Dr. Folkman's Proposal
March 22, 2005
Dr. Michael Dodd
Co-founder
Friends for Life
Dear Dr. Dodd:
I enclose a brief summary of a plan for a sponsored post-doctoral fellowship dedicated to researching the possibility of diagnosing and treating
recurrent neuroblastoma before it becomes symptomatic and before it can be anatomically located by conventional methods.
I. Background.
a) Platelet uptake of angiogenesis regulatory proteins produced by tumors.
We have recently discovered that circulating platelets carry about 30 proteins that regulate angiogenesis. More than half of these proteins stimulate angiogenesis and the others inhibit it. These proteins include for example, the angiogenesis stimulators VEGF and bFGF and the angiogenesis inhibitors endostatin and angiostatin, among others.
We have further found that human cancers secrete these same angiogenesis regulatory proteins, which are then selectively taken up by platelets and sequestered. We have developed a rapid analytical method that quantifies these proteins in the platelet lysate from less than a milliliter of blood. We are therefore able to “read” the profile of angiogenesis regulatory proteins in a sample of platelets and this reflects precisely the output of angiogenic proteins from a tumor.
We have found the same profile of angiogenesis regulatory proteins, or the “platelet angiogenesis proteome” (as we now call it), in human platelets from normal volunteers.
What has been most surprising is that analysis of the platelet angiogenesis proteome can detect human cancers (breast cancer, colon cancer, liposarcoma, glioblastoma, etc.), when they are still non-angiogenic and at a microscopic size of less than a millimeter (pinhead size) in a mouse. All of the angiogenesis regulatory proteins secreted by such a tiny tumor are taken up by platelets and continue to increase their concentration or stay at a high concentration in the platelets as long as the tumor is present. None of the angiogenesis regulatory proteins secreted by early tiny tumors are found in the plasma.
A predictable percentage of non-angiogenic human tumors eventually switch to the angiogenic phenotype at a predictable time. For example, human liposarcoma becomes angiogenic at approximately a median of 133 days ± 2 to 3 weeks. Human osteosarcoma does not become angiogenic until after more than a year. Once a tumor has become angiogenic it grows rapidly and can metastasize. Furthermore, after a tumor has grown to approximately 1 cm3 platelets become saturated with angiogenesis regulatory proteins and these proteins begin to spill into the plasma.
References: Achilles et al (Folkman) J Nat’l Cancer Institute 2001, 93:1075.
Udagawa et al, (Folkman) FASEB Journal, 2002, 16:1631.
Klement et al (Folkman) Blood 2004 (November), 104:239a
(Abstract 839).
b) Detection of metalloproteinases in urine.
In the Vascular Biology Program which I direct, Marsha Moses, PhD., (Associate Professor), and her post-doctoral fellows have found that during the onset of angiogenesis in human tumors, specific enzymes, called metalloproteinases are elaborated by the new blood vessels growing toward a tiny tumor. These enzymes are produced by the cells that make up the blood vessels (called endothelial cells), to facilitate the elongation of new blood vessel sprouts through tissues toward a tumor colony.
Dr. Moses has also found that these metalloproteinases spill into the urine soon after a tumor has become angiogenic. In contrast, in the absence of cancer, these proteins are not usually found in the urine. Marsha Moses’ laboratory has analyzed the urine of 5000 patients and has reported the detection of very early recurrences of breast cancer and prostate cancer, before these tumors are detectable by conventional methods such as magnetic resonance imaging or mammogram.
Reference: Roy et al. (Moses) J Biol Chem 2004; 279:51323.
II. Proposed Experimental Plan:
a) Animal studies.
We will obtain specimens of human neuroblastoma from the operating room and from the American Tissue Culture Collection. We will separate and clone non-angiogenic and angiogenic tumor cells and both will be implanted into immunodeficient mice. We will transfect tumor cells with green fluorescent protein so that we can see them at a size of approximately 0.1 millimeter (100 microns). We will then follow the course of these tumors with our two most sensitive and specific “forecasting” biomarkers: (i) the platelet angiogenesis proteome, and (ii) urinary metalloproteinase.
We expect to be able to detect human neuroblastoma in these mice many months before it could be detected by any other conventional method. This would be equivalent to detecting a recurrent neuroblastoma in a human, one or two years before any conventional method could detect it, and long before any symptoms would appear.
We will use the positive biomarkers to guide antiangiogenic therapy with non-toxic angiogenesis inhibitors, . . . until the platelet angiogenesis proteome and the urinary metalloproteinase tests return to normal levels.
