report has recently been published in which antibiotics (erythromycins, tetracyclines, and 
chloramphenicol – but in particular doxycycline) were also used to selectively target cancer stem 
cells. 4  
2.3 
Metakaryotes Are Present in Resected Pancreatic Cancers 
Our primary collaborators, Dr. Elena Gostjeva and Professor William Thilly of MIT have 
published a series of peer-reviewed papers that have advanced and supported the hypothesis that 
the stem cells of organogenesis and carcinogenesis are non-mitotic metakaryotic cells that can be 
easily recognized by their pronounced hollow, bell shaped nuclei in mononuclear and tubular 
syncytial structures by ordinary light microscopy. The MCW Pancreatic Cancer Research Group 
has collaborated with Dr. Gostjeva over the past four years to supply additional pancreatic tissue 
from resected specimens for analysis. (Figure 3) Together they have demonstrated the presence 
of mononuclear metakaryotes in primary pancreatic tumors and both mononuclear and tubular 
syncytia (aka Indian file figures) in peritoneal metastases of pancreatic adenocarcinomas. 
Widespread pyknotic (dead) eukaryotic (non-stem) cells were observed in the presence of many 
rapidly dividing 
metakaryotic cells in 
primary pancreatic 
adenocarcinomas excised 
3-6 weeks after the 
completion of neoadjuvant 
therapy. Insofar as no 
dead or visibly disrupted 
metakaryotic nuclei were 
discovered in these 
specimens, they serve as 
a set of negative controls 
for the proposed trials of 
drugs that have shown 
specific metakaryocidal 
activity in cell culture 
assays at MIT.  
 
2.4 
Determining metakaryocidal activity 
The MIT group grew and inspected a series of cell lines derived from human tumors, e.g. Capan-
1 (pancreas) and HT-29 (colon) in which metakaryotic cells were required for continuous 
propagation. In a series of single cell experiments in microtitre dishes, they demonstrated that 
Figure 3: Metakaryotic cells present in surgical specimens. Post-resection 
analysis of pancreatectomy specimen demonstrates necrotic cells present with 
adjacent intact metakaryote. 


 
 
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PRO 28944  1/12/18 
immortal colonies arose only from single cells that expressed the metakaryotic phenotype in the 
first few doublings. All single cells that did not express the metakaryotic phenotype gave rise to 
terminal colonies that did not grow further on passaging. Using these two cell lines they have 
devised means to measure metakaryocidal activity of test agents by either destruction of 
immortal colonies (clonal assays) or disruption of metakaryotic nuclei as observed by light 
microscopy (cytometric assays).  
 
Clonal assays  
Cells in this form of assay are treated for the desired period of exposure starting 24 hours after 
being seeded in T-flasks dispersed as single cells. Most single cells are non-stem, eukaryotic 
cells but under the conditions developed at MIT about 5-15% of single cells give rise to 
colonies containing metakaryotes that on continued passage are shown to be immortal. But 
large colonies, of ~200 to 2000 cells are formed from single non-stem eukaryotic cells which 
appear to be the earliest "transition" cells of a stable "turnover unit" of 8000 cells similar to the 
8000 cell crypts observed in human colonic adenocarcinomas2. Colonies derived from 
metakaryotic stem cells, in contrast, exceed 4000 cells three weeks post-treatment and, on 
trypsinized transfer, continue to grow, "immortalized" by the presence of the metakaryotic stem 
cells. Thus large colonies obtained in this clonal assay contain a metakaryotic cell which allows 
for continued division of the cells beyond what would be possible for a colony derived from a 
eukaryotic cell. 
Figure 4a  shows decreasing large colonies observed/flask exposed to a range of metformin 
concentrations for 5 weeks. Varying both concentration and duration of exposure revealed 
conditions specifically toxic to metakaryotic stem cells between ~500-2000- microMolar 
weeks. Replating colonies after exposures between 500 and 2000 micromolar weeks showed 
that no immortal cells survived treatment. This is interpreted as a specific metakaryocidal 
effect. Above 2000 micromolar weeks the eukaryotic cells that form mortal colonies were also 
killed (Figure 4b). Some nine drugs have been identified in this way.   
 
 
Figure 4: Metakaryote clonal assay. HT-29 cells after 5 week exposure to metformin.  (a) Clonal assay - cells were 
initially seeded at ~800 total cells resulting in an average of ~375 total large colonies. (b) Five weeks at ~100 microM 
or two weeks at~250 microM reduced the total number of large colonies by ~ 10% 
 


 
 
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PRO 28944  1/12/18 
Cytometric assays  
Following a prolonged (at least 2 week) period of time of drug exposure at concentrations well 
below plasma levels associated with frank toxicity, dead metakaryotic cells could be identified 
and enumerated by the appearance of the chromatin of the metakaryotic nuclei, well dispersed in 
healthy growing cells (Figure 5a) condensed into thick rope-like structures easily identified by 
cytometry (Figure 5b, 5d). With verapamil an additional marker of cytotoxicity was observed 
(Figure 5c) when treated cultures were stained with the dye Hoechst 33342. First a single bright 
blue bar then a cross (shown) and a three bar asterisk appeared in one to three weeks of 
verapamil treatment. For all metakaryocides found to date exposure of at least two weeks has 
been required to eliminate all immortal colony forming metakaryotic cells. 
 
2.5 
Identification of metakaryocidal agents   
The investigators at MIT began to test a variety of drugs on the cell cultures from pancreas 
(CAPAN-1) and colon (HT-29) cells. Initial investigation included several classes of 
chemotherapeutic agents, including antimetabolites, alkylating agents, and anti-tumor 
antibiotics. As expected, these agents had no effect on metakaryotic cells. The 
chemotherapeutic agent, gemcitabine, was effective killing eukaryotic but not metakaryotic 
Figure 5: Cytometric appearance of failed metakaryotic division. (a) Untreated metakaryotic nuclei undergoing 
symmetrical amitosis (human fetal hindgut, (b) representative appearance of metakaryote after treatment with 200 
micromolar metformin for two weeks (HT-29 cells). Cells were fixed and stained with Feulgen reagent that renders 
dsDNA purple. (c) Peculiar condensed form of Hoechst 33342 stained chromatin in the cytoplasm of a metakaryotic 
stem cell after two weeks treatment of HT-29 cells with verapamil. At this point metakaryotic cells do not further 
divide and bell shaped nuclei collapse. (d) Capan-1 cells treated with verapamil for two weeks. Feulgen staining 
demonstrates pyknotic nuclear forms.  Further degradation of metakaryotic nuclei is observed. 
 
 
d


 
 
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PRO 28944  1/12/18 
stem cells. However, nine drugs were identified that produced clearly specific metakaryocidal 
effects when treated for five or more weeks at concentrations in the range of FDA 
recommended plasma concentrations or somewhat higher plasma concentrations. In all cases 
the effective concentrations required to kill metakaryotic stem cells were well below the 
concentrations associated with clinical toxicity. Importantly, the eukaryotic cells were not 
immediately killed by conditions that killed metakaryotes, offering the possibility that they 
might be used in patients without the toxicities associated with chemotherapy and radiation 
therapy.  
The concentrations of most 
metakaryocidal drugs required to 
eliminate metakaryotes after a five week 
exposure are well below the levels 
reported as toxic to humans but are 
above the FDA recommended level for 
ordinary use (acetaminophen, celecoxib, 
metformin, verapamil).  Roughly 2 to 8 
fold higher plasma levels might be 
required to create a potent 
metakaryocidal regimen for these drugs. 
One drug appears to be metakaryocidal 
within the approved plasma 
concentration range, doxycycline. 
(Figure 6 and 7) 
 
 
Figure 6. Doxycycline Clonal Assay. Doxycycline maintained 
at a concentration as low as 3 - 10 micromolar for aduration of 
two weeks, reduced the fraction of large HT-29 cell colonies to 
about 90% indicative of a metakaryocidal effect. At 
concentrations at or above 40 micromolar colonies initiated by 
a eukaryotic cell were also killed. (Pink symbol indicates small 
surviving colonies.) 
Figure 7. Cytometric Response to Doxycycline. HT-29 cells after 2 weeks in the presence of (a) 11.3 or (b) 22.6 
micromolar doxycycline. Metakaryotic (bell shaped) show contracted, "pyknotic" metakaryotic nuclei (red arrow) 
amidst unchanged eukaryotic nuclei (blue arrow).  
a
b


 
 
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PRO 28944  1/12/18 
Dr. Elena Gostjeva (MIT) has received some ninety (90) deidentified samples of pancreatic 
adenocarcinomas and metastases from MCW colleagues in the past several years. Using her 
protocol for detecting and imaging metakaryotic cells and distinguishing between live and dead 
metakaryotic cells she has observed many hundreds of metakaryotic cells in samples from those 
patients who had received radiotherapy and or chemotherapy prior to surgery. In none of these 
samples has she detected a dead or dying metakaryotic cell, a result extending and confirming 
her original observations in human lung tumor samples after radiotherapy and chemotherapy. 
 
