Chemotherapy Drug Sensitivity Microculture (MiCK) Assay for Apoptosis
Chemotherapy Drug Sensitivity Microculture (MiCK) Assay for Apoptosis
DiaTech is a private company performing patient specific cancer chemosensitivity testing for
 patients and physicians. DiaTech Oncology is doing this clinical study to see if an
 experimental new technology called the microculture kinetic (MiCK) assay will predict
 treatment outcome and can help to direct the chemotherapy of cancer subjects. This study is
 focused on subjects diagnosed with breast, ovarian, lung, and colon malignancies and
 low-grade lymphomas.

 Study Objectives:

 -  To evaluate the ability of the MiCK assay to predict the outcome of chemotherapy of
 cancer patients.

 -  To evaluate the ability of the MiCK assay to guide chemotherapy of cancer patients.
Application of the Microculture Kinetic (MiCK) Assay for Apoptosis to Testing Drug
 Sensitivity of Solid Tumors DiaTech Oncology, LLC

 ----------------------------------------------------------- 1.0 Background and Rationale:
 Despite the use of aggressive treatment protocols, less than 10% of cancer patients with an
 advanced disease respond to the therapy. There is a variety of different cancer drug
 regimens, all of which have approximately the same probability of clinical effectiveness.
 Identification of those patients who will or will not respond to a specific chemotherapy is
 important for making decisions regarding chemotherapy regimens as well as alternative
 management approaches. A laboratory test that could help to determine the sensitivity of an
 individual patient's tumor cells to specific chemotherapeutic agents would be valuable in
 choosing the optimal chemotherapy regimen for that patient with an expectation of increasing
 the response rate to the therapy. Several types of in vitro assays that measure tumor cell
 survival following exposure to cytotoxic agents have been evaluated for their ability to
 predict chemotherapy outcomes. As a group, these assays are referred to as drug resistance
 assays. In a resistance assay, the surviving tumor cells can be detected directly by their
 exclusion or metabolism of specific dyes. Alternatively, since some of tumor cells are
 proliferating, their survival can be detected by measurement of DNA synthesis by radiolabeled
 precursor incorporation or demonstration of clonogenic potential by growth into colonies in
 semi-solid culture medium. In several clinical studies, these assays were useful in detecting
 drug resistance and in predicting a poor prognosis for cancer patients. However, these
 resistance assays cannot detect sensitivity of an individual patient's tumor cells to a
 specific drug. Therefore, new methods determining drug-sensitivity of the tumor cells of an
 individual patient and, thus, capable of both predicting a positive treatment outcome and
 guiding chemotherapy, would be of significant value.

 Recently, Dr. Kravtsov has developed an automated microculture kinetic (MiCK) assay for
 measuring drug induced apoptosis in tumor cells1-4. Apoptosis is a distinct mode of cell
 death which occurs under physiological conditions and yet can be induced in malignant cells
 by chemical and physical factors including antitumor drugs5-7. During the last decade, it has
 been recognized that chemotherapeutic agents exert their antitumor activity by triggering
 apoptosis in susceptible tumor cells8-17. This implies that the MiCK assay for apoptosis
 provides a mechanism-based approach to studying effects of cytotoxic agents on tumor cells.
 Unlike "resistance" assays that measure a fraction of cells surviving drug exposure, the MiCK
 assay measures a fraction of tumor cells killed by a chemotherapeutic agent via mechanism of
 apoptosis. Therefore the MiCK assay determines drug sensitivity, rather than resistance.
 Recently the MiCK assay has been shown to predict complete remission rate and survival in
 acute myeloid leukemia patients better than clinical criteria did18-20. In a limited study,
 the MiCK assay has been used to direct chemotherapy of the leukemia patients 21.