In the mice we will carry out preclinical studies using FDA approved angiogenesis inhibitors that have proven to be safe in thousands of patients. These will include doxycycline, zolendronate (Zometa), rosiglitazone (Avandia), very low doses of interferon alpha, and possibly Avastin. These angiogenesis inhibitors will not all be used together. Instead we will start with a single agent, such as doxycycline and later add one or two other angiogenesis inhibitors until the platelet and urine tests become normal. We already have experience with this regimen in at least 5 patients.
Alternatively, we could use Mark Kieran’s 4-drug metronomic regimen which has been underway at the Dana Farber Cancer Center in Children with brain tumors. It has had very good results in the first few children with recurrent neuroblastoma in Europe.
Based on preliminary data we expect that the blood and urine tests may return to normal after 2-3 months of treatment. Visualization of the tiniest tumors by bioluminescence (green fluorescent protein or luciferase) may reveal that they have become dormant or have disappeared.
b) Human clinical trials.
In parallel with the animal studies we may begin to validate the platelet angiogenesis proteome biomarker and the urinary metalloproteinase biomarker (in collaboration with Marsha Moses), in children with the potential for recurrent neuroblastoma. If we collect an ounce of urine every 2-3 months and less than a milliliter of blood any time that a patient has blood drawn for other tests, we may learn if it is possible to detect a recurrent neuroblastoma months, or more than a year before conventional methods can make this diagnosis.
If early detection is possible, then it may be feasible to employ non-toxic angiogenesis inhibitors guided by these biomarkers, without waiting for the recurrent tumor to be located anatomically. In other words, our goal would be to treat an early recurrence of neuroblastoma before waiting to “see” it, analogous to the way that cardiologists treat with Lipitor guided by lipid biomarkers in the blood to prevent a heart attack years in advance.
III. Post-doctoral Fellowship:
I would plan to do this study with a post-doctoral fellow who is an M.D., Ph.D. (tentatively Akash Patnaik) who is coming from the Mayo Clinic. We would also enlist the help of Susan Connors, who is our data manager for the angiogenesis inhibitor trials at the Dana Farber Cancer Institute.
I greatly appreciate your offer to help support our efforts. I would be happy to discuss this with.
Sincerely yours,
Judah Folkman, M.D.
JF/wpf
cc: Marsha Moses, Ph.D.
Ms. Susan Connors
Mrs. Bonnie Brodowski
Ms. Joan Romanition
Dr. Michael Dodd
Co-founder
Friends for Life
Dear Dr. Dodd:
I enclose a brief summary of a plan for a sponsored post-doctoral fellowship dedicated to researching the possibility of diagnosing and treating
recurrent neuroblastoma before it becomes symptomatic and before it can be anatomically located by conventional methods.
I. Background.
a) Platelet uptake of angiogenesis regulatory proteins produced by tumors.
We have recently discovered that circulating platelets carry about 30 proteins that regulate angiogenesis. More than half of these proteins stimulate angiogenesis and the others inhibit it. These proteins include for example, the angiogenesis stimulators VEGF and bFGF and the angiogenesis inhibitors endostatin and angiostatin, among others.
We have further found that human cancers secrete these same angiogenesis regulatory proteins, which are then selectively taken up by platelets and sequestered. We have developed a rapid analytical method that quantifies these proteins in the platelet lysate from less than a milliliter of blood. We are therefore able to “read” the profile of angiogenesis regulatory proteins in a sample of platelets and this reflects precisely the output of angiogenic proteins from a tumor.
We have found the same profile of angiogenesis regulatory proteins, or the “platelet angiogenesis proteome” (as we now call it), in human platelets from normal volunteers.
What has been most surprising is that analysis of the platelet angiogenesis proteome can detect human cancers (breast cancer, colon cancer, liposarcoma, glioblastoma, etc.), when they are still non-angiogenic and at a microscopic size of less than a millimeter (pinhead size) in a mouse. All of the angiogenesis regulatory proteins secreted by such a tiny tumor are taken up by platelets and continue to increase their concentration or stay at a high concentration in the platelets as long as the tumor is present. None of the angiogenesis regulatory proteins secreted by early tiny tumors are found in the plasma.
A predictable percentage of non-angiogenic human tumors eventually switch to the angiogenic phenotype at a predictable time. For example, human liposarcoma becomes angiogenic at approximately a median of 133 days ± 2 to 3 weeks. Human osteosarcoma does not become angiogenic until after more than a year. Once a tumor has become angiogenic it grows rapidly and can metastasize. Furthermore, after a tumor has grown to approximately 1 cm3 platelets become saturated with angiogenesis regulatory proteins and these proteins begin to spill into the plasma.
