A Validation of a Genomics Based Prognostic in Severe Trauma

Specific Aim: To prospectively validate a rapid genomic test obtained from blood leukocyte
subpopulation of severely traumatized patients in the first 24 hours after admission, that
can be used to discriminate those patients who will have a complicated clinical trajectory
and would, therefore, be good candidates for interventional, immunomodulatory therapies.

Following severe blunt traumatic injury and during recovery, alterations in the leukocyte
transcriptome occur. Thus, we propose that a leukocyte transcriptome signal based on the
expression of the validated 63 total leukocyte or 181 PMN genes can be used to identify
patients who are at risk of a complicated clinical outcome, either alone or when combined
with anatomical or physiological predictors. The goal of this clinical trial is to validate
the predictive ability of this transcriptome‐based prognostic.

This is a non‐interventional, observational study of 200 severely injured patients with blunt
trauma and 40 healthy volunteers. The entry criteria will be the same as has been used for
the past eight years with the Glue Grant. Severe blunt injured patients that meet the
inclusion criteria will undergo rapid leukocyte genomic screening at admission and the
following morning(~24 hrs). Additional blood samples will be collected at 4, 7, 14, 21 and 28
days, or as long as the patients are in the trauma or surgical intensive care unit, for
banking.

Over a four year period, we will enter 240 subjects from the University of Florida and from
the University of Washington at Harborview Medical Center. In the trauma subjects, blood
samples will be collected at admission, days 1, 4, 7, 14, 21 and 28, or until discharge from
the ICU or death. A total of 4 to 5 mLs of blood will be collected at each time point. In the
healthy volunteers, a one-time blood sample will be collected.

Screening and Consent: Trauma patients will be identified by study personnel following
presentation to the emergency room. The subject or legal next-of-kin will be approached for
consent within 96 hours if the patient meets entry criteria. We ask for delayed consent in
this patient population for 96 hours due to the severity of the injuries, the vulnerable
nature of the patient at this early time, and the challenge in reaching the patient's legal
representatives. Should informed consent not be obtained within 96 hours, all blood samples
and data collected will be discarded.

Healthy volunteer subjects will be identified from the general population by way of
advertisement flyers posted at the respective universities. The flyers will provide contact
information for the laboratories. Healthy subjects will be screened via inclusion/exclusion
criteria upon contact. If appropriate for participation, the subject will be consulted for
consent.

Blood will be collected from existing access, or by venipuncture, if required. Blood will be
promptly placed on ice and transferred to the laboratory where it will be processed for cell
lysates and plasma.

All other relevant clinical data and hemodynamic measurements will be collected daily while
patient is hospitalized.

- Demographic information.

- Past and present medical records

- Laboratory, microbiology, and all other test results

- X-ray, CT, MRI, US and all other imaging test results

- Records about any medication received during admission

- Records of physical exam during admission

- Records of all vital signs and hemodynamic monitoring during admission

- Records of any procedure or intervention during admission

- Records of any procedure or intervention during hospital admission

- Condition at the discharge and discharge facility

Each of the two clinical sites will maintain responsibility for the collection and storage of
clinical data. REDCap (Research Electronic Data Capture) will be used for the collection of
clinical data. It is a secure, Web-based application designed to support traditional case
report form data capture. Cell lysates obtained at the University of Washington‐Harborview
Medical Center will be shipped to the University of Florida for genomic analyses, while
plasma samples collected at Shands Hospital at the University of Florida will be shipped to
the University of Washington for cytokine analyses by Luminex™. At yearly intervals,
electronic data transfers from the University of Florida to the University of Washington of
both non-identifiable, coded clinical data and cytokine and genomic data will occur.

Blood draws related to the study will be done at the bedside in a manner compatible with the
standard of clinical care or in our laboratories for healthy volunteers. Tubes used for the
blood draws related to study will be labeled with the unique study number and will not carry
any patient identifier. The key linking unique patient number and patients personal
identifiers will be stored in a password protected and encrypted file and will be only
accessible for the PI and the research team. All patient collected clinical data will be
stored in a cabinet that is located in the office of the PI or research team. The computer
used for the storage of data will be located in the office of the PI or research staff that
is always locked. The computer will be password protected and encrypted.

