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).
Purpose