 The MiCK assay has also been used to study drug-induced apoptosis in solid tumors, including
 neuroblastoma and colon adenocarcinoma cell lines22-23. More recent data accumulated by
 DiaTech has demonstrated that the MiCK assay can detect drug induced apoptosis in primary
 cultures of tumor cells isolated from patients with ovarian carcinoma, gastric carcinoma,
 metastatic breast cancer and high grade soft tissue sarcoma. Based on these data, we suggest
 that the MiCK assay may be used to detect drug sensitivity profiles of individual patients
 with various types of solid tumors. This, in turn, may provide a way to tailor chemotherapy
 to an individual patient's drug sensitivity profile, and, thus, improve treatment outcomes,
 decrease adverse effects of the chemotherapy, increase the quality of patient's life, and
 reduce the treatment cost.

 2.0 Study Objectives: 2.1 To evaluate the ability of the MiCK assay to predict the outcome of
 chemotherapy of cancer patients.

 2.2 To evaluate the ability of the MiCK assay to guide chemotherapy of cancer patients.

 3.0 Patient Population:

 3.1 Inclusion criteria: 3.1.1 Patients with pathological diagnoses of breast, lung, and
 ovarian adenocarcinomas and soft tissue sarcoma.

 3.1.2 Patients with de novo malignancies and no previous chemotherapy 3.1.3 Patients with
 advanced refractory malignancies who received no more than 2 standard chemotherapy treatment
 protocols.

 3.1.4 Patients of any age group. 3.1.5 Patients must have tumor which is accessible and agree
 to undergo biopsies, or drainage of effusions.

 3.1.6 Patients for whom chemotherapy is a treatment option. 3.2 Explanations: We anticipate
 that newly diagnosed patients will be mostly used to evaluate the ability of the MiCK assay
 to predict the outcome of the chemotherapy (Objective #2.1) and to establish criteria
 correlating numerical response in the MiCK assay with probability of the clinically
 established complete remission. The patients with refractory malignancies will be mostly used
 to evaluate the ability of the MiCK assay to guide cancer chemotherapy (Objective #2.2).
 Patients will be seen and managed as outpatients or inpatients, depending on a clinical
 standard of the institution.

 3.3 Exclusion criteria: 3.3.1 Patients with symptomatic/uncontrolled parenchymal brain
 metastasis and not accessible tumors.

 3.3.2 Patients with meningeal metastasis. 3.3.3 Patients for whom chemotherapy clinically is
 not indicated. 3.3.4 Pregnancy. During the course of the study, all patients of childbearing
 potential should be instructed to contact the treating physician if they suspect they might
 have conceived a child; for females, a missing or late menstrual period should be reported to
 the treating physician. If pregnancy is confirmed by a pregnancy test, the patient must not
 receive study medication and must not be enrolled into the study or, if already enrolled,
 must be withdrawn from the study. If a male patient is suspected of having fathered a child
 while on the study drugs, the pregnant female partner must be notified and counseled
 regarding the risk to the fetus. Pregnancy during the course of this study will be reported
 to the Principal Investigator as a serious adverse event. Women of child bearing potential
 are defined to include any female who has experienced menarche and has not undergone
 successful surgical sterilization (hysterectomy, bilateral tubal ligation, or bilateral
 oophorectomy) or is not post-menopausal (defined as amenorrhea for more than 12 consecutive
 months); these includes also females using oral, implanted, or injectable contraceptive
 hormones, mechanical devices, or barrier methods to prevent pregnancy.

 4.0 Treatment Plan: 4.1 For the patients involved under the Study Objective (2.1), treatment
 protocol for an individual will not be influenced by the results of the MiCK assay for that
 patient. Oncologists will be blinded of the results of the MiCK assay and the patients will
 be treated according to a standard treatment protocol. When available, actual clinical
 response to the treatment will be compared with that predicted by the MiCK assay.

 4.2 For the patients involved under the Study Objective (2.2), treatment protocol for an
 individual patient will be designed based on the results of the MiCK assay for that patient.
 The drug with the best activity in the assay will be used to treat the individual patients.
 If more than one drug is equally active, the patient will receive the active drug in a class
 he/she has not received before. If no drug is found to have activity (insensitivity in the
 assay), the patients will be treated on an empiric basis with a class of drug he/she never
 received in the past.

