Patent Grant
10494677
Field of the Invention
This document relates to methods and materials involved in assessing prostate cancer in male mammals. For example, this document provides methods and materials for determining whether a mammal having prostate cancer is susceptible to a good or poor outcome.
Background Information
Two of the major decision-making points related to therapy for patients with prostate cancer are at the time of biopsy and after radical prostatectomy. Positive biopsies contain a small portion of the tumor for diagnosis of aggressiveness. Based on morphological parameters such as Gleason score, patients are given three choices. In the case of benign or less aggressive disease, the choices are either watchful waiting or surgery. Immediate surgery is recommended in intermediate situations to prevent further advance. Radiation or hormonal therapies are recommended if the cancer has metastasized and surgery would not help. After surgery, when the bulk of the tumor is available for more thorough diagnosis, decisions still have to be made concerning additional therapies such as radiation or hormonal treatment that could depend on the aggressiveness of the disease. Determining disease aggressiveness is important for clinical decisions towards the management of prostate cancer patients.
This document provides methods and materials related to assessing prostate cancer in mammals (e.g., human males). For example, this document provides methods and materials for determining whether or not a mammal has an aggressive form of prostate cancer.
Gleason grade, stage, ploidy, and margin status are major descriptors of prostate cancer aggressiveness and are thought to be important in determining cancer management. However, these clinicopathological parameters have significant drawbacks. For example, Gleason grading is heavily dependent upon the visual interpretation (Gleason, Hum. Pathol., 23:273-279 (1992); Gleason and Vacurg (1977) Histologic grading and clinical staging of prostatic carcinoma. In Urologic Pathology: The Prostate (Tannenbaum M., ed.) Lea & Febiger, Philadelphia, Pa. 171-213). In addition, the level of resolution of this grading system appears to be too coarse as it is dependent on architectural pattern rather than cytologic changes. This concern is supported by the high level of uncertainty regarding the clinical outcome of patients with intermediate Gleason grades. Identifying cancer patients who have a poor prognosis can allow such patients, who are at risk for progression, to be offered more aggressive therapy earlier. In addition, identifying cancer patients who do not have a poor prognosis can avoid unnecessary treatment and suffering.
This document is based in part on the discovery of nucleic acids, referred to herein as aggressive prostate cancer biomarkers, having variant over-expression in aggressive tumors. Such nucleic acids, as well as polypeptides encoded by such nucleic acids, can be analyzed to assess prostate cancer in mammals. Analysis of the nucleic acids, or polypeptides encoded by the nucleic acids, can allow prostate cancer to be assessed in mammals based on an elevated level of one or more of the nucleic acids or polypeptides in a biological sample (e.g., a prostate biopsy specimen) from the mammal. The levels of multiple nucleic acids or polypeptides can be detected simultaneously using nucleic acid or polypeptide arrays.
In one aspect, a method for assessing prostate cancer is provided. The method comprises, or consists essentially of, determining whether or not a mammal having prostate cancer comprises an elevated level of expression of a KHDRBS3, NRP1, COL1OA1, C20orf102, SSTR1, RRM2, F5, HSPC150, CDC2, TOP2A, SERPINI1, TDO2, GRIN3A, COL2A1 or PCDHB10 nucleic acid, or a polypeptide encoded by the nucleic acid, where the presence of the elevated level indicates that the mammal is susceptible to a poor outcome. The method can comprise determining whether or not a mammal having prostate cancer comprises an elevated level of an NRP1, SSTR1, KHDRBS3, or RRM2 nucleic acid, or a polypeptide encoded by the NRP1, SSTR1, KHDRBS3, or RRM2 nucleic acid. The mammal can be a human. The level can be determined in prostate tissue. The level can be determined using PCR or in situ hybridization. The level can be determined using immunohistochemistry. The poor outcome can comprise systemic progression within five years of prostatectomy.
In another aspect, a method for assessing prostate cancer is provided. The method comprises, or consists essentially of, (a) determining whether or not a mammal has an aggressive prostate cancer profile, and (b) classifying the mammal as susceptible to a poor outcome if the mammal has the aggressive prostate cancer profile and classifying the mammal as not susceptible to a poor outcome if the mammal does not have the aggressive prostate cancer profile. The mammal can be a human. The aggressive prostate cancer profile can be determined in prostate tissue. The aggressive prostate cancer profile can be determined using PCR or a nucleic acid array. The aggressive prostate cancer profile can be determined using immunohistochemistry or an array for detecting polypeptides. The poor outcome can comprise systemic progression within five years of prostatectomy.