In this proposed clinical trial, she and a cytopathologist (B. Hunt) will evaluate each patient 
sample until one hundred metakaryotic nuclei have been observed per sample. Each image of a 
live or dead metakaryotic cell will be electronically recorded so that (a.) other observers may 
confirm or dispute each reported observation and (b.) the images may be used in training new 
personnel. 
 
The ultimate goal of the use of drugs found to kill human metakaryotic cancer stem cells in 
culture is to eradicate cancer stem cells in the patient to affect a cure. However the effect of 
doxycycline cannot be predicted based on its plasma concentration alone. There may be 
pharmacokinetic barriers to doxycycline penetrance into tumor or particular areas of tumors due 
to low vascularization, e.g. mesenchyme, or extracellular matrices that slow or prevent drug 
diffusion to the target stem cells.  
 
The target level of 20% is one that would yield at least 10 dead metakaryotic nuclei in a tumor 
from which 100 total metakaryotic cells had been observed and imaged. As it is proposed to scan 
each tumor for 100 metakaryotic cells, no data of a sighting of one (1) or more dead 
metakaryotic nuclei will be lost to analysis. 
 
It should be clearly noted that a clinical correlation such as significantly extended lifespan is 
possible but not expected at this stage of research.  Here the experiment seeks to discover if the 
metakaryocidal drug doxycycline kills any significant fraction of the metakaryotic cells found in 
treated pancreatic tumors.  Such a result would suggest the next steps to be taken including 
testing of increased levels and duration of doxycycline treatment, testing of other metakaryocidal 
drugs commonly used for other medical purposes and testing of methods to increase penetrance 
of metakaryocidal drugs to the target cancer stem cells in tumors and metastases. 
 
In summary, our collaborators at MIT have demonstrated that metakaryotic cells are self-
renewing, pluripotent, and resistant to chemotherapy and radiotherapy. Importantly, the 
metakaryotic process of amitotic division offers a logical explanation for the resistance of these 
cells to anti-mitotic therapies (chemotherapy and radiotherapy). Therefore targeting metakaryotic 
cells may decrease cancer relapse and metastases and the development of anti-metakaryotic is 
vital particularly for pancreatic cancer patients who are at risk for disease recurrence and cancer-
related death. Development of a targeted cancer stem cell therapy potentially benefits patients at 
all stages of disease from operable to metastatic. By targeting the majority of tumor mass 
(eukaryotic cells) with the increasingly effective cytotoxic therapies in addition to targeting 
cancer stem cells (metakaryotic cells) we expect a resultant prolongation of survival and possibly 
durable eradication of disease.  


 
 
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PRO 28944  1/12/18 
In this proposal, we propose to test doxycycline for in vivo efficacy. We propose to discover if 
doxycycline will kill the metakaryotic stem cells in human pancreatic tumors. We hypothesize 
that doxycycline can be administered to pancreatic cancer patients in regimens that deplete and 
possibly eliminate their cancer stem cell populations and result in prolonged disease-free and 
overall survival.  To do this, we propose to examine the efficacy of doxycycline to effect 
metakaryocidal death in a primary pancreatic cancer tumor.  We propose to administer 
doxycycline during the 8 week period of neoadjuvant therapy for patients with resectable 
pancreatic cancer and analyze the resected pancreatic specimen for cytologic evidence of 
metakaryocidal death. A time frame of 8-10 weeks was selected because following neoadjuvant 
chemoradiation, surgery is usually performed at a minimum of 4 weeks from the end of 
radiation. To avoid a rebound effect of metakaryotic proliferation during this period, we plan to 
continue doxycycline during this period, allowing a 5-7 day washout period prior to surgery. 
Pancreatic cancer patients will receive oral doses of doxycycline for 8-10 weeks to maintain 
their plasma concentrations at the upper level of the FDA recommended plasma concentrations. 
Sections of tumors will be removed at the time of surgery and studied using the cytometric 
assays previously described by Dr. Gostjeva2,5 to discover if the treatments have killed or 
disrupted the metakaryotic cells.  
Because the metakaryotic stem cells of human organogenesis have been found to have very high 
mutation rates6,7 it is anticipated that any pancreatic tumor will contain cancer stem cells will 
develop resistance to any single metakaryocidal drug. It is anticipated that treatment with a series 
or a combination of metakaryocidal drugs (e.g. 4, 5 or 6) would be required to kill all of the 
cancer stem cells in a pancreatic or other form of tumor. However, the effectiveness of each 
potentially metakaryocidal drug must be tested and authenticated before it could be expected to 
be useful in such anticipated multi-metakaryocide therapy.  Thus in a first series of trials each 
drug found to be metakaryocidal to tumor stem cells in adenocarcinoma derived cell lines at 
levels expected to be well tolerated by patients will be individually tested in order to discover if 
they are in fact killing metakaryotic cancer stem cells in resected pancreatic tumors.   
 
2.6 
Doxycycline Hyclate Doxycycline hyclate is a member of the tetracycline class of 
antibiotics.  It has a chemical designation of 4-(dimethlamino)-1,4,4a,5,5,6,11,12A-
octahydro-3,5,10,12,12A-pentahydroxy-6-methy-1,11-dioxo-2-naphthacenecarboxamide 
monohydrochlorde.  The empirical formula is (C22H24N2O8HCl)2C2H6OH2O and the 
molecular weight is 1025.89.  It is a yellow to light-yellow crystalline powder which is 
soluble in water. 
Pharmacology of Doxycycline 
Tetracyclines are readily absorbed and are bound to plasma proteins in varying degree. They are 
concentrated by the liver in the bile, and excreted in the urine and feces at high concentrations 
and in a biologically active form. Doxycycline is primarily a bacteriostatic antibiotic. The main 
mechanism of action of doxycycline is on protein synthesis. Doxycycline passes directly through 
the lipid bilayer of the bacterial cell wall and an energy dependent active transport system pumps 
the drug through the inner cytoplasmic membrane. Once inside the cell doxycycline inhibits 
protein synthesis by binding to 30S ribosomes and prevents the addition of amino acids to the 
growing peptide chain. Doxycycline will impair protein synthesis in the 50S ribosomes of 
mammalian cells at very high concentrations but these cells lack the active transport system 


 
 
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PRO 28944  1/12/18 
found in bacteria. However, the mammalian mitochondrial 30S ribosomes may preserve a 
doxycycline binding site and its action may be mediated by inhibition of mitochondrial protein 
synthesis in stem cells. 
Pharmacokinetics 
Absorption and Bioavailability 
Doxycycline is virtually completely absorbed after oral administration and is not subject to 
presystemic metabolism.  The mean bioavailability is approximately 93%. Following a 200 mg 
dose, normal adult volunteers averaged peak serum levels of 2.6 mcg/mL of doxycycline at 2 
hours, decreasing to 1.45 mcg/mL at 24 hours.  
Distribution 
Tissue distribution is good and doxycycline has a strong affinity for renal and lung tissue. Plasma 
protein binding is in the range 82-93% and doxycycline is transferred into breast milk. The 
volume of distribution for doxycycline ranges from 0.9-1.8 lkg-1 and the plasma half-life ranges 
from 18-22 hours. 
Metabolism and Elimination 
Excretion of doxycycline by the kidney is about 40% in 72 hours in individuals with normal 
function (creatinine clearance about 75 mL/min.). This percentage excretion may fall as low as 
1–5% in 72 hours in individuals with severe renal insufficiency (creatinine clearance below 10 
mL/min.). Studies have shown no significant difference in serum half-life of doxycycline (range 
18–22 hours) in individuals with normal and severely impaired renal function. 
 