 5.0 Definition of Clinical Response 5.1 All patients will be evaluated for clinical response
 to chemotherapy as per standard protocol established for a specific malignancy.

 5.1.1 Complete Remission is defined as total disappearance of clinically and radio logically
 detectable disease for at least four weeks.

 5.1.2 Partial Response is defined as at least 50% reduction of all measurable tumor lesions
 as measured by the sum of products of the perpendicular diameters of the greatest dimensions
 of measurable lesions, with no new lesions appearing for at least four weeks.

 5.1.3 Stable Disease is defined as a reduction by less than 50% or increase by less than 25%
 in the size of the tumor lesions, with no new lesions appearing.

 5.1.4 Progressive Disease is defined as appearance of any new tumor lesions or increase of
 25% or more in the sum of products of the perpendicular diameters of the greatest dimensions
 of measurable existing lesions.

 6.0 Definition of the drug response in the MiCK assay. 6.1 In the MiCK assay, the extent of
 drug-induced apoptosis is measured in Kinetic Units (KU) on a scale from 0 to 16 (Blood,
 1998). Apoptotic responses in KU will be calculated for each dose of a tested drug or drug
 combination. The maximal numerical response induced by a drug will be considered the "best
 apoptotic response" to the drug.

 6.1.1 Under the Study Objective (2.1), the "best apoptotic response" to the drug will be
 compared to the clinical response to the drug to establish a numerical value of the in vitro
 response discriminating drug-sensitive and drug-insensitive tumors (establishing a numerical
 cut-off of sensitivity). From our experience with establishing cut-off values of drug
 sensitivity for acute leukemia, 25-30 patients should provide a sufficient statistical power
 for getting a significant discriminator. These 25-30 patients will be fed from both patients
 participating under Study Objective (2.1) and Study Objective (2.2). After the numerical
 cut-off for sensitivity is established, it will be applied to a cohort of patients who
 received treatment with the drug to compare responses predicted in the MiCK assay to the
 actual clinical responses. This second part of the study will have two legs: (a)
 "prospective" (using 30-35 patients entering the study after establishing the discriminator,
 and (b) "retrospective" (using an available database of the patients who received treatment
 in the past and who's clinical response to the chemotherapy is known).

 6.1.2 For the patients participating under the Study Objective (2.2), a drug inducing the
 highest "best apoptotic response" will be considered the best active drug and will be
 recommended for including in the treatment protocol. Only drugs with the numerical value of
 the "best apoptotic response" exceeding a certain threshold should be expected to induce a
 complete remission. In the beginning of the study, such a threshold will be judged based on
 the results of the previous leukemia study19-22. As first 25-30 patients enter the study, the
 thresholds will be established for solid tumor patients as described in (6.1.1) and used
 thereafter. Considering that these 25-30 patients will be fed from both patients
 participating under Study Objective (2.1) and Study Objective (2.2), we expect to have first
 statistical correlations available no later than 3-4 months after the study begins.

 6.1.3 Drug selection for testing against tumor cells will be based on the previous
 chemotherapy history of an individual patient with consideration of the future treatment
 plans. The drugs will be selected from a compendium of agents recommended for the treatment
 of the patient's malignancy.

 7.0 Statistics 7.1 We estimate that sufficient statistical power will be obtained with
 approximately 50-60 patients with successful MiCK assays. A successful MiCK assay is defined
 as an assay for which a sufficient number of viable tumor cells could be isolated from the
 submitted pathologic material. For correlations between the patient's sensitivity profiling
 in the MiCK assay and clinical responses, the patients will be evaluated as the whole group
 as well as stratified based on the type of malignancy. The latter will depend on the number
 of patients with specific tumor types available for the study.

