Biomoda, Inc.

Internal Biomoda Studies

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Internal Validation Studies

Constance Dorian
Vice President, Technical Operation
Biomoda Inc.

Completed in June 2008

The assay as currently practiced was optimized from 2007 through 2008. This optimization improved discrimination during the microscopic scoring of the slides. A Biomoda internal study completed in June 2008, included 27 study samples from a variety of sources. Biomoda performed this in-house validation study using sputum samples from 15 patients diagnosed with lung cancer and 12 participants who had no lung cancer diagnosis. Cohort 1 consisted of 15 patients who had recently been diagnosed with primary lung cancer.  Cohort 2 included 12 subjects who did not have a history or diagnosis of lung cancer.

This study was initiated with an approved protocol and an informed consent document that was reviewed and approved by a duly constituted Institutional Review Board (IRB). Subjects aged 18 and above were included in the study. Patients with a history of angina after minimal exertion, with severe obstructive lung diseases with predicted Forced Expiratory Volume in one second (FEV1) less than 20%, and with uncontrolled asthma (defined as a hospitalization or emergency room visit within the last year, > two nocturnal morning dip index per month, or daily wheezing), along with those on supplemental oxygen or resting saturation of peripheral oxygen < 90%, were excluded from the study. The rationale for these exclusion criteria was to avoid circumstances that could aggravate the medical conditions of these patients, since a certain amount of exertion is required to produce an adequate quantity of sputum.

A sputum sample was collected from each participant following either a triple morning cough procedure or an inhalation saline-induced procedure. The sputum sample collection required two collection cups, a sterile cup and a second collection cup that contained a fixative solution. Subjects who followed the triple morning cough procedure were given the materials and instructions for sputum collection at the doctor’s office. Participants were instructed to cough up sputum in the morning after getting out of bed over a period of three days. They were told to note any adverse event that occurred up to 15 minutes after sputum collection and to report the same to the doctor.  After three days of sputum collection, subjects returned the collection cups containing the samples to the doctor’s office. For samples obtained by the inhalation saline-induced method, a one-time collection was performed at the doctor’s office. Pulmonary personnel performed sputum induction on subjects who could not spontaneously produce sputum on their own. These subjects were monitored for adverse events for 15 minutes after sputum induction.   After collection all samples were sent to the Biomoda Laboratory for processing.

At the Biomoda Laboratory, the samples were processed onto a microscope slide using a ThinPrep T2000 processor. The fixed sputum sample was processed to separate the cells from the mucous and cell fragments.  Each prepared slide contained a monolayer of the sputum cells. After preparing the labeling reagents containing TCPP (Biomoda CyPath® Early Detection Lung Cancer Assay), the slide was immersed in the TCPP labeling solution, rinsed, air-dried, and cover-slipped. The completed slide was viewed under a medium energy light utilizing a fluorescent microscope containing a fluorescein isothiocyanate (FITC) filter to observe the presence of fluorescing red cells and other cellular metrics.

The CyPath®-labeled slide was evaluated under the microscope, and the results were interpreted based on the characteristics of the cells on each slide. Normal cells appear white, green-yellow or pale orange in color and are transparent. Cancer cells appear dark crimson red and are opaque and intact. The appearance of both cancer and normal cells is shown below (figure 1, 2).

FIGURE 1- Picture of TCPP-labeled cancer cells demonstrating red fluorescence under a microscope with medium energy light being observed with a FITC (fluorescein isothiocyanate) filter.

FIGURE 2- The appearance of cancer cells along with other cells in a sputum sample collected from a lung cancer patient.

Internal Validation Testing Results

In this study, the TCPP compound revealed 100% specificity (12/12) and 100% sensitivity (15/15).