Safety Experience 
Doxycycline is used in the treatment of a variety of infections cause by susceptible strains of 
Gram-positive and Gram-negative bacteria and certain other microorganisms. 
Clinical experience with doxycycline 100 mg BID 
Review of treatment regimens using doxycycline shows that use of 200 mg/day or 2 x 100 
mg/day in adults is a widely used therapeutic strategy that has been recommended by the US 
CDCP and international public health authorities for treatment of: anthrax, chlamydia, Lyme 
disease, malaria and tick borne rickettsial diseases for which direct citations from texts are 
provided here. No evidence of increased adverse effects were noted in treatment of Lyme disease 
patients for up to one month. Other than clear warnings regarding drug induced solar sensitivity, 
there appear to be no reports (Pub MED, ToxLine) of acute human toxicity associated with a 
prolonged exposure to doxycycline or doxycycline hyclate.  This is notable as it has been in 
worldwide use since the 1940s. Cited immediately below is a report of an adult who was treated 
with 1000 mg/day of doxycycline for twelve years whose various systemic symptoms resolved 
after the drug was discontinued. However, the literature records several cases of doxycycline 
associated psychotropic responses including suicidal feelings, an example of which is cited 
below.  
   
Chronic Doxycycline Intoxication (J Intern Med. 1999 246:591-2) 


 
 
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PRO 28944  1/12/18 
"We report the clinical case of a 12-years' intoxication by doxycycline. A patient with a 
depersonalization and derealization syndrome took 1 g doxycycline per day. In addition to 
hepatocellular necrosis with cholestasis, nephrotoxicity, leukopenia, anaemia and skin 
hyperpigmentation he suffered from hitherto unreported adverse cardiac events as intermittent 
supraventricular tachycardia and sporadic Wenckebach heart block. Despite a long period of 
self-medication these side-effects were reversible." 
 
Anthrax (FDA Safety Alerts for Human Medical Products):  
"The currently recommended dosage regimen of doxycycline for severe disease is 100 mg every 
12 hours for adults and 1 mg per pound (2.2mg per kilogram) every 12 hours for children less 
than 100 pounds."  
 
Chlamydial Infections (2010 CDCP Treatment Guidelines): 
"Chlamydia treatment should be provided promptly for all persons testing positive for 
infection..."  Recommended regimens: Doxycycline 100 mg orally twice a day for 7 days. 
 
Lyme disease (J Am Acad Dermatol. 1995 32(2 Pt 1):223-7) 
"Doxycycline is widely used to treat Lyme disease associated with erythema migrans. Whether it 
is comparable to tetracycline is unknown 
We conducted a two-part retrospective analysis of (1) the safety and efficacy of doxycycline 
compared with tetracycline and (2) the safety and efficacy of a 14-day versus a 20-day course of 
doxycycline. Twenty-seven patients given tetracycline (500 mg four times a day for 14 days 
[group 1]) were compared retrospectively with 21 patients who received doxycycline (100 mg 
two or three times a day for 14 days [group 2]). The results for group 2 were also compared with 
that of 38 patients who received doxycycline for 20 days (100 mg three times daily) in a 
prospective treatment trial (group 3). There was no significant difference in the incidence of 
adverse drug effects or in efficacy at 1 month..." 
 
Malaria (Am J Trop Med Hyg. 2011 84: 517–531): 
"Doxycycline, a synthetically derived tetracycline, is a partially efficacious causal prophylactic 
(liver stage of Plasmodium) drug and a slow acting blood schizontocidal agent highly effective 
for the prevention of malaria. ... Although not recommended for pregnant women and children < 
8 years of age, severe adverse events are rarely reported for doxycycline. This report examines 
the evidence behind current recommendations for the use of doxycycline for malaria and 
summarizes the available literature on its safety and tolerability." "Treatment: 100 mg twice a 
day for 7 days; must be used in conjunction with a fast acting schizontocide." 
 
Polycystic Ovarian Syndrome (Completed randomized clinical trial: NCT01788215; University 
of Rochester). Assess the effectiveness of daily doxycycline use vs. placebo in the primary 
outcome of reduction of serum testosterone in women with PCOS at 12 weeks on study 
medication and upon conclusion of the study at week 24. Doxycycline dose 100 mg BID 
Clinical experience with doxycycline 200 mg BID 
Lyme Neuroborreliosis (Antimicrob Agents Chemother, 1989; p1078-1080) 10 patients treated 
with doxycycline 200 mg BID for 10 days. “Two patients complained of mild phototoxic 
reactions, none suffered adverse gastrointestinal reactions.” 


 
 
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Lyme Disease with Associated Facial Palsy and Meningitis (Clin Infect Dis. 1999; p569-74). 
Prospective clinical trial with 29 patients with Lyme disease and facial palsy. “Mean durations of 
treatment was 10.8 days (range: 9-17).  Doxycycline dose was 200 mg BID for 28 of 29 patients 
and 100 mg BID for an 11 year old child.” 
Relapsed NHL (Open clinical trial: NCT02086591; University of Rochester). Assess the overall 
response rate to doxycycline monotherapy in patients with relapsed indolent and aggressive NHL 
at three months.  Dose of doxycycline is 200 mg BID. 
Warnings and Precautions 
Consult the package insert for comprehensive toxicity information.  Listed below are common 
drug associated toxicities. 
Photosensitivity 
Photosensitivity manifested by an exaggerated sunburn reaction has been observed in some 
individuals taking tetracyclines, including doxycycline. Patients likely to be exposed to direct 
sunlight or ultraviolet light should be advised that this reaction can occur with tetracycline drugs 
and treatment should be discontinued at the first evidence of skin erythema.  
Use in patients with impaired hepatic function 
 Doxycycline should be administered with caution to patients with hepatic impairment or those 
receiving potentially hepatotoxic drugs. Abnormal hepatic function has been reported rarely and 
has been caused by both the oral and parenteral administration of tetracyclines, including 
doxycycline.  
Use in patients with renal impairment 
Excretion of doxycycline by the kidney is about 40%/72 hours in individuals with normal renal 
function. This percentage excretion may fall to a range as low as 1-5%/72 hours in individuals 
with severe renal insufficiency (creatinine clearance below 10ml/min). Studies have shown no 
significant difference in the serum half-life of doxycycline in individuals with normal and 
severely impaired renal function. Hemodialysis does not alter the serum half-life of doxycycline. 
Microbiological overgrowth 
The use of antibiotics may occasionally result in over-growth of non-susceptible organisms, 
including Candida. If a resistant organism appears, the antibiotic should be discontinued and 
appropriate therapy instituted. Pseudomembranous colitis has been reported with nearly all 
antibacterial agents, including doxycycline, and has ranged in severity from mild to life-
threatening. It is important to consider this diagnosis in patients who present with diarrhea 
subsequent to the administration of antibacterial agents. 
Ionizing Radiation Sensitivity 
Of some 46,523 PubMed citations for doxycycline or tetracyclines, none were found to indicate 
any additive effects with x-rays or any other form of ionizing radiation. The UV sensitivity of 
patients treated with doxycycline appears to be mediated by a direct photochemical reaction of 
non-ionizing UV radiation with the drug that would not be expected with ionizing radiation such 
as x-rays.  
 