 7.2 In order to discriminate between the drug-sensitive and drug insensitive tumor cell
 populations, the best responses to each agent will be compared in a receiver operator
 characteristic (ROC) analysis as well as logistic regression analysis with attainment of CR
 (CR vs No CR). Based on the best combination of statistical sensitivity, specificity, and p
 value, thresholds demarcating drug sensitivity and insensitivity will be established. The
 Fisher's exact test, chi-square test and analysis of variance method will be employed to test
 the correlation between the clinical response results and other clinical variables. For
 lifetime data analyses, Kaplan-Meier estimates of survival will be used. Other statistical
 techniques may also be implied if found useful.

 8.0 Specimen collection, purification of tumor cells and sensitivity testing 8.1 Specimen
 collection must be performed as per related DiaTech Standard Operating Procedures (SOP).

 8.1.1 Collection of a Solid Tumor Biopsy Specimen is performed under sterile conditions,
 using excision biopsy technique, to obtain at least 2 cm3 of viable tumor tissue. The more
 viable tumor tissue is submitted for the study, the more chemotherapeutic agents can be
 tested against the tumor cells. Effusion specimen (peritoneal fluid, pleural fluid and
 effusions from other anatomical sites) should be collected in a commercial sterile
 container/bag with added Sodium Heparin (10 U/ml) to prevent clotting. The sample size should
 not be less than 500 ml. The specimen should not be fixed, or frozen.

 8.1.2 Transportation: seal the transport tube/container/bag tightly. Label specimen with
 institution, patient name, date and time of collection, and anatomical site of collection.
 Place the transport tube/container/bag on ice pack (blue ice). Ice pack must be frozen before
 placing it to the container. Fill out the Study Requisition form and include with the
 transportation container.

 8.1.3 Place the 50 mL tubes or a container inside the zip-lock bag and seal. Place specimen
 and Cold Pak (blue ice) in a transport box. Place completed patient information forms and
 specimen transport box into FedEx plastic "Diagnostic Specimen Envelope" and seal. Complete
 and affix the FedEx Airbill to the outside of the Diagnostic Specimen Envelope. Be sure that
 the airbill is marked "FedEx Priority Overnight" delivery. Put $5 value on the FedEx Airbill.
 Indicate "Human tissue for diagnostic studies" in the appropriate section of the FedEx
 Airbill. Call DiaTech Oncology at (514)-398-5174 (Steve Moisan, lab manager) or
 (514)-398-5154 (general lab) with the Fed Ex Tracking Number. DiaTech must receive the
 specimen within 24 hours of collection. Specimens sent on Friday must be marked for Saturday
 delivery.

 8.2 Tumor cell purification and their chemosensitivity testing will be performed as per
 related DiaTech SOPs.

 8.2.1 Tissue biopsy and effusion specimens will be treated to obtain a suspension containing
 single tumor cells and/or small cell aggregates composed of 2-20 tumor cells. Using gradient
 centrifugation, red blood cell lysing, cell strainers, magnetic beads and other appropriate
 techniques, the tumor cell suspension will be enriched to at least 80% purity and no less
 than 90% viability. Immunocytochemical stains or, when applicable, flow cytometry will be
 used to confirm the presence of specific tumor markers on purified cells After purification,
 selected chemotherapeutic agents will be tested against purified tumor cells in the MiCK
 assay.

 8.2.2 The purified tumor cells will be suspended in culture medium and plated in 96-well
 microtiter plates. Multiple concentrations of chemotherapeutic agents will be achieved by
 adding each respective agent to wells in 5 μL aliquots The ranges of final drug
 concentrations will be based on reports of pharmacokinetic studies of the drugs and their
 active metabolites in patients. Data processing and quantification of drug-induced apoptosis
 will be performed by a proprietary ProApoTestTM software. The extent of apoptosis will be
 determined and expressed as kinetic units (KU) of apoptosis.

 9.0 Efficacy measurement 9.1 For patients participating under Study Objective (2.1), the goal
 of the study is to establish a statistically significant discriminator of sensitivity which
 would correlate with the rate of CR. Such discriminator should be applicable to patient's
 populations participating under Study Objectives (2.1) and (2.2).

