blood-based diagnostic assay that indicates the presence or likelihood of breast cancer through measurement of molecular/protein
markers that are uniquely expressed in malignant tumors.
therapeutic use of a p21-based protein to suppress malignant proliferation of cancer cells, inhibit the allo-immune activation
that results in transplant rejection, and limit the progression of arteriosclerosis.
A blood-based diagnostic assay to monitor the success (acute rejection, nephrotoxicity, or chronic rejection)
of renal transplants.
A National Cancer Institute
(NCI) report estimates that about 1 in 8 women in the United States (approximately 13.3 percent) will develop breast cancer
during her lifetime. The American Cancer Society estimated that in 2003, 211,300 new cases of invasive breast cancer were
diagnosed, along with an additional 55,700 cases of in-situ breast cancer. In 2004, 41,800 women were expected to have died
from breast cancer (2nd only to lung cancer for cancerous deaths).
X-ray mammography is by far the most prevalent diagnostic technique. Ultrasound and MRI are used when
x-ray mammography is likely to be inconclusive because of highly dense breasts. Sensitivity of mammography is high, ~90%,
but specificity is poor, ~40% (leading to biopsies in ~50% of overall cases that turn out to be benign).
Digital mammography and computer-aided detection is gaining ground only slowly
(the American Cancer Society lists 300 machines in the US) due to high cost and limited gains in effectiveness.
Experimental noninvasive techniques include magnetic resonance elastography,
PET scans, electrical impedance spectral imaging, microwave imaging spectroscopy, near infrared spectral imaging, and terahertz
Biopsy is required when a breast image
reveals a suspicious lesion. Between 800,000 and 1.25 million biopsies are performed each year with a cost of $800 to
$3,000, depending on the type of biopsy performed. (USFDA Center for Devices – Radiological Heath & Office
of Women’s Health). According to the American Cancer Society, approximately 80% of breast biopsies are benign.
Since biopsies are generally performed when a breast image reveals a suspicious lesion, sensitivity is not applicable, but
specificity is biopsies is excellent (~100%?).
molecular and genetic tests are under development, but only one test is widely accepted: if a woman carries a hereditary mutation
in genes BRCA1 or BRCA2, she has a 36%-85% chance of developing cancer before age 70 (National Cancer Institute). However,
this test is of limited value since only a small percentage of breast cancers are hereditary in nature (to be reviewed).
Will need to assess for new multiplex PRC assay the specificity and sensitivity
compared to conventional tests (mammography and biopsy) and evaluate as appropriate for different types of cancer (DCIS, fibrosis
and benign). If a new test can show greater sensitivity than mammography, it could replace mammography. If sensitivity is
not high, but specificity is good, it could be used in conjunction with mammography to eliminate unnecessary biopsies.
In breast cancer cells, some
genes are overexpressed while others are under expressed; the associated mRNA can be measured using polymerase chain reaction
(PCR) techniques and overexpressed proteins can be measured using ELISA and Western blot analysis techniques. OncoDetect Inc.
will develop diagnostic assay kits to accurately diagnose the presence and/or likelihood of breast cancer by determining the
unique pattern of expression using the following molecular/protein markers. These markers are particularly useful because
they can be measured non-invasively, via blood lymphocytes rather than biopsy.
Mammaglobin B (protein) – positively correlated ; Skp2 (mRNA)
– positively correlated; PPAR-alpha (mRNA) – positively correlated;
PPAR- gamma (mRNA) – negatively correlated; TIMP-2 (mRNA) – negatively correlated; TNF-alpha ( mRNA) –
positively correlated); Cycline E (protein or mRNA?) – positively correlated;
MMP-9 (mRNA) – positively correlated; MMP-2
(mRNA) – negatively correlated (Figure below)
Research to Date:
Research by Dr. Khanna has demonstrated the following results significant to the development of breast
cancer diagnostic assays:
Application of standard
PCR techniques to malignant and non-malignant tissues from 50 human breast tissue pairs has confirmed the statistically significant
difference in expression of molecular and protein markers between diseased and non-diseased states. (Results for Mammaglobin
B, PPAR-alpha, PPAR-gamma, MMP-9, Skp2, TNF-alpha and TIMP-2.)
