SEQUENCE LISTING

Sequence listing filename ITP452-Sequencelisting, created on Dec. 20, 2016 and having a 1.22 kbyte file size in hereby incorporated by reference herein.

BACKGROUND OF THE INVENTION

Field of the Invention

The invention is directed to a risk stratification method for a patient and particularly to a patient having a polymorphism and a tumor, such as a hemangioma and for determination of placenta health, including prediction of diseases such as an intrauterine growth restriction, pre-eclampsia, eclampsia and placental insufficiency in a patient.

Background

Infantile hemangioma (IH) is the most common tumor of the pediatric age group, affecting up to 4% of newborns ranging from inconsequential blemishes, to highly aggressive tumors. Following well defined growth phases (proliferative, plateau involutional) IH usually regress into a fibro-fatty residuum. Despite the high prevalence of IH, little is known regarding the pathogenesis of disease.

Infantile hemangioma (IH) is the most common tumor of the pediatric age group, affecting up to 4% of newborns with nearly 60% localized to the head and neck. These vascular lesions range from inconsequential blemishes to highly aggressive tumors that can cause eye obstructions or blindness, blockage of airways, facial deformations and ulcerations. During the first year, hemangiomas demonstrate both histology and behavior that are also commonly noted in malignancy: immature vascular channels, high mitotic indices, and strong positivity for proliferative markers such as Ki-67.

Despite these ominous beginnings, IH remain benign. The growth velocity slowly reverses leading to a “quiescent or plateau” phase of non-growth (1-2 years) and then transitions into a regressive or “involuting” phase by replacing the once proliferative endothelium with a fibrofatty residuum (2-10 years). However, these growth phases are a matter of clinical judgment alone and the exact timing of each varies considerably among studies. Despite its prevalence, little is known regarding the pathogenesis of disease. Insulin Like Growth Factor 2 (IGF2) has been implicated as an important player in driving the growth of these lesions. IGF2 decreases over six fold from proliferative to involuting IH samples. Furthermore, Beckwith-Wiedmann Syndrome (BWS), where hemangiomas are considered a supportive finding of the diagnosis, is associated with duplications or a loss of imprinting of the IGF2/H19 locus that leads to IGF2 overproduction. Moreover, explant hemangioma cultures grow strongly in response to exogenous IGF2.

IGF2 is an imprinted gene that is usually only expressed from the paternal copy. Commonly, DNA methylation of cytosines preceding guanines (CpG's) reinforce DNA imprinting. These so called epigenetic marks in part determine and are determined by the array of DNA binding proteins capable of interacting with specific chromatin structures. The end result of this process is diploid DNA that is potentially identical in sequence but chemically, transcriptionally and architecturally distinct in a parent of origin specific manner. This leads to activation of one given parental allele and reciprocal silencing of another. The IGF2/H19 region of chromosome 11p15.5 serves as a model for the production of multiple imprinted transcripts.

There is evidence to support the existence of a common genetic program between placental and hemangioma endothelium. The idea of a placental origin of hemangioma is evaluated through molecular profiling and genome-based analysis as described in “Evidence by Molecular Profiling For A Placental Origin of Infantile Hemangioma”, Proceedings of the National Academy of Sciences of the United States, Dec. 19, 2005, Carmen M. Barnes et al.; the entirety incorporated by reference herein. The transcriptomes of human placenta and infantile hemangioma are shown in these studies to be sufficiently similar and therefore suggest a placental origin for this tumor. The unique hemangioma cycle is marked by rapid endothelial cell proliferation, endothelial cell apoptosis, and tumor involution which may mirror the lifetime of a placental endothelial cells.

SUMMARY OF THE INVENTION

The invention is directed to a risk stratification method for a patient and particularly to a patient having a polymorphism and a tumor, such as a hemangioma and for determination of placenta health, including prediction of diseases such as an intrauterine growth restriction, pre-eclampsia, eclampsia and placental insufficiency in a patient and prediction of development of postnatal Infantile hemangioma. Identification of the expression of CTCF with parent of origin specific determination from the mother and/or from the maternal placenta and/or from the fetus or fetal placenta may be used to predict placental health and stratify patients for diseases such as an intrauterine growth restriction, pre-eclampsia, eclampsia and placental insufficiency.