 9.2 For patients participating under Study Objective (2.2), we would expect no greater than a
 10% response rate in refractory solid tumor patients to any standard chemotherapy. A response
 rate to the MiCK assay-guided therapy of less than 10% would be of no further interest. A
 response rate of 10-30% will suggest the MICK assay needs further study. A response rate
 greater than 30% would justify usefulness of the MiCK assay in guiding chemotherapy of
 patients with refractory solid tumors.

 10.0 Specimen's left over 10.1 If a specimen contains more tumor cells than needed for
 testing their sensitivity to the drugs specified by the patient's oncologist, an excess of
 the tumor cells may be considered for use for other research studies conducted by DiaTech
 Oncology. Patients will be asked for their permission to use specimen's left over for
 research purposes by signing the following release included in the requisition form: "You are
 participating in the Research Program which may result in improvements of the treatment
 outcome for cancer patients. There is no direct benefit for you at present time. Your
 physician is submitting to DiaTech a specimen containing your tumor cells. At DiaTech, we
 purify the tumor cells, count them, check their viability, and store them in the DiaTech
 Tissue Bank. All these procedures are performed at no cost to you or your family. Your cells
 may be used for the chemosensitivity testing for research purposes or in other studies.
 Results of the research may be published, or used commercially in the area of new anti-cancer
 drug or treatment protocol development. To assure your privacy, should the results of the
 studies be published, you will be referred to only by number. Your signature below indicates
 that you agree to these terms".

 If a patient refuses to sign the above release, the specimen's left over will be
 decontaminated using 10% formaldehyde for 24h and discarded as per related SOP. If a patient
 grants his/her permission for use on the specimen's left over in a future research, an excess
 of the tumor cells will be frozen and stored in liquid nitrogen indefinitely.

 10.2 Specimen's left over may be used to study anti-tumor effects of chemotherapeutic drugs
 or drug combinations, to study mechanisms of drug resistance, to correlate phenotypic
 features of the tumor cells with their drug sensitivity profile.

 11.0 Plan of communication between Principal Investigator and co-Principal Investigator.

 To insure proper communication between Vladimir Kravtsov, MD, the Study Principal
 Investigator, and participating co-Principal Investigator the following communication means
 will be used: FedEx delivery of the printed materials, Phone & Fax, E-mail,
 Video-conferencing

 11.1 At the time of submission of the patient's specimen for the study, relevant clinical
 information will be submitted to DiaTech in the form of a study requisition form prepared by
 the participating co-PI (or designated staff nurse) using FedEx courier service.

 11.2 Upon receiving the specimen and requisition form and after purification of tumor cells
 from the specimen, Dr. Kravtsov will place a telephone call to the referring oncologist
 (co-PI) to discuss the case.

 11.3 Upon completion of the patient's tumor drug sensitivity testing, results will be scored
 and introduced to the study data base. Dr. Kravtsov will issue a study report and it will be
 faxed to the referring oncologist no later than 96h after receiving the specimen. After
 faxing the report, Dr. Kravtsov will call the referring oncologist to discuss the results.

 11.4 After each 2 cycles of therapy, the patient's treatment response will be evaluated by
 the referring oncologists (co-PI). A study Response Evaluation Form (see attached) will be
 filled out and faxed to DiaTech to be included in the study data base.

 11.5 After data from first ten consecutive patients are collected, a telephone or video
 conference will be scheduled between Dr. Kravtsov and the co-PI to summarize to discuss the
 preliminary results.

 11.6 After data from first 30 consecutive patients are collected, an interim statistical
 analysis will be performed and Dr. Kravtsov will visit the participating hospital to present
 and discuss the results with the co-Principal investigator.

 11.7 At the end of the first year, statistical analysis of the accumulated data will be
 performed and all participating co-PIs will be invited to a conference (place to be
 determined) to discuss the results.

 11.8 DiaTech Study coordinator (Mr. Garry Latimer) will contact each site co-PI or a
 designated nurse monthly to assure proper supply of transportation containers and to address
 administrative issues.