References: Achilles et al (Folkman) J Nat’l Cancer Institute 2001, 93:1075.
Udagawa et al, (Folkman) FASEB Journal, 2002, 16:1631.
Klement et al (Folkman) Blood 2004 (November), 104:239a
(Abstract 839).
b) Detection of metalloproteinases in urine.
In the Vascular Biology Program which I direct, Marsha Moses, PhD., (Associate Professor), and her post-doctoral fellows have found that during the onset of angiogenesis in human tumors, specific enzymes, called metalloproteinases are elaborated by the new blood vessels growing toward a tiny tumor. These enzymes are produced by the cells that make up the blood vessels (called endothelial cells), to facilitate the elongation of new blood vessel sprouts through tissues toward a tumor colony.
Dr. Moses has also found that these metalloproteinases spill into the urine soon after a tumor has become angiogenic. In contrast, in the absence of cancer, these proteins are not usually found in the urine. Marsha Moses’ laboratory has analyzed the urine of 5000 patients and has reported the detection of very early recurrences of breast cancer and prostate cancer, before these tumors are detectable by conventional methods such as magnetic resonance imaging or mammogram.
Reference: Roy et al. (Moses) J Biol Chem 2004; 279:51323.
II. Proposed Experimental Plan:
a) Animal studies.
We will obtain specimens of human neuroblastoma from the operating room and from the American Tissue Culture Collection. We will separate and clone non-angiogenic and angiogenic tumor cells and both will be implanted into immunodeficient mice. We will transfect tumor cells with green fluorescent protein so that we can see them at a size of approximately 0.1 millimeter (100 microns). We will then follow the course of these tumors with our two most sensitive and specific “forecasting” biomarkers: (i) the platelet angiogenesis proteome, and (ii) urinary metalloproteinase.
We expect to be able to detect human neuroblastoma in these mice many months before it could be detected by any other conventional method. This would be equivalent to detecting a recurrent neuroblastoma in a human, one or two years before any conventional method could detect it, and long before any symptoms would appear.
We will use the positive biomarkers to guide antiangiogenic therapy with non-toxic angiogenesis inhibitors, . . . until the platelet angiogenesis proteome and the urinary metalloproteinase tests return to normal levels.
In the mice we will carry out preclinical studies using FDA approved angiogenesis inhibitors that have proven to be safe in thousands of patients. These will include doxycycline, zolendronate (Zometa), rosiglitazone (Avandia), very low doses of interferon alpha, and possibly Avastin. These angiogenesis inhibitors will not all be used together. Instead we will start with a single agent, such as doxycycline and later add one or two other angiogenesis inhibitors until the platelet and urine tests become normal. We already have experience with this regimen in at least 5 patients.
Alternatively, we could use Mark Kieran’s 4-drug metronomic regimen which has been underway at the Dana Farber Cancer Center in Children with brain tumors. It has had very good results in the first few children with recurrent neuroblastoma in Europe.
Based on preliminary data we expect that the blood and urine tests may return to normal after 2-3 months of treatment. Visualization of the tiniest tumors by bioluminescence (green fluorescent protein or luciferase) may reveal that they have become dormant or have disappeared.
b) Human clinical trials.
In parallel with the animal studies we may begin to validate the platelet angiogenesis proteome biomarker and the urinary metalloproteinase biomarker (in collaboration with Marsha Moses), in children with the potential for recurrent neuroblastoma. If we collect an ounce of urine every 2-3 months and less than a milliliter of blood any time that a patient has blood drawn for other tests, we may learn if it is possible to detect a recurrent neuroblastoma months, or more than a year before conventional methods can make this diagnosis.
If early detection is possible, then it may be feasible to employ non-toxic angiogenesis inhibitors guided by these biomarkers, without waiting for the recurrent tumor to be located anatomically. In other words, our goal would be to treat an early recurrence of neuroblastoma before waiting to “see” it, analogous to the way that cardiologists treat with Lipitor guided by lipid biomarkers in the blood to prevent a heart attack years in advance.
III. Post-doctoral Fellowship:
I would plan to do this study with a post-doctoral fellow who is an M.D., Ph.D. (tentatively Akash Patnaik) who is coming from the Mayo Clinic. We would also enlist the help of Susan Connors, who is our data manager for the angiogenesis inhibitor trials at the Dana Farber Cancer Institute.
I greatly appreciate your offer to help support our efforts. I would be happy to discuss this with.
Sincerely yours,
Judah Folkman, M.D.
JF/wpf
cc: Marsha Moses, Ph.D.
Ms. Susan Connors
Mrs. Bonnie Brodowski
Ms. Joan Romanition
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