The placenta functions as a fetomaternal organ with two components, the fetal placenta, Chorion frondosum, which develops from the same blastocyst that forms the fetus, and the maternal placenta, Decidua basalis, which develops from the maternal uterine tissue. A blood sample from the mother may be taken for DNA analysis of both the mother and the fetus. In some cases, a simply check swap may be used to provide a sample for DNA analysis of the mother. Both of these techniques are non-invasive. In some cases, a blood sample may be taken from the placenta or from the fetus for DNA analysis as described herein.

Brother of the regulator of imprinted sites or BORIS, also known as transcriptional regulator transcriptional repressor CTCFL, is a protein that in humans is encoded by the CTCFL gene.

With respect to infantile hemangiomas, a reported six fold decrease in IGF2 expression (correlating with transformation of proliferative to involuted lesions) prompted a study of the IGF-2 axis further. As described herein, it has been discovered that IGF2 expression in IH is strongly related to the expression of a cancer testes and suspected oncogene BORIS (paralog of CTCF), placing IH in the unique category of being the first known benign BORIS positive tumor. IGF2 expression was strongly and positively related to BORIS transcript expression. Furthermore, a stronger association was made when comparing BORIS levels against the expression of CTCF via either a percentage or difference between the two, or a rate of increase of % CTCF. A patient with a reducing rate of % CTCF may be a higher risk patient, as this may indicate a more aggressive tumor, as described herein. A common C/T polymorphism at CTCF BS6 appeared to modify the correlation between CTCF/BORIS and IGF2 expression in a parent of origin specific manner. Moreover, these effects may have phenotypic consequences as tumor growth also correlates with the genotype at CTCF BS6. This may provide a framework for explaining the clinical variability seen in IH and suggests new insights regarding CTCF and BORIS related functionality in both normal and malignant states.

Investigations into the differential regulation and potential role of IGF2 as it pertains to IH, as well as the expression of CTCF, a known chromatin insulator element for IGF2 and its antagonist, BORIS (also known as CTCFL—for CTCF like) at both the transcript and protein levels are described herein. The nearby imprinted and maternally expressed H19 gene, which shares enhancers with IGF2, was also quantified. These results were then correlated with methylation analysis of key regulatory regions in the IGF2 and H19 locus. This analysis suggests that a common polymorphism within CTCF Binding Site Six, the critical imprinting control region of H19/IGF2, may have both cellular and phenotypic consequences in a parent of origin specific manner. These findings may serve as a predictor of clinical behavior of IH and may enable risk stratification for patients having a tumor, and specifically IH.

In an exemplary embodiment, a risk stratification method for a patient in a disease state is accomplished by first determining whether said patient is a homozygote patient or a heterozygote patient with respect to CTCF biding site 6. When the patient is a homozygote patient it is then determined if the patient has a Thymine/Thymine, (TT) allelic expression or a Cytosine/Cytosine (CC) allelic expression for said CTCF biding site 6. When the patient is a heterozygote, it is then determined whether the cytosine is methylated; whereby when the cytosine is not methylated the patient has a Thymine/Cytosine, (TC) allelic expression for said CTCF biding site 6; and whereby when the cytosine is methylated the patient has a Cytosine/Thymine (CT) allelic expression for said CTCF biding site 6. A patient with a TC allelic expression has a maternal cytosine and a patient with a CT allelic expression has a paternal cytosine. Risk stratifying can then be determined based on the patient's allelic expression for CTCF binding site 6, wherein the patient with a TT allelic expression is classified as a highest risk patient, the patient with a TC allelic expression is classified as a second highest risk patient, the patient with a CT allelic expression is classified as a third highest risk patient and the patient with a CC allelic expression is classified as a lowest risk patient. Furthermore, unique to the disease of vascular tumors in general and infantile hemangioma specifically is the risk of ulceration. A painful condition where the epithelium of the lesion degrades, creating an open wound prone to bleeding. The TT allelic expression is classified at the highest risk for ulceration.