 11.9 All communication between the study coordinator and the participating oncology practice
 will be documented, reported to the Principal Investigator, and discussed with the
 co-Principal Investigator.

 APPENDIX A

 TREATMENT PLAN ALGORITHM FOR STUDY OBJECTIVE 2.1

 Study Objective 2.1: To evaluate the ability of the MiCK assay to predict the outcome of
 chemotherapy of patients with solid tumors for first- and second-line treatments.

 Chemotherapeutic agent(s) to be included in the treatment protocol are selected by a medical
 oncologist based on published standards and treatment guidelines.

 Chemotherapy of the patient is initiated as per clinical treatment protocol and the
 oncologist discretion.

 Name(s) of the drug(s) selected for the treatment is provided to the DiaTech lab along with
 the tumor material obtained from the patient.

 Tumor cells purified from the patient's specimen are tested for their sensitivity to the
 drugs selected for the patient's treatment.

 Results of the drug sensitivity testing are scored and introduced to the study database.

 After each cycle of therapy, the patient is evaluated for the response to the treatment as
 per established criteria (clinical exam, CT, tumor markers).

 Actual treatment response to the drug is compared with the drug scoring in the in vitro drug
 sensitivity test.

 APPENDIX B

 TREATMENT PLAN ALGORITHM FOR STUDY OBJECTIVE 2.2

 Study objective 2.2: To evaluate the ability of the MiCK assay to guide chemotherapy of
 cancer patients in a third line, refractory treatment setting.

 Chemotherapeutic agents available for the treatment are identified by a medical oncologist
 based on published standards and treatment guidelines.

 Names of the drugs are provided to the DiaTech lab along with the tumor material obtained
 from the patient.

 Tumor cells purified from the patient's specimen are tested for their sensitivity to the
 drugs selected by the patient's oncologist.

 Results of the drug sensitivity testing are scored, introduced to the study database, and
 reported to the medical oncologist.

 The drug with the best anti-tumor activity in the assay is used to treat the patient.

 After each cycle of therapy, the patient is evaluated for the response to the treatment as
 per established criteria (clinical exam, CT, tumor markers).
Inclusion Criteria:

 -  Patients with pathological diagnoses of breast, lung, and ovarian adenocarcinomas and
 soft tissue sarcoma.

 -  Patients with de novo malignancies and no previous chemotherapy

 -  Patients with advanced refractory malignancies who received no more than 2 standard
 chemotherapy treatment protocols.

 -  Patients of any age group.

 -  Patients must have tumor which is accessible and agree to undergo biopsies, or
 drainage of effusions.

 -  Patients for whom chemotherapy is a treatment option.

 Exclusion Criteria:

 -  Patients with symptomatic/uncontrolled parenchymal brain metastasis and non-accessible
 tumors.

 -  Patients with meningeal metastasis.

 -  Patients for whom chemotherapy is not clinically indicated.

 -  Pregnancy. During the course of the study, all patients of childbearing potential
 should be instructed to contact the treating physician if they suspect they might have
 conceived a child; for females, a missing or late menstrual period should be reported
 to the treating physician. If pregnancy is confirmed by a pregnancy test, the patient
 must not receive study medication and must not be enrolled into the study or, if
 already enrolled, must be withdrawn from the study. If a male patient is suspected of
 having fathered a child while on the study drugs, the pregnant female partner must be
 notified and counseled regarding the risk to the fetus. Pregnancy during the course of
 this study will be reported to the Principal Investigator as a serious adverse event.
 Women of childbearing potential are defined to include any female who has experienced
 menarche and has not undergone successful surgical sterilization (hysterectomy,
 bilateral tubal ligation, or bilateral oophorectomy) or is not post-menopausal
 (defined as amenorrhea for more than 12 consecutive months); this also includes
 females using oral, implanted, or injectable contraceptive hormones, mechanical
 devices, or barrier methods to prevent pregnancy.