In another aspect, this document provides a method for assessing prostate cancer. The method comprises, or consists essentially of, determining whether or not a mammal having prostate cancer comprises a reduced level of expression of a CDH10 nucleic acid, or a polypeptide encoded by the nucleic acid, wherein the presence of the reduced level indicates that the mammal is susceptible to a poor outcome. The mammal can be a human. The level can be determined in prostate tissue. The level can be determined using PCR or in situ hybridization. The level can be determined using immunohistochemistry. The poor outcome can comprise systemic progression within five years of prostatectomy.
In another aspect, this document provides a method for assessing prostate cancer. The method comprises, or consists essentially of, determining whether or not a mammal having prostate cancer comprises an elevated level of expression of a ST6GALNAC5, DIRAS2, or BIRC5 nucleic acid, or a polypeptide encoded by the nucleic acid, wherein the presence of the elevated level indicates that the mammal is susceptible to a poor outcome. The mammal can be a human. The level can be determined in prostate tissue. The level can be determined using PCR or in situ hybridization. The level can be determined using immunohistochemistry. The poor outcome can comprise systemic progression within five years of prostatectomy.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
This document provides methods and materials related to assessing prostate cancer in mammals. For example, this document provides methods and materials for determining whether or not a biological sample (e.g., prostate tissue sample) from a mammal (e.g., a male human) contains an elevated level of one or more than one aggressive prostate cancer biomarker. An aggressive prostate cancer biomarker can be a KHDRBS3, NRP1, COL10A1, C20orf102, SSTR1, RRM2, F5, HSPC150, CDC2, TOP2A, SERPINI1, TDO2, GRIN3A, COL2A1, PCDHB10, ST6GALNAC5, DIRAS2, or BIRC5 nucleic acid or polypeptide, or a nucleic acid or polypeptide listed in Table 2 or Table 3. As described herein, if the level of an aggressive prostate cancer biomarker in a sample from a mammal is elevated, then the mammal can be classified as being susceptible to a poor outcome. If the level of an aggressive prostate cancer biomarker in a sample from a mammal is not elevated, then the mammal can be classified as not being susceptible to a poor outcome. In some cases, a reduced or low level of expression of a cancer biomarker (e.g., CDH10) can indicate the presence of aggressive prostate cancer. As described herein, an elevated level of CDH10 can indicate the presence of prostate cancer, and a reduced level of CDH10 can indicate the presence of aggressive prostate cancer.
In some cases, a mammal can be classified as being susceptible to a poor outcome if it is determined that a sample (e.g., prostate tissue) from the mammal has an aggressive prostate cancer profile. For the purpose of this document, the term “aggressive prostate cancer profile” as used herein refers to a nucleic acid or polypeptide profile in a sample where one or more than one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more) of a KHDRBS3, NRP1, COL10A1, C20orf102, SSTR1, RRM2, F5, HSPC150, CDC2, TOP2A, SERPINI1, TDO2, GRIN3A, COL2A1, PCDHB10, ST6GALNAC5, DIRAS2, or BIRC5 nucleic acid or polypeptide, or fragment thereof, or a nucleic acid or polypeptide listed in Table 2 or Table 3, or fragment thereof, is present at an elevated level. In some cases, the aggressive prostate cancer profile can be a profile in a sample where a majority of a KHDRBS3, NRP1, COL1OA1, C20orf102, SSTR1, RRM2, F5, HSPC150, CDC2, TOP2A, SERPINI1, TDO2, GRIN3A, COL2A1, PCDHB10, ST6GALNAC5, DIRAS2, or BIRC5 nucleic acid or polypeptide, and the nucleic acids or polypeptides listed in Table 2 and Table 3, is present at an elevated level.
The term “aggressive” as used herein refers to the invasive and metastatic activity of a cancer. For example, an aggressive prostate cancer is more invasive and metastatic than a less aggressive prostate cancer. Aggressive cancers can produce adverse changes in a mammal's overall health to a greater extent than if that cancer were not aggressive. A mammal with an aggressive prostate cancer can, for example, experience bladder obstruction problems to a greater extent than if that prostate cancer were not aggressive. Other adverse changes in overall health include, without limitation, edema, mineral and vitamin deficiencies, increased risk of infection, loss of appetite, depression, enlargement of organs such as lymph nodes, and pain associated with metastasis. Aggressive cancers can increase mortality to a greater extent than less aggressive cancers. For example, aggressive prostate cancer can cause a poor outcome such as systemic progression within five years of prostatectomy.