We have based our assessment for the number of subjects on our preliminary data set from 1637
severe trauma subjects. In that study, of the 1637 subjects, 31% or 507 patients had a time
to recovery of less than five days, and could be identified as having an uncomplicated
outcome. In contrast, 442 patients or 27% had complicated outcome, as defined by either late
death or MOF lasting longer than 14 days. Assuming a similar frequency of complicated and
uncomplicated patients in the 200 subjects, we would expect 62 of these subjects to be
discharged within 5 days without organ injury, and 54 subjects to have prolonged organ
failure and/or late death. These numbers are well within the ranges required for statistical
validation of these different models.

We anticipate that our overall consent rate for this observational study with minimal risk
will be 75% meaning that to achieve an accrual rate of 25 trauma subjects per year at each
institution, we will have to have 34 subjects per year meeting entry criteria. We expect no
difficulty in accruing these 200 subjects over a four year period at the participating sites.
They are both Level 1 trauma centers that have excess capacity to complete the proposed
studies.

In this protocol, the only study specific intervention will be the collection of blood. The
risks of drawing blood from a vein include discomfort at the site of puncture; possible
bruising and swelling around the puncture site; rarely an infection; and, uncommonly,
faintness from the procedure. Invasion of privacy will be negated with the use of coding for
all data and specimens collected.

There are no direct benefits to study subjects for participation. This research may benefit
society in the future. This study may one day result in new tests or treatments, or may help
to prevent or cure diseases. No conflict of interest exists for the PI or sub-investigator.

Inclusion Criteria Trauma Patients:

- All adults (age ≥18)

- Blunt trauma patients with hemorrhagic shock, defined by either a systolic BP (SBP)
<90 mm Hg or base deficit (BD) <-6 meq

- Ability to obtain Informed Consent within 96 hours of injury.

- Ability to obtain the first lab draw within 12 hours of presentation to the Emergency
Department.

Exclusion Criteria Trauma Patients:

- Patients not expected to survive greater than 48 hours.

- Patients with severe head injury.

- Severe pre-existing organ dysfunction

- Those that we are unable to obtain the first blood sample within 12 hours of injury

- Subjects who have received oncolytics within 14 days

- Subjects who are HIV + and have a CD4 count of <200/mm3

- Subjects not expected to survive 28 days due to pre-existing, uncorrectable medical
condition

- Total body surface burns >40%

- Prisoners

- Current, chronic steroid use

If it is uncertain what the patient's past medical history is at the time of enrollment,
they can be enrolled in the study and subsequently removed if they fail to meet criteria.
This will be done because there are many circumstances with this patient population due to
the severity of the injuries, the vulnerable nature of the patient at this early time, and
the challenge in reaching the patient's legal representatives that we are unaware of their
past medical history. The purpose of these exclusions is to ensure an adequate allocation
of resources. Specifically, our goal is to evaluate patients at high risk for Multiple
Organ Dysfunction Syndrome (MODS). Those with anticipated early death will add little to
achieving this objective. Severe head injuries are excluded as well since the mortality in
these subjects is usually attributable to their head injury rather than severe organ
dysfunction. In addition, steroid use is known to affect the immunologic response to
injury.

Inclusion Criteria Healthy Volunteers:

1. all adults (age ≥18)

2. Ability to obtain Informed Consent prior to blood collection.

Exclusion Criteria Healthy Volunteers:

1. Severe pre-existing organ dysfunction

2. Subjects who have received oncolytics within 14 days

3. Subjects who are HIV + and have a CD4 count of <200/mm3

4. Subjects not expected to survive 28 days due to pre-existing, uncorrectable medical
condition

5. Total body surface burns >40%

6. Prisoners

7. Current, chronic steroid use