A highest risk patient having an IH may be prone to a rapidly growing tumor, ulceration of the tumor, facial deformations, eye obstructions, blockage of airways or any combination of these conditions. A second highest risk patient having IH may be prone to moderately to rapidly growing tumors, facial deformations, eye obstructions, blockage or airways or any combination of these conditions. A third highest risk patient having IH may have a slow to moderately growing tumor, facial blemishes. A lowest risk patient having IH may have a slow growing tumor, facial blemishes.

Any suitable method may be used to for determining for a heterozygote patient whether the cytosine is methylated including, bisulfite conversion and quantitative methylation sensitive pyrosequencing or directly sequencing parental DNA, for example.

A risk stratification method, as described herein, may be initiated for a patient in a diseased state, wherein the patient is in a diseased state by identification of a patient tumor. A patient may be considered to be in a diseased state when the identified tumor is an infantile hemangioma, or any other tumor such as a cancerous tumor including, but not limited to, breast cancer, ovarian cancer, testicular cancer, liver cancer, lung cancer, brain cancer, skin cancer, esophageal cancer, throat cancer, bladder cancer, cervical cancer, colorectal cancer, kidney cancer, leukemia, melanoma, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, thyroid cancer, uterine cancer and any other cancer.

The risk stratification as described herein, may further comprise the step of identification of an abnormal expression or mutation/function of a gene regulated by CTCF biding site 6. The abnormal expression or mutation/function of a gene product regulated by CTCF biding site 6 may be IGF2, H19, H19 antisense, IGF2 antisense, a micro ma within the gene locus, or the gene product may be an isoform.

The risk stratification as described herein, may further comprise the step of identification of an abnormal expression or mutation/function of a binding gene product that binds to the CTCF biding site 6. The abnormal expression or mutation/function of a binding gene product that binds to the CTCF biding site 6 may be CTCF, BORIS or a biding isoform.

The risk stratification as described herein, may further comprise the step of identification of an abnormal expression or mutation/function of a binding partner of CTCF or BORIS.

In an exemplary embodiment, a risk stratification method further comprising the steps of determining an expression level of BORIS and an expression level of CTCF with respect to CTCF biding site 6, and then determining a percentage of CTCF transcript, such as the percentage of CTCF with respect to the total of CTCF and BORIS. In an alternative embodiment, a difference in the amount of CTCF to the amount of BORIS may be used. A risk stratification method may take into account the allelic expression, as described herein, and may further take into account the % CTCF, wherein, when the % CTCF is less than 20%, the patient is classified as a highest % CTCF risk patient, wherein when the % CTCF is less than 50% but greater than 20%, the patient is classified as a second highest % CTCF risk patient, wherein when the % CTCF is less than 80% but greater than 50% the patient is classified as a third highest % CTCF risk patient and wherein when the % CTCF ratio is less than 100% or less and greater than 80% the patient is classified as a lowest % CTCF risk patient. Furthermore, the risk stratification method may include the rate of reduction of % CTCF, wherein a rapid drop in % CTCF may indicated a higher risk of an aggressive tumor, or a tumor that grows quickly.

In an exemplary embodiment, a risk stratification method further comprising the steps of determining an expression level of BORIS and an expression level of CTCF with respect to CTCF biding site 6, and then determining a CTCF-BORIS difference which is a difference in the expression level of CTCF to the expression level of BORIS. The patient may then be risk stratified according to said CTCF-BORIS difference wherein, when the CTCF-BORIS difference is less than zero, the patient is classified as a highest CTCF-BORIS risk patient; and when the CTCF-BORIS difference is greater than zero, the patient is classified as a lowest CTCF-BORIS risk patient.

In an exemplary embodiment, the risk stratification method as described herein, may be used to predict a response of medication to the tumor. For example, a patient with a TT allelic expression and classified as a highest risk patient may have a predicted medication response that is most effective, a patient with a TC allelic expression and classified as a second highest risk patient may have a predicted medication response that is second most effective, a patient with a CT allelic expression and classified as a third highest risk patient may have a predicted medication response that is third most effective and a patient with a CC allelic expression and classified as a lowest risk patient may have a predicted medication response that is least effective. Patients classified as the highest risk patients may have the most effective response to medication whereas patients classified as the lowest risk patients may have the least effective response to medication. Medication may be beta blockers, cortical steroids, alpha interferon, and/or IGF2 receptor blockers. Preferred medications are beta blockers and cortical steroid. A most effective response or highly effective response to medication includes slowing of the growth of the tumor, reduction in size of the tumor without rebound or relapse, closure of ulceration or healing or ulceration. A second most effective response to medication includes reduction in size of the tumor without rebound or relapse. T third most effective response to medication includes reduction in size of the tumor without rebound or relapse. A least effective response to medication includes reduction in size of the tumor, or no reduction in size of the tumor.