To assess the aggressiveness of prostate cancer in a mammal, the level of one or more than one aggressive prostate cancer biomarker can be analyzed in a sample from the mammal. The level of an aggressive prostate cancer biomarker can be determined by measuring any aggressive prostate cancer biomarker including, without limitation, native, truncated, and mutant aggressive prostate cancer biomarkers, as well as any fragments thereof. Examples of aggressive prostate cancer biomarkers include, without limitation, human KHDRBS3 (GenBank Accession No. NM_006558, NP_006549), NRP1 (GenBank Accession No. NM_003873.3, NP_003864), COL10A1 (GenBank Accession No. NM_000493, NP_000484.2), C20orf102 (GenBank Accession No. NM_080607, NP_542174.1), SSTR1 (GenBank Accession No. NM_001049, NP001040.1), RRM2 (GenBank Accession No. NM_001034, NP_001025.1), F5 (GenBank Accession No. NM_000130, NP_000121.2), CDC2 (GenBank Accession No. NM_001786, NP_001777.1), TOP2A (GenBank Accession No. NM_001067, NP_001058.2), CDH10 (GenBank Accession No. NM_006727, NP_006718.2), SERPINI1 (GenBank Accession No. NM_005025.2, NP_005016.1), TDO2 (GenBank Accession No. NM_005651.1, NP_005642.1), GRIN3A (GenBank Accession No. NM_133445, NP_597702.1), COL2A1 (GenBank Accession No. NM_001844, NP_001835.2), PCDHB10 (GenBank Accession No. NM_018930, NP_061753.1), ST6GALNAC5 (GenBank Accession Nos. AA462934, AB028840, AB030836, AI851940, AK034387, AK038434, AK042683, and BC055737), DIRAS2 (GenBank Accession Nos. AA920095, AI414999, AI425960, AK019341, AK019342, AK041534, AK136096, AK136101, BC086799, BF449664, and BM114282), or BIRC5 (GenBank Accession Nos. AB036741, AF077349, AF077351, AF115517, AI413910, AK018022, AK038229, AK142768, AL591433, BC004702, BG063957, BG077309, BY023910, CN724527, and W34764) nucleic acids and polypeptides.
The term “elevated level” as used herein with respect to the level of an aggressive prostate cancer biomarker is any level that is greater than a reference level for that aggressive prostate cancer biomarker. The term “reference level” as used herein with respect to an aggressive prostate cancer biomarker is the level of that aggressive prostate cancer biomarker typically expressed by mammals free of aggressive prostate cancer. For example, a reference level of an aggressive prostate cancer biomarker can be the average level of that aggressive prostate cancer biomarker that is present in samples obtained from a random sampling of 50 males with prostate cancer who did not have systemic progression for at least seven years after having a prostatectomy. In some cases, a reference level can be the average level of an aggressive prostate cancer biomarker that is present in samples obtained from a random sampling of 50 males with a prostate cancer of Gleason score 7 who did not have systemic progression for at least seven years after having a prostatectomy.
It will be appreciated that levels from comparable samples are used when determining whether or not a particular level is an elevated level. For example, the average level of an aggressive prostate cancer biomarker present in bulk prostate tissue from a random sampling of mammals may be X units/g of prostate tissue, while the average level of the aggressive prostate cancer biomarker present in isolated prostate epithelial cells may be Y units/number of prostate cells. In this case, the reference level for the aggressive prostate cancer biomarker in bulk prostate tissue would be X units/g of prostate tissue, and the reference level for the aggressive prostate cancer biomarker in isolated prostate epithelial cells would be Y units/number of prostate cells. Thus, when determining whether or not the level of an aggressive prostate cancer biomarker in bulk prostate tissue is elevated, the measured level would be compared to the reference level for the aggressive prostate cancer biomarker in bulk prostate tissue. In some cases, the reference level of an aggressive prostate cancer biomarker can be a ratio of an expression value of the biomarker in a sample to an expression value of a control nucleic acid or polypeptide in the sample. A control nucleic acid or polypeptide can be any polypeptide or nucleic acid that has a minimal variation in expression level across various samples of the type for which the nucleic acid or polypeptide serves as a control. For example, GAPDH, HPRT, NDUFA7, and RPS16 nucleic acids or polypeptides can be used as control nucleic acids or polypeptides, respectively, in prostate samples.