Correlation of Mammaglobin B in breast tissues and lymphocytes of patients known to have malignant breast cancer
(based on 25 tissue/lymphocyte pairs) indicates that mammaglobin B levels can be detected in blood samples, not only tissue
samples requiring biopsy.
of Mammaglobin B in the lymphocytes can be amplified using 10 to 40 PCR cycles, greatly lowering the detection threshold.
Correlation of multiple molecular markers
with in tissue and lymphocytes (done for a single patient, to be completed for remaining sample of 25 patients for which complete
Development of intellectual property for the breast cancer diagnostics will focus on: This is based on our
PATENT APPLICATION "Patterns of gene expression and uses therefeof for the detection and diagnosis of breast cancer.
US PROVISIONAL PATENT APPLICATION No. 60/642,481"
Diagnostic kit using multiplex PCR assay(s) to diagnose breast cancer and assess genetic predisposition toward breast
Multiplex PCR assays that includes
a combination of genetic markers that have been statistically selected to maximize the selectivity and specificity of the
Diagnostic kit that includes a combination
of PCR assays where one assay is comprised of genetic markers that increase with the likelihood and/or presence of breast
cancer and another assay is comprised of genetic markers that decrease with the likelihood and/or presence of breast cancer
Diagnostic kit using ELISA techniques to diagnose presence of breast
cancer based on increased/decreased expression of proteins
Diagnostic kit based on applying multiplex PCR to blood lymphocytes for the purpose of diagnosing breast cancer
and/or assessing the genetic predisposition toward breast cancer
Diagnostic kit based on applying multiples PCR to nipple discharge and/or ductile lavage
Unique primers used in development of PCR assays for specific
best target markers for multiplex assay (part of phase I feasibility testing)
Submit SBIR Phase II grant(s) for prototyping of breast cancer diagnostic assay
Develop real-time multiplex PCR assay using best candidate markers (part
of Phase II prototyping)
Phase III commercialization
will include: development of diagnostic kit and quality system.
Determine FDA regulatory strategy and submit appropriate documentation – 510k submission (market to labs as
research product) or PMA (requiring additional testing allowing product to be marketed as diagnostic product)
p21 as a potent immunosuppressive
Thes studies demonstrate
that by virtue of its potent anti-proliferative effects, cyclin kinase inhibitor p21 can render host lymphocytes less responsive
to allo- and mitogenic stimuli; induce tolerance and prevent alloimmune activation enabling a prolonged graft survival. Additionally,
its known inhibitory effects on tumor cells, protective effects on renal function and abrogation of viral replication will
allow p21 to be an attractive alternate strategy to achieve immunosuppression for organ transplant recipients without side
effects associated with current immunosuppressive drugs.
p21 protein when injected to mice results in the inhibition of lymphocyte proliferation and markers of inflammation and does
not cause any toxicity Western blot analysis has confirmed that p21 enters cell nucleus within 4 hours interact
with transcription factors and inhibits gene and protein expression of proinflammatory cytokines.Recombinant
p21 protein inhibits lymphocyte proliferation similar to the currently clinically used immunosuppressive agents; Cyclosporine,
Tacrolimus and Sirolimus Overexpression of p21 in recipients of rat cardiac transplants prolongs graft survival
without immunosuppressive agents by inhibiting intragraft expression of pro-inflammatory cytokines.
Intellectual Property Development:
Development of intellectual property for the p21 based therapies will
focus on: Intellectual Property Development: Development of intellectual property for the P21 based therapies will focus on
based on our Patent application" Cyclin inhibitor p21: potent inhibitor of cellular proliferation; potential therapeutic
applications in Transplantation, Cancer and atherosclerosis. US PROVISIONAL PATENT APPLICATION No. 60/638,930"
Use of p21 as a therapeutic to address abnormal cell proliferation
Use of p21 as an anti-proliferation applied to cancer, transplant rejection
Use of the protein transducing
domain properties of p21 that allow p21 to penetrate the cell cytoplasm and the cell nucleus without the cumbersome process
of attaching the appropriate protein transducing domain that is required to utilize other cyclin kinase inhibitors such as
p16, p23, p27, p53, p73
Use of p21 with targeted
mutations to selectively increase or decrease cell proliferation by creating proteins that can enter The prototyping phase of development will include statistical analysis to examine which combination of
markers provides the best diagnostic results with respect to sensitivity and specificity