The medication response prediction method may further comprise the step of determining the disease state of said patient by identification of an abnormal expression or mutation/function of a gene product produced by CTCF biding site 6. The abnormal expression or mutation/function of a gene product regulated by CTCF biding site 6 may be IGF2, H19, H19 antisense, IGF2 antisense, a micro ma within the gene locus, or the gene product may be an isoform.

The method of determining this polymorphism and allelic expression of the gene product regulated by CTCF binding site 6 for the fetus or for an infant, may also be used to predict placental health and risk stratify mothers for placental diseases including an intrauterine growth restriction, pre eclampsia, eclampsia, placental insufficiency. DNA analysis of the mother and/or the fetus may be used in this predictive model. As with the risk stratification model described herein, a predicted model may be based on the allelic expression for CTCF binding site 6 of the mother, the fetus or some combination thereof. A mother with a TT allelic expression may be classified as a highest stratified risk patient, a mother with a TC allelic expression may be classified as a second highest risk patient, a mother with a CT allelic expression may be classified as a third highest risk patient and a mother with a CC allelic expression may be classified as a lowest risk patient. The various combinations of allelic expression of the mother and fetus may further be used in a risk stratification method. When the mother and the fetus both have a TT allelic expression, this may correlate with the highest risk patient for placental health issues, the mother and fetus have a CT or TC allelic expression, this may correlate with a second highest risk patient for placental health issues and when both the mother and fetus have a CC allelic expression, this may correlate with the lowest risk patient for placental health issues. It is to be noted that data to determine correlations between the allelic expression of CTCF binding site 6 of the mother and/or the fetus may alter the risk stratification categories as provided herein.

A G/A polymorphism approximately 130 base pairs downstream of rs10732516 was found to be in strong linkage disequilibrium with the (C/T) polymorphism at CTCF BS6. This polymorphism is rs2107425. This polymorphism was detected by using differential primer binding, see incorporated reference Tost et al, for details. The A polymorphism at rs2107425 was strongly associated with the T polymorphism at CTCF BS 6 (rs10732516) in the samples. Thus, it is conceivable that nearby polymorphisms within the CTCF BS6 locus would be used as a surrogate for a test at rs10732516. This concern is underscored by the fact that large areas of linkage disequilibrium of over 500 base pairs have been documented, shoemaker et al1. This has included regions in IGF2, Shoemaker et al. Given the high level of linkage disequilibrium we observed at CTCF binding six between re10732516 and rs2107425 we propose that the sequence between CTCF binding site 5 and CTCF binding site seven (this includes CTCF binding site six and rs10732516) be considered one quantitative trait locus. Therefore, for the purposes of this invention, a polymorphism as described herein, may be determined by analysis of any sequence between CTCF binding site 5 and CTCF binding site 7. The placental health predictive indicators as well as the risk stratification methods can be based on analysis of any sequence between CTCF binding site 5 and CTCF binding site 7.

Allele-specific methylation is prevalent and is contributed by CpG-SNPs in the human genome. The risk stratification method, as described herein, may be characterized from CTCF binding site 6, or a range upstream and downstream, because of high linking disequilibrium, as described in Robert shoemaker, jie Deng, Wei Wang and Kun Zhang Genome Research 2010 July; 20(7): 883-889, the entirety of which is hereby incorporated by reference. The risk stratification method of the present invention may be performed on base pairs up to 500 base pairs from CTCF binding site 6, or up to about 350 base pairs from CTCF binding site 6, or from about 150 base pairs from CTCF binding site 6, or from CTCF binding site 5 to CTCF binding site 7.

The summary of the invention is provided as a general introduction to some of the embodiments of the invention, and is not intended to be limiting. Additional example embodiments including variations and alternative configurations of the invention are provided herein.