 
 
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PRO 28944  1/12/18 
Drug Interactions 
1. Patients who are on anticoagulation therapy may require downward adjustment of their 
anticoagulant dose 
2. Absorption of doxycyline may be impaired by antacids or other drugs containing 
aluminum, calcium, or magnesium, bismuth subsalicylate; iron-containing products and 
dairy projects.  It is recommended that the doxycycline administration is separated by at 
least 2 hours before or after any of these products. 
3. Concurrent use of doxycycline may render oral contraceptives less effective and a back-
up method of birth control, such as a condom or a diaphragm with spermicide, or a 
contraceptive sponge is recommended. 
4. Barbiturates, carbamazepine, and phenytoin decrease the half-life of doxycycline 
5. Doxycycline may interfere with the bactericidal action of penicillin and co-administration 
should be avoided 
6. Concurrent use of tetracyclines and methoxyflurane (Penthrane) has been reported to 
result in fatal renal toxicity 
7. False elevations of urine catecholeamines may occur due to interference with the 
fluorescence test. 
8. No drug interaction has been reported with gemcitabine. 
 
3. 
PATIENT SELECTION 
3.1 Eligibility Criteria  
Patients must have histologically or cytologically confirmed pancreatic adenocarcinoma which 
may be acquired using a fine needle aspiration and who have not received any prior therapy. 
Patients must undergo either a dual phase pancreatic protocol CT scan or pancreatic protocol 
MRI to determine the clinical stage. Based on radiographic imaging, resectable pancreatic cancer 
will be defined by: (a) no radiographic evidence of arterial abutment of the celiac, superior 
mesenteric, or hepatic arteries, and (b) <50% tumor-induced narrowing of the superior 
mesenteric vein or portal vein. Patients with resectable pancreatic cancer will be eligible for the 
study. 
Additional eligibility criteria include: 
1. Patients will be receiving neoadjuvant therapy for resectable pancreatic cancer in 
anticipation of surgical resection. Resectable stage will be defined by radiographic 
criteria including; (a) no radiographic evidence of arterial abutment of the celiac, superior 
mesenteric, or hepatic arteries, and (b) <50% tumor-induced narrowing of the superior 
mesenteric vein or portal vein. 
2. Patients who will receive neoadjuvant therapy (chemoradiation) are eligible.   
3. Age ≥18 years.  
4. ECOG performance status ≤2 (Karnofsky ≥60%). 
5. Life expectancy of greater than 6 months 
 
 
 
 


 
 
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PRO 28944  1/12/18 
 
6. Patients must have normal organ and marrow function as defined below: 
a. leukocytes 
≥3,000/mcL 
b. absolute neutrophil count ≥1,500/mcL 
c. platelets 
≥100,000/mcL 
d. creatinine clearance ≥60 mL/min or creatinine ≤1.5 mg/dL 
e. Bilirubin ≤2mg/dL: If the subject’s serum bilirubin is greater than two at the time 
of enrollment but the patient has demonstrated a progressive decline and is 
anticipated to be ≤2mg/dL prior to start of therapy they are eligible, at the 
discretion of the trial investigators and their treatment assignment.  
7. Have no active or chronic infection with HIV, Hepatitis B or Hepatitis C 
8. Ability to understand and the willingness to sign a written informed consent document. 
3.2 
Exclusion Criteria 
1. Patients with more clinically advanced pancreatic cancer (borderline resectable, locally 
advanced, or metastatic) 
2. Uncontrolled intercurrent illness including, but not limited to, ongoing or active infection, 
symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, or 
psychiatric illness/social situations that would limit compliance with study requirements. 
3. Pregnant women are excluded from this study because the effects of metakaryocidal 
agents have the potential for teratogenic or abortifacient effects.   
4. Previous history of other malignancy (other than cured basal or squamous cell carcinoma 
of the skin or cured in-situ carcinoma of the cervix) within 2 years of study enrollment. 
5. Active or chronic HIV, hepatitis B or hepatitis C 
6. Patients who are receiving other investigational drugs or enrolled in other clinical trials 
7. Inability to undergo scheduled blood acquisition per protocol. 
8. Drug specific exclusion including history of allergic reactions to tetracyclines. 
9. Prior treatment with doxycycline within a 7 day washout period prior to initiating 
treatment with alternate antimetakaryocidal medication. 
3.3 Inclusion of Women and Minorities 
Both men and women of all races and ethnic groups are eligible for this trial. 
4. REGISTRATION PROCEDURES 
4.1 General Guidelines 
Eligible patients will be entered on study centrally at the Medical College of Wisconsin by the 
Study Coordinator. Patients must have a signed informed consent form prior to registration on 
study. Following registration, patients should begin protocol treatment within 28 days – allowing 
for coordination of radiation therapy. Issues that would cause treatment delays should be 
discussed with the Principal Investigator.  If a patient does not receive protocol therapy 
following registration, the patient’s registration on the study may be canceled.  The Study 
Coordinator should be notified of cancellations as soon as possible. 


 
 
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PRO 28944  1/12/18 
4.2 Registration Process 
Investigators or their appropriate designees will identify potentially eligible patients from their 
clinics, patient self-referrals, or referrals from other clinicians. Inclusion/exclusion criteria will 
be reviewed and patient eligibility will be confirmed after consent is signed.  
If the patient (or LAR, legally authorized representative) agrees to study participation, first, 
he/she must provide signed informed consent for study screening.  After signing a screening 
consent form, each patient will be assigned a unique subject number. The investigator will then 
review the patient’s medical history, cardiac performance, and medication list to determine if 
there  any contraindication to doxycycline therapy. If all criteria are met, the subject will undergo 
study treatment.   Treatment must be initiated 0-4 weeks after registration.  
To register a patient, the following documents should be completed by the study coordinator  
• Copy of required laboratory tests, medication list 
• Signed patient consent form 
• Eligibility Screening Checklist 
 
At the point of registration, the study staff will register the patient in the secure electronic 
database (OnCore), including on-study information. Any additional source documentation with 
personal health information will be stored on a secure folder maintained and accessed only by 
cancer center or study personnel with restricted access to the study.  
5. STUDY DESIGN 
5.1 Treatment Plan 
Treatment with doxycycline hyclate will be administered as an oral agent on an outpatient basis. 
Patients will receive doxycycline 100 mg twice daily for a period of 8-10 weeks (56-70 days).  
We plan to enroll 12 patients with anticipation of 10 patients completing all therapy and surgery. 
Following consent, patient will be referred to a radiation oncologist for consultation and 
treatment planning.  A simulation CT scan will be performed and radiation planning will be 
performed at the discretion of the treating physician. Neoadjuvant therapy will consist of 
concurrent chemoradiation with gemcitabine 400 mg/m2 given intravenously at a fixed dose rate, 
infused over 40 minutes on Day 1 (day-2 to +1), and then weekly x 6 during radiation.  Radiation 
will be delivered using external-beam radiation.  It will be delivered 5 days/week over 5.5 weeks 
with 6-18 meV photons.  Using 3D conformal or IMRT techniques, patients will receive a total 
dose of 50.4Gy prescribed to the 95% isodose at 1.8 Gy/fraction (28 fractions). Doxycycline will 
be made available through the investigational pharmacy at MCW prior to the initiation of 
radiation therapy. Doxycycline hyclate 100 mg PO Q12 hours will be administered to the patient 
for a period of 8-10 weeks starting on the first day of radiation therapy. 
Restaging imaging with either pancreatic protocol CT or MRI will be performed week 8 (+1) 
week to assess disease status.  Patients will generally undergo surgery week 9-11.  Doxycycline 
will be discontinued 5-7 days prior to surgery.   
Upon registration (baseline) and at the first, third, and fifth days of doxycycline therapy, patients 
will be seen to obtain trough serum concentrations for pharmacokinetic studies.  Additional 
serum levels will be checked on days 8, 15, 22, 29, and at restaging imaging to assess drug 
compliance and a final level will be checked at the time surgery, allowing for +/- 1 day window 
for any timepoint after the first week (days 7-9, 14-16, 21-23, 28-30, restaging +/- 1 day, surgery 
+/- 1 day). (Figure 9)The patient will be requested to maintain a medication diary of each dose of 


 
 
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PRO 28944  1/12/18 
medication to assess compliance.  Reported adverse events and potential risks are described in 
Section 6.  No investigational or commercial agents or therapies other than those described 
below may be administered with the intent to treat the patient's malignancy.  
 