An elevated level of an aggressive prostate cancer biomarker can be any level provided that the level is greater than a corresponding reference level for that aggressive prostate cancer biomarker. For example, an elevated level of an aggressive prostate cancer biomarker can be 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, 6, 7, 8, 9, 10, 15, 20, or more times greater than the reference level for that aggressive prostate cancer biomarker. In addition, a reference level can be any amount. For example, a reference level for an aggressive prostate cancer biomarker can be zero. In this case, any level of the aggressive prostate cancer biomarker greater than zero would be an elevated level.
Any appropriate method can be used to determine the level of an aggressive prostate cancer biomarker present within a sample. For example, quantitative PCR, in situ hybridization, or microarray technology can be used to determine the level of an aggressive prostate cancer biomarker in a sample. In some cases, the level of an aggressive prostate cancer biomarker can be determined using polypeptide detection methods such as immunochemistry techniques. For example, antibodies specific for an aggressive prostate cancer biomarker can be used to determine the polypeptide level of the aggressive prostate cancer biomarker in a sample.
Any appropriate type of sample can be used to evaluate the level of an aggressive prostate cancer biomarker including, without limitation, prostate tissue, seminal vesicle tissue, lymphatic tissue, serum, urine, and other body fluids. In addition, any appropriate method can be used to obtain a sample. For example, a prostate tissue sample can be obtained by a tissue biopsy or following surgical resection. Once obtained, a sample can be processed prior to measuring the level of an aggressive prostate cancer biomarker. For example, a prostate tissue sample can be processed to extract RNA from the sample. Once obtained, the RNA can be evaluated to determine the level of one or more than one aggressive prostate cancer biomarker present. In some embodiments, nucleic acids present within a sample can be amplified (e.g., linearly amplified) prior to determining the level of one or more than one aggressive prostate cancer biomarker (e.g., using array technology). In another example, a prostate tissue sample can be frozen, and sections of the frozen tissue sample can be prepared on glass slides. The frozen tissue sections can be stored (e.g., at −80° C.) prior to analysis, or they can be analyzed immediately (e.g., by immunohistochemistry with an antibody specific for an aggressive prostate cancer biomarker). In some cases, the level of a KHDRBS3, NRP1, COL10A1, C20orf102, SSTR1, RRM2, F5, HSPC150, CDC2, TOP2A, SERPINI1, TDO2, GRIN3A, COL2A1, PCDHB10, ST6GALNAC5, DIRAS2, or BIRC5 nucleic acid or polypeptide, or a nucleic acid or polypeptide listed in Table 2, or any combination thereof, can be evaluated in a prostate tissue sample obtained following a prostatectomy procedure. In some cases, the level of one or more than one nucleic acid or polypeptide listed in Table 3 can be evaluated in a prostate tissue sample obtained by a tissue biopsy.
Once the level of an aggressive prostate cancer biomarker in a sample from a mammal is determined, then the level can be compared to a reference level for that aggressive prostate cancer biomarker and used to evaluate the susceptibility of the mammal to a poor outcome. A level of one or more than one aggressive prostate cancer biomarker in a sample from a mammal that is higher than the corresponding one or more than one reference level can indicate that the mammal is susceptible to a poor outcome. In contrast, a level of one or more than one aggressive prostate cancer biomarker in a sample from a mammal that is equal to or lower than the corresponding one or more than one reference level can indicate that the mammal is not susceptible to a poor outcome.
In some cases, the aggressiveness and outcome of prostate cancer can be assessed based on the numbers and/or levels of aggressive prostate cancer biomarkers in a sample from a mammal. The greater the number of aggressive prostate cancer biomarkers present at an elevated level in a sample from the mammal, the more aggressive is the prostate cancer in the mammal, and the more susceptible is the mammal to a poor outcome. In addition, the greater the differences between the levels of the aggressive prostate cancer biomarkers in a sample from a mammal and the corresponding reference levels, the more likely the prostate cancer is to progress in the mammal.
In some cases, the levels of aggressive prostate cancer biomarkers in a sample can be used in combination with one or more other factors to determine whether or not a mammal having prostate cancer is susceptible to a poor outcome. For example, levels of aggressive prostate cancer biomarkers in a sample from a mammal having prostate cancer can be used in combination with the clinical stage, the serum PSA level, and/or the Gleason score of the prostate cancer to determine whether or not the mammal is likely to have to a poor outcome. Additional information about the mammal, such as information concerning genetic predisposition to develop cancer, SNPs, chromosomal abnormalities, gene amplifications or deletions, and/or post translational modifications, can also be used in combination with the level of one or more aggressive prostate cancer biomarkers to assess the aggressiveness and outcome of prostate cancer. In some cases, the level of one or more aggressive prostate cancer biomarkers in a sample from a mammal can be used in combination with the Gleason score, preoperative PSA, seminal vesicle invasion, and margin status to determine whether or not the mammal is susceptible to a poor outcome (Blute et al., J Urol., 165(1): 119-25 (2001)).
This document also provides methods and materials to assist medical or research professionals in determining whether or not a mammal having prostate cancer is susceptible to a poor outcome. Medical professionals can be, for example, doctors, nurses, medical laboratory technologists, and pharmacists. Research professionals can be, for example, principle investigators, research technicians, postdoctoral trainees, and graduate students. A professional can be assisted by (1) determining the level of one or more than one aggressive prostate cancer biomarker in a sample, and (2) communicating information about that level to that professional.
Any method can be used to communicate information to another person (e.g., a professional). For example, information can be given directly or indirectly to a professional. In addition, any type of communication can be used to communicate the information. For example, mail, e-mail, telephone, and face-to-face interactions can be used. The information also can be communicated to a professional by making that information electronically available to the professional. For example, the information can be communicated to a professional by placing the information on a computer database such that the professional can access the information. In addition, the information can be communicated to a hospital, clinic, or research facility serving as an agent for the professional.
This document also provides nucleic acid arrays. The arrays provided herein can be two-dimensional arrays, and can contain at least two different nucleic acid molecules (e.g., at least three, at least five, at least ten, at least 20, at least 30, at least 40, at least 50, or at least 60 different nucleic acid molecules). Each nucleic acid molecule can have any length. For example, each nucleic acid molecule can be between 10 and 250 nucleotides (e.g., between 12 and 200, 14 and 175, 15 and 150, 16 and 125, 18 and 100, 20 and 75, or 25 and 50 nucleotides) in length. In some cases, an array can contain one or more cDNA molecules encoding, for example, partial or entire polypeptides. In addition, each nucleic acid molecule can have any sequence. For example, the nucleic acid molecules of the arrays provided herein can contain sequences that are present within aggressive prostate cancer biomarkers.
In some cases, at least 25% (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 95%, or 100%) of the nucleic acid molecules of an array provided herein contain a sequence that is (1) at least 10 nucleotides (e.g., at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or more nucleotides) in length and (2) at least about 95 percent (e.g., at least about 96, 97, 98, 99, or 100) percent identical, over that length, to a sequence present within an aggressive prostate cancer biomarker. For example, an array can contain 60 nucleic acid molecules located in known positions, where each of the 60 nucleic acid molecules is 100 nucleotides in length while containing a sequence that is (1) 90 nucleotides is length, and (2) 100 percent identical, over that 90 nucleotide length, to a sequence of an aggressive prostate cancer biomarker. A nucleic acid molecule of an array provided herein can contain a sequence present within an aggressive prostate cancer biomarker where that sequence contains one or more (e.g., one, two, three, four, or more) mismatches.
The nucleic acid arrays provided herein can contain nucleic acid molecules attached to any suitable surface (e.g., plastic, nylon, or glass). In addition, any appropriate method can be used to make a nucleic acid array. For example, spotting techniques and in situ synthesis techniques can be used to make nucleic acid arrays. Further, the methods disclosed in U.S. Pat. Nos. 5,744,305 and 5,143,854 can be used to make nucleic acid arrays.
This document also provides arrays for detecting polypeptides. The arrays provided herein can be two-dimensional arrays, and can contain at least two different polypeptides capable of detecting polypeptides, such as antibodies (e.g., at least three, at least five, at least ten, at least 20, at least 30, at least 40, at least 50, or at least 60 different polypeptides capable of detecting polypeptides). The arrays provided herein also can contain multiple copies of each of many different polypeptides. In addition, the arrays for detecting polypeptides provided herein can contain polypeptides attached to any suitable surface (e.g., plastic, nylon, or glass).