5.2 Surgery  
Since surgery is usually performed 3-5 weeks after the completion of chemoradiation, there will 
be no delay in the timing for surgery for the patients who are enrolled.  We anticipate that they 
will complete the study drug 2-4 weeks after the completion of chemoradiation and will undergo 
surgery 3-5 weeks after completion of chemoradiation. 
Extent of surgical resection will be performed at the discretion of the treating surgeon. 
Pancreatectomy will be performed at the Medical College of Wisconsin by hepatobiliary 
surgeons (DBE, KKC, ST,) as previously described.8   Approximately 75% of the tumor will be 
removed for evaluation of doxycycline treatment effect, the remaining 25% of the pancreatic 
tumor tissue except for margins and lymph nodes will be used for pathologic assessment and 
immediately fixed in Carnoy’s solution. (See 10.4 Specimen Collection) 
5.3 Doxycycline pharmacokinetics 
Blood drawn for pharmacokinetics will be drawn in 8mL purple top tubes with EDTA and 
immediate processed within 2 hours of blood draw into plasma components.  Plasma will be 
frozen at -80°C and shipped to an external contractor(s) for analysis using mass spectrometry 
(Wisconsin State Hygiene laboratory, Hepatochem). Twelve serial samples will be acquired for 
each patient.  The data regarding serum concentrations of doxycycline will be reported to the 
data manager.  
5.4 Pathologic Assessment 
Tissues will be sent to either Dr. Tsai laboratory on the MCW campus for  tissue processing as 
previously described by Gostjeva et al.(3) or  assessment of metakaryote protocol will be 
performed by a cytogeneticist  at MIT.  Digital images will be made with annotation of the 
location and number of dead/dying metakaryotic nuclei. Images will be sent back to the MCW 
and an independent blinded pathologic examination will be performed by a board-certified 
cytopathologist. In addition, the histopathologic response of the primary tumor will be assessed 
using the College of American Pathology criteria for residual tumor response following 
neoadjuvant therapy for the exocrine pancreas. All slides will be stored at the Medical College of 
Wisconsin. 
5.5 General Concomitant Medication and Supportive Care Guidelines 
Supportive care for doxycycline 
Figure 9: Study Schema 
 
Blood Draws
Tissue 
Acquisition
5-7 day 
washout
Week 5.5-10
Gemcitabine based chemoradiation
Surgery
Enroll
Week 1
Week 2-5.5
Doxycycline 100 mg po BID
Restaging


 
 
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PRO 28944  1/12/18 
Anticipated toxicities with doxycycline include gastrointestinal discomfort and/or diarrhea and 
photosensitivity.  If patient develops diarrhea, a stool specimen will be collected to evaluate for 
clostridium difficile, ova and parasite infection.  In the absence of infection, anti-diarrheal agents 
may be initiated.  Loperamide 4 mg may be taken after loose stools every 12 hours titrating up to 
16 mg/24 hr as needed. In anticipation of photosensitivity, patients will be advised to avoid sun 
exposure and consistently use sun protection with protective clothing and broad-spectrum 
sunscreens with coverage in the UV-A and UV-B ranges.  Topical corticosteroids and cool 
compresses may alleviate drug-induced photosensitivity.  The use of systemic corticosteroids 
will be reserved for the most severe cases. 
Supportive care during radiation therapy 
1. Proton pump inhibitor BID throughout treatment up to 1 month post treatment.   
2. Zofran 8 mg PO 1 hour prior to each radiation treatment.  May take 8 mg TID if needed. 
3. Intravenous fluids prn 
4. Creon, simethicone, Reglan, Decadron on a per patient basis 
5. Short or long acting analgesia for upper abdominal discomfort 
6. Gas-X or Beno prn in addition to Creon for increased gas 
If patient develops gastritis or duodenitis near end of treatment or within 2 weeks of completion, 
IV fluids, histamine receptor blockers, or antiemetics may be administered.  Gaviscon liquid 
between meals or Carafate slurry may be added as needed. 
 
Concomitant treatment with other tetracyclines should be avoided unless there is no appropriate 
alternative medication for the patient to use.  
5.6 Duration of Therapy 
In the absence of treatment delays due to adverse event(s), treatment with the study drug may 
continue for up to 10 weeks or until one of the following criteria applies: 
Disease progression which precludes surgical exploration 
Intercurrent illness that prevents further administration of treatment, 
Unacceptable adverse event(s) 
Patient decides to withdraw from the study 
General or specific changes in the patient's condition render the patient unacceptable for further 
treatment in the judgment of the investigator. 
5.7 Duration of Follow Up 
Patients will be followed up to 60 days from the time of study termination using review of 
medical records or telephone contact to assess for adverse events.  Adverse events up to 60 days 
after the last dose of investigational agent will only be recorded if deemed possibly, probably, or 
definitely related to the investigational agent.  Patients removed from study for unacceptable 
adverse event(s) will be followed until resolution or stabilization of the adverse event. 
5.8 Patient Discontinuation 
Patients may discontinue treatment if any of the following occur: (1) recurrent or metastatic 
disease, (2) unacceptable toxicity, (3) patient withdrawal of consent, (4) investigator decision 
(see section 6; dose delays), or (5) non-compliance. The reason for study removal and the date 
the patient was removed must be documented in the Case Report Form if an investigator 
removes a patient from the study or if the patient declines further participation, a final 
assessment of the patient’s disease status should be performed prior to any therapeutic 
intervention. These results, along with a reason for study discontinuation will be recorded 
Patients will be monitored for up to 60 days after discontinuation for adverse events as long as 


 
 
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PRO 28944  1/12/18 
the patient agrees.The entire trial will be stopped if evidence emerges that makes the study 
continuation unnecessary or unethical, or when the stated objectives are achieved. 
6. DOSE DELAYS/DOSE MODIFICATIONS 
If toxicities occur, it may be difficult to identify whether this is related to the doxycycline or the 
radiation therapy.  However, if the severity of the toxicity exceeds the level commonly observed 
during pancreatic cancer irradiation, the toxicity will be ascribed to doxycycline and the drug 
reductions or discontinuation will be managed as below.   
 
 
 
 
Dose Level 
Doxycycline Dose 
-1 
50 mg po Q12H 
0 
100mg po Q12H 
Diarrhea 
Management/Next Dose for Doxycycline 
 
 
≤ Grade 1 
No change in dose 
Grade 2 
Hold until ≤ Grade 1.  Resume at same dose level. 
Grade 3 
Hold* until < Grade 2.  Resume at one dose level lower, if indicated.** 
Grade 4 
Off protocol therapy 
*Patients requiring a delay of >2 weeks should go off protocol therapy. 
**Patients requiring > two dose reductions should go off protocol therapy.  
Recommended management:  Loperamide antidiarrheal therapy 
Loperamide dosage schedule:  4 mg at first onset, followed by 2 mg with each loose motion 
until diarrhea-free for 12 hours (maximum dosage:  16 mg/24 hours) 
Adjunct anti-diarrheal therapy is permitted and should be recorded when used. 
Liver Function 
Management/Next Dose for Doxycycline 
≤ Grade 1 
No change in dose 
Grade 2 
Hold until ≤ Grade 1.  Resume at same dose level. 
Grade 3 
Hold* until < Grade 2.  Resume at one dose level lower, if 
indicated.** 
Grade 4 
Off protocol therapy 
*Patients requiring a delay of >2 weeks should go off protocol therapy. 
**Patients requiring > two dose reductions should go off protocol therapy.  


 
 
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PRO 28944  1/12/18 
 
 
7. ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS  
7.1 Definitions 
Adverse Event 
Adverse event is defined as any undesirable sign, symptom or medical condition occurring after 
starting the investigational drug even if the event is not considered to be related to the study. 
Medical conditions/diseases present before starting the study treatment are only considered 
adverse events if they worsen after starting the study treatment.  Adverse events occurring before 
the start of treatment but after the signing of consent will be recorded.  Abnormal laboratory 
values or test results constitute adverse events only if they include clinical signs or symptoms or 
require therapy. 
Serious Adverse Events 
 A serious adverse event is an undesirable sign, symptom, or medical condition which: 
 
Results in death 
 
Is life-threatening (an event in which the subject was at risk of death at the time of the 
event) 
 
Requires inpatient hospitalization or causes prolongation of existing hospitalization 
 
Is a congenital anomaly or birth defect 
 
An important medical event, e.g. intensive treatment in an emergency room 
 
 
Unexpected adverse event  
An adverse event, which varies in nature, intensity, or frequency from information on the 
investigational drug provided in the Investigator’s brochure, package insert, or safety reports.  
Any adverse event that is not included in the informed consent is considered “unexpected”. 
Expected adverse event  
An adverse event, which has been reported in the Investigator’s Brochure.  An adverse event is 
considered “expected” if it is included in the informed consent document as a risk. 
7.2 Relationship 
Attribution of the AE: 
- 
Definite – The AE is clearly related to the study treatment. 
- 
Probable – The AE is likely related to the study treatment. 
Phototoxicity 
Management/Next Dose for Doxycycline 
≤ Grade 1 
No change in dose 
Grade 2 
Hold until ≤ Grade 1.  Resume at same dose level. 
Grade 3 
Hold* until < Grade 2.  Resume at one dose level lower, if 
indicated.** 
Grade 4 
Off protocol therapy 
*Patients requiring a delay of >2 weeks should go off protocol therapy. 
**Patients requiring > two dose reductions should go off protocol therapy.  


 
 
30 
PRO 28944  1/12/18 
- 
Possible – The AE may be related to the study treatment. 
- 
Unlikely – The AE is doubtfully related to the study treatment. 
- 
Unrelated – The AE is clearly NOT related to the study treatment. 
 
7.3 Reporting of Adverse Events 
All grade 3-4 adverse events (AE) occurring during the study protocol will be recorded. The 
National Cancer Institute Common Toxicity Criteria for Adverse Events (NCI-CTCAE) version 
4.0 will be used to grade adverse events. Grade 3-4 events must be recorded noting severity and 
causality. Any consequent changes to the dosage schedule or any corrective therapy should be 
recorded. 
Adverse events will be recorded on electronic Case Report Forms (eCRFs) in OnCore, our 
clinical trial management system.  SAEs requiring expedited reporting to the IR (unexpected and 
at least possibly related to the intervention) will be entered into OnCore and reported to the IRB 
within 5 calendar days of knowledge of the event. SAEs not requiring expedited reporting will be 
entered into OnCore for annual review by the IRB.  SAEs will be followed until resolution and 
the eCRFs will be updated. 
The trial will be stopped if more than 5 patients have to discontinue the therapy due to treatment 
related adverse events.   
 
7.4 Study Risks 
The known risk associated with participating in this study is related to the administration of 
doxycycline in conjunction with neoadjuvant chemoradiation. The administration of radiation 
therapy is per a standard protocol and not considered experimental. During the neoadjuvant 
treatment period, patients are monitored closely for treatment related toxicity, including a weekly 
radiation review.  During this time, unanticipated toxicities related to the combination therapy of 
doxycycline and radiation therapy will be monitored. There is an additional risk of serial blood 
draws for pharmacokinetic monitoring which will involve obtaining up to 10 blood draws of 5-
10 cc of extra blood at several time points during the study. 
7.5 Risk Discussion 
 
This study is assessing the efficacy of doxycycline as a potential selective cancer stem cell 
therapeutic. The risks with prolonged therapy with doxycycline include gastrointestinal 
discomfort (abdominal discomfort, cramping, diarrhea), infectious colitis, and photosensitivity. 
These risks will be reviewed with the subject at the time of consent and symptom monitoring 
will be performed on a weekly basis for the first 5weeks, then every other week until week 9, and 
at the first postoperative visit (30-60 days following discontinuation of investigational agent).  
7.6        Study Benefits 
 
This is a new treatment approach, and the exact degree of efficacy is unknown. The patient 
may benefit from participating in this study if doxycycline is effective at killing cancer stem 
cells, however, since it is anticipated that several such agents will be necessary to prevent future 
relapse, it is uncertain what benefit participation in the trial may bring.  The knowledge gained 
from this study may benefit future patients with pancreatic adenocarcinoma.  
8. STUDY CALENDAR 
Schedules shown in the Study Calendar below are provided as an example and should be 


 
 
31 
PRO 28944  1/12/18 
modified as appropriate. 
Pre-study evaluations are to be conducted within 6 weeks prior to start of protocol therapy.  
Scans and x-rays must be done <6 weeks prior to the start of therapy.  Weekly monitoring will 
include medication review, physical examination, and laboratory studies as described below and 
will be performed in conjunction with the weekly radiation therapy review or at the time of the 
hematology oncology assessment within 7 days of the previous assessment.  In the event that the 
patient’s condition is changes and adverse events occur as described in section 7, laboratory 
evaluations should be repeated prior to the resumption of therapy. 
 
Pre- 
Study 
Wk 
1 
Wk 
2 
Wk 
3 
Wk 
4 
Wk 
5 
Wk 
6 
Wk 
7 
Wk 
8!! 
Wk 9-11 
Surgery 
 
Routine 
postop 
Visit 
 
Off Study 
Doxycycline 100 mg PO BID & Diary 
 
X 
X 
X 
X 
X 
X 
X 
X 
X# 
 
 
Radiation therapy 
 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
 
 
 
Height 
X 
 
 
 
 
 
 
 
 
 
 
 
Cross-sectional Imaging Study (CT or 
MRI) 
X 
 
 
 
 
 
 
 
X 
 
 
 
Pathologic Assessment of Surgical 
Specimen 
 
 
 
 
 
 
 
 
 
X 
 
 
Concurrent Meds* 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Physical exam* 
X 
Xx3† 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Vital signs* 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Weight* 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Performance status* 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
CBC w/diff, plts* 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Basic Chemistry and LFT* 
X 
X-† 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Investigational Blood Draw§ 
X 
Xx3† 
X 
X 
X 
X 
 
 
X 
X 
 
 
Adverse event evaluation* 
 
X 
X 
X 
X 
X 
 
X 
 
X 
X 
 
 
* With the exception of the first week, serial assessments of medications, PE, performance status, labs, and adverse event evaluation 
will coincide with weekly radiation therapy assessments and occur within a 7 day window of the previous assessment. Additional 
assessments may be performed as clinically indicated at the discretion of the treating physician. Final adverse event assessment will 
occur at the  routine postop visit between  (30-60 days from last dose of investigational agent). 
 †During the first week, serum concentrations of doxycycline will be measured every other day for the first 5 days. 
§ Investigational blood draws must be performed at baseline and on days 1, 3, 5, 8, 15, 22, 29, at the time of restaging prior to surgery 
and at the time of surgery.  A window of +/- 1 day will be allowed for assessment of serum concentrations of doxycycline after the 
first week. 
# Doxycycline is tied to surgery and will be completed 5-7 days prior 
!! Assessments due on week 8 will be allowed within +1 week window of week 8. 
 
 
 


 
 
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PRO 28944  1/12/18 
9. DRUG PROCUREMENT 
9.1 Drug Dispensing 
The study drug will be provided by the investigational pharmacy at the clinical cancer center of 
Froedtert Hospital.  The study drug will be labeled and handled as open-lab material, and 
packaging labels will fulfill all requirements by governing regulations. Drugs will be dispensed 
in child-resistant containers.  Upon receipt of the study drug, patients will be instructed to take 
the medication as directed. All study drug costs will be covered by the study (funding provided 
by the Batterman Foundation) and patients will not be responsible for the cost of the study drug.   
 
10. MEASUREMENT OF EFFECT 
Based on the previous assessments of resected surgical samples from patients treated with 
neoadjuvant radiation, no metakaryotic treatment has been previously observed.  Assessment of 
the efficacy of doxycycline will be performed utilizing cytometric characteristics as outlined in 
section 2.4-2.5.  Quantitation of the number of dead/dying metakaryotic cells per 100 
metakaryotic cells in at least 1 gram of tissue will be performed. Digital image acquisition is 
mandatory for every dead/dying metakaryotic cell identified.  
10.1 
Definitions 
Evaluable for toxicity.   
All patients will be evaluable for toxicity from the time of their first treatment with the study 
drug. 
Evaluable for metakaryote cell death: 
Only patients who undergo surgical resection of the primary tumor will be considered evaluable 
for study drug effect.  These patients will have their response classified according to the 
definitions below. 
Evaluable non-target disease response.   
Patients may be taken to the operating room without radiographic evidence of metastatic disease 
but at the time of diagnostic laparoscopy are found to have liver or peritoneal metastases.  In 
such patients, resected metastatic lesions will be evaluable for non-target disease response per 
the definitions below. 
A patient will be deemed unevaluable if: 1) patient has an insufficient amount of tissue removed 
for analysis or 2) patient experienced disease progression while on therapy and could not 
complete the designated therapy. Based on our experience, ~90% of patients are expected to 
complete all neoadjuvant therapy and undergo surgery.   
10.2 
Methods for Evaluation of Measurable Disease 
Histologic confirmation of pancreatic adenocarcinoma of adjacent tissue will be confirmed by a 
histopathologist at the time of tissue acquisition.  Only specimens with confirmed 
adenocarcinoma will be assessed for study drug response. Metakaryotes will be enumerated in 
serial sections of pancreatic parenchyma.  The nuclei will be assess for changes consistent with 
cell death (pyknotic nuclei, nuclear fragmentation) and enumerated. The number of dead/dying 
metakaryotes per 100 metakaryotes will be assessed for each specimen. 


 
 
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PRO 28944  1/12/18 
10.3 
Response Criteria 
Evaluation of Metakaryote Cell Death 
Cytometric criteria of dead/dying metakaryotic cells are based on assessment of the nuclear 
structure. Specifically, metakaryotic cells will be identified by previously described cellular 
morphologic criteria: (1) presence of an unusual large, hollow, bell-shaped nuclei, and (2) 
eccentric placed nuclei in relation to the cytoplasm. Characteristics of dead/dying metakaryotes 
include: (1) demonstration of pyknotic nuclear forms using Feulgen staining (figure 7a,b) or (2) 
failure of nuclear separation (figure 5b).  Drug response will be assessed as the proportion of 
dead/dying metakaryotes out of 100 metakaryotic cells. 
10.4 
Specimen Collection  
Blood collection and tissue collection is mandatory.  The following must be provided in order for 
the case to be evaluable. 
1. Serum 
a. One Red Top tube A at all investigational blood time points 
i. Within 1-2 hours of collection, spin in a standard clinical centrifuge at 
~2500 RPM for 10 minutes at 4C.  
ii. Aliquot 1 mL serum into as many cyrovials as are necessary labeled with 
study ID, collection date, and clearly marked as serum 
iii. Freeze cyrovials at -70 to -90 C until ready to ship. 
iv. Ship specimens on dry ice to Wisconsin State Hygiene Lab and/or 
Hepatochem. 
2. Tissue 
a. All pancreatic tumor tissue except for margins and lymph nodes for clinical 
evaluation  will be removed from the pathologic specimen at the time of tissue 
accession 
i. One H&E stained slide will be provided from the adjacent specimen 
ii. Tissue will be placed in Carnoy’s fixative (ethanol: acetic acid) for 24 
hours 
iii. Tissue will then be placed in 70% methanol for storage 
iv. Specimens will be labeled with study ID, collection date, and clearly 
marked as pancreatic tumor. 
v. Specimens will be shipped on ice to the Thilly/Gostjeva Laboratory 
3. Specimen Collection Summary 
 
Specimens Collection 
Specimens taken 
from patient 
Collected when: 
Submitted as: 
Shipped: 
Serum: ~ 3mL 
Pre-treatment, Day 
1, 3, 5, then week 
2-5, restaging, and 
at surgery 
Frozen serum 
samples containing 
1mL per aliquot in 
1 mL cryovials 
Serum sent frozen 
on dry ice via 
overnight carrier 
TISSUE: ~1gram 
At the time of 
surgery 
In 70% methanol 
following Carnoy’s 
fixation for 24 
hours with 
Shipped on ice via 
overnight carrier 


 
 
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PRO 28944  1/12/18 
corresponding H&E 
slide 
11. DATA REPORTING / REGULATORY REQUIREMENTS 
Adverse event lists, guidelines, and instructions for AE reporting can be found in Section 7.0 
(Adverse Events: List and Reporting Requirements). 
11.1 
Data Reporting 
11.1.1 Method 
Study data will be recorded in OnCore, and study coordinators are responsible for keeping 
information in OnCore up to date. Every 6 months, the Data and Safety Monitoring Committee 
will download a summary report from OnCore and review the study for scheduled monitoring.   
11.1.2 Responsibility for Data Submission 
Study coordinators are responsible for submitting CDUS data and/or data forms to DMSB on the 
study quarterly.  When setting the dates, allow time for Coordinating Center compilation, 
Principal Investigator review, and timely submission to DSMB by the quarterly deadlines (see 
Section 12.1.1) 
11.1.3 
Retention of Study Documentation 
The Investigator must maintain adequate and accurate records to enable the conduct of the study 
to be fully documented and the study data to be subsequently verified. The patient’s involvement 
in the study should be clearly documented in the clinical records. Details are to include the 
protocol number, the patient’s identification number, the patient’s consent(s) to take part in the 
study, the date of all study visits and the dates of treatment. Copies of CRFs must be retained by 
investigators, for as long as legally required after completion of the study. All study data 
including master files, CRFs and source data will be stored for at least 10 years according to 
MCW Office of Research SOP (see Appendix E). 
11.2 
Data Monitoring 
This study will be reviewed by the Medical College of Wisconsin Cancer Center Data and Safety 
Monitoring Committee (MCWCC DSMC). A summary of the MCWCC DSMC activities are as 
follows:  
1. Review the clinical trial for data integrity and safety.  
2. Review all unexpected grade 3, and all grade 4, and 5 adverse events, as well as any 
others requiring expedited reporting as defined in this protocol. (Grades 4 & 5 events 
must be reported to the DSMC within 5 calendar days of study staff’s knowledge.)  
3. Review all DSM reports.  
4. Submit a summary of any recommendations related to study conduct.  
5. Terminate the study if deemed unsafe for patients.  
A copy of the MCWCC Data and Safety Monitoring Plan and membership roster will be 
maintained in the study research file and updated as membership changes. The committee will 
review reports from the study PI twice annually (or more frequently if needed) and provide 
recommendations on trial continuation, suspension or termination as necessary.  
Any available DSMC letters will be submitted to the IRB of record as required. 
11.3 
Collaborative Agreements Language 
Agent(s) may not be used for any purpose outside the scope of this protocol, nor can Agent(s) be 


 
 
35 
PRO 28944  1/12/18 
transferred or licensed to any party not participating in the clinical study.  Collaborator(s) data 
for Agent(s) are confidential and proprietary to Collaborator(s) and shall be maintained as such 
by the investigators.  The protocol documents for studies utilizing Agents contain confidential 
information and should not be shared or distributed without the permission of the NCI.  If a copy 
of this protocol is requested by a patient or patient’s family member participating on the study, 
the individual should sign a confidentiality agreement.  A suitable model agreement can be 
downloaded from: http://ctep.cancer.gov. 
11.4 
Ethics Consideration 
The study must be approved by an appropriate institutional review committee as defined by 
Federal Regulatory Guidelines and the Office of Human Research Protection. The investigators 
will inform the IRB of subsequent protocol amendments. 
The investigator must inform the patient about the background and present knowledge of 
treatment with doxycycline. The investigator must ensure the following items are discussed:  
1. The patient must be provided the patient information and informed consent form consistent 
with the protocol version used and approved by the IRB. The patient must be informed that 
this is a new treatment approach and that the exact degree of efficacy is unknown, and that 
treating him/her will contribute to further knowledge. 
2. The patient will be given time to discuss his/her participation with the investigator and 
members of their family. Before the patient is entered into the study, the patient’s written 
consent must be obtained. A copy of the signed consent form will be provided to the patient 
and the original scanned into the electronic database created for this study. 
3. The patient may refuse to provide a biopsy or may refuse treatment proposed in the context of 
the study before or at any time during the study. Refusal to participate will involve no penalty 
or loss of benefits to which the patient is otherwise entitled. 
4. An explanation of whom to contact for answers to pertinent questions about the research and 
the patient’s rights, and who to contact in the event of a research-related injury must be given 
to the patient. 
11.5 
Publication Policy 
All the results are the property of the Principal investigators. Investigators may not submit for 
publication or presentation study results without allowing the PIs 30 working days to review and 
comment on the pre-publication manuscript. Co-authorship will be discussed and mutually 
agreed upon before submission of a manuscript to a publisher. 
 
12. STATISTICAL CONSIDERATIONS 
12.1 
Study Design/Endpoints 
The primary endpoint of the study is to evaluate for the presence of dead or dying metakaryotic 
cells as manifested by the cytometric characteristics. 
Secondary objectives:  
1. To determine the plasma drug concentrations of the study drug at days 1, 3, 5, 8, 15, 22, 
29, and at restaging and at the time surgery. 
2. To assess the histopathologic treatment response of the primary tumors which have 
undergone neoadjuvant gemcitabine based chemoradiation and concurrent doxycycline 
therapy. 


 
 
36 
PRO 28944  1/12/18 
3. To enumerate the number of observed dead/dying metakaryotes per 1 gram of resected 
pancreatic tissue. 
 
12.2 
Sample Size/Accrual Rate 
Assessment of Primary Endpoint 
At the moment the number of dead metakaryotic cells observed by our MIT collaborator, Dr. 
E.V. Gostjeva in more than ten pancreatic and/or lung tumors is still zero (0) in about 1000 
metakaryotic nuclei observed.  
To be of clinical utility, we set up our hypothesis as follows: 
H0: π=ζ vs H1: π>ζ 
where π is the average proportion of cell death and ζ is determined a prior with clinical utility.  
Considering the heterogeneous response to treatment between individuals, we assume that the 
true number of cell death with doxycycline follows a beta-binomial distribution with shape 
parameters (α,β)=(k,100-k), where k≥31 (i.e, a mean of ≥31 per 100 cells and an over-dispersion 
parameter 𝜌𝜌= 9.9). For a one-sided test, a sample size of 10 will achieve 80% power to detect 
an average proportion of cell deaths > ζ =20% at a type I error 0.05 level. The following figure 
show the statistical power for three ζ values, (10%, 15%, and 20%) and the corresponding true 
proportion of dead cells.  
 
 
For data analysis, the maximum likelihood estimation (by R package: VGAM) will be used to 
estimate the shape parameters (α,β) and calculate the mean and standard error based on a beta-
binomial distribution. The normal approximation will be used to construct the 95% confidence 


 
 
37 
PRO 28944  1/12/18 
interval for the average proportion of cell death.  
  
We plan a 1-year recruitment period with an anticipated attrition of 10% due to disease 
progression or drug toxicity. With an enrollment target of 12 patients, we anticipate that 10 
patients will be able to complete the assigned therapy and surgery.  Enrollment will be ongoing 
until the primary endpoint has been achieved. 
 
Assessment of Secondary Endpoints 
Plasma doxycycline concentrations and number of identified dead/dying metakaryotic cells will 
be reported as continuous variables. We will perform descriptive analysis of the continuous 
variables, including mean (SD) and median (IQR). We will further compare patients by the 
primary outcome (presence/absence of metakaryote death). We anticipate that our data will not 
be normally distributed and we will use nonparametric testing (Mann Whitney) to evaluate our 
results. The frequency of the number of dead/dying metakaryotic cells will provide a preliminary 
data on the expected distribution for the treatment dose. 
Histologic treatment response is part of the standard pathologic reporting.  Using the College of 
American Pathologists (CAP) criteria, which is a 4 tier grading system, chi-squared analysis will 
be used to compare observed histologic treatment response with reported treatment response 
from historical controls. 
 
 
 
 


 
 
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PRO 28944  1/12/18 
13. REFERENCES 
 
1. 
Gostjeva EV, Thilly WG. Stem cell stages and the origins of colon cancer: a 
multidisciplinary perspective. Stem cell reviews. 2005;1(3):243-251. 
2. 
Gostjeva EV, Zukerberg L, Chung D, Thilly WG. Bell-shaped nuclei dividing by 
symmetrical and asymmetrical nuclear fission have qualities of stem cells in human 
colonic embryogenesis and carcinogenesis. Cancer genetics and cytogenetics. Jan 1 
2006;164(1):16-24. 
3. 
Gostjeva EV, Koledova V, Tomita-Mitchell A, et al. Metakaryotic stem cell lineages in 
organogenesis of humans and other metazoans. Organogenesis. Oct 2009;5(4):191-200. 
4. 
Lamb R, Fiorillo M, Chadwick A, et al. Doxycycline down-regulates DNA-PK and 
radiosensitizes tumor initiating cells: Implications for more effective radiation therapy. 
Oncotarget. Jun 10 2015;6(16):14005-14025. 
5. 
Sudo H, Li-Sucholeiki XC, Marcelino LA, et al. Distributions of five common point 
mutants in the human tracheal-bronchial epithelium. Mutation research. Apr 11 
2006;596(1-2):113-127. 
6. 
Sudo H, Li-Sucholeiki XC, Marcelino LA, et al. Fetal-juvenile origins of point mutations 
in the adult human tracheal-bronchial epithelium: absence of detectable effects of age, 
gender or smoking status. Mutation research. Nov 10 2008;646(1-2):25-40. 
7. 
Kini LG, Herrero-Jimenez P, Kamath T, et al. Mutator/Hypermutable fetal/juvenile 
metakaryotic stem cells and human colorectal carcinogenesis. Frontiers in oncology. 
2013;3:267. 
8. 
Christians KK, Tsai S, Tolat PP, Evans DB. Critical steps for pancreaticoduodenectomy 
in the setting of pancreatic adenocarcinoma. Journal of surgical oncology. Jan 
2013;107(1):33-38. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

ICF_001.pdf: 
Informed Consent Form 
 
Clinical Trials.gov number: NCT02775695  
 
Protocol Title: Efficacy of doxycycline on metakaryote cell death in patients with 
resectable pancreatic cancer 
 
Document Date:  02/14/2018