A polypeptide capable of detecting a polypeptide can be naturally occurring, recombinant, or synthetic. The polypeptides immobilized on an array also can be antibodies. An antibody can be, without limitation, a polyclonal, monoclonal, human, humanized, chimeric, or single-chain antibody, or an antibody fragment having binding activity, such as a Fab fragment, F(ab′) fragment, Fd fragment, fragment produced by a Fab expression library, fragment comprising a VL or VH domain, or epitope binding fragment of any of the above. An antibody can be of any type, (e.g., IgG, IgM, IgD, IgA or IgY), class (e.g., IgG1, IgG4, or IgA2), or subclass. In addition, an antibody can be from any animal including birds and mammals. For example, an antibody can be a mouse, chicken, human, rabbit, sheep, or goat antibody. Such an antibody can be capable of binding specifically to an aggressive prostate cancer biomarker. The polypeptides immobilized on the array can be members of a family such as a receptor family, protease family, or an enzyme family.
Antibodies can be generated and purified using any suitable methods known in the art. For example, monoclonal antibodies can be prepared using hybridoma, recombinant, or phage display technology, or a combination of such techniques. In some cases, antibody fragments can be produced synthetically or recombinantly from a nucleic acid encoding the partial antibody sequence. In some cases, an antibody fragment can be enzymatically or chemically produced by fragmentation of an intact antibody. In addition, numerous antibodies are available commercially. An antibody directed against an aggressive prostate cancer biomarker can bind the polypeptide at an affinity of at least 104 mol−1 (e.g., at least 105, 106, 107, 108, 109, 1010, 1011, or 1012 mol−1).
Any method can be used to make an array for detecting polypeptides. For example, methods disclosed in U.S. Pat. No. 6,630,358 can be used to make arrays for detecting polypeptides. Arrays for detecting polypeptides can also be obtained commercially, such as from Panomics, Redwood City, Calif.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Gene expression was profiled in cancerous and non-cancerous prostate epithelial cells using microarray technology. The cells were collected from 101 frozen tissue samples using laser capture microdissection. Benign epithelial cells adjacent to tumor tissue were collected from 19 tissue samples (cases). Cells were also collected from 10 benign prostatic hyperplasia (BPH) cases, 5 prostatic intraepithelial neoplasia (PIN) cases, 30 cases with only Gleason pattern 3 (Gleason score 6), 20 cases with only Gleason pattern 4 (Gleason score 8), and 10 cases with only Gleason pattern 5 (Gleason score 10). In addition, prostatic adenocarcinoma cells were collected from 7 cases with lymph node metastases. Total RNA was extracted from cells of each type collected. Each RNA sample was linearly amplified, labeled, and hybridized to a U133 Plus 2.0 array (Affymetrix, Santa Clara, Calif.). The arrays were washed, stained, and scanned. The gene expression data were analyzed using dChip and GCOS 6.0 software. In analyzing the data with dChip, invariant set normalization and PM/MM Model Based Expression values were generated.
The gene expression data generated as described in Example 1 were analyzed using bioinformatics. The bioinformatics analysis was designed to account for the heterogeneity of prostate cancer better than other methods that require more consistent differential expression of nucleic acids in tumor samples. The analysis included three steps (
In the first step of the analysis, VOGs were identified from the microarray data. The maximum expression levels of probesets in non-neoplastic cases were determined (Bmax,
In the second step of the analysis, the percentage of aggressive (Pag) and non-aggressive (Pnag) cases over-expressing each VOG was determined. Aggressive tumors were represented by Gleason patterns 4 and 5 cases, and non-aggressive tumors were represented by Gleason pattern 3 cases. To identify probesets that are over-expressed in higher percentages of aggressive tumors and are therefore likely to have prognostic value, VOGs were ranked by Δp, where Δp=Pag−Pnag. Mean differences in expression were plotted against Δp for probesets selected by VOG (
In the third step of the analysis, the size of the dataset was reduced by grouping probesets that had a similar pattern of expression across the 101 cases analyzed as described in Example 1. A Pearson's correlation was calculated for each probeset against all other probesets, and probesets that had a correlation coefficient greater than 0.5 were grouped into separate clusters.
The gene expression data were also analyzed using the p-value and fold change (pFC) approach, and results of this analysis were compared to results of the VOG analysis. Two sets of analyses were performed using fold change and p-values generated using dChip software. Probesets were selected that were detected as present in at least 40% of tumor cases, and that were over-expressed by at least 2.5-fold in tumor cases compared to non-neoplastic cases, with a p-value less than 0.0005. This comparison identified 248 probesets with a 90th percentile false discovery rate (FDR) of 0%. Probesets were also selected that were detected as present in at least 40% of aggressive tumor cases (Gleason pattern 4 and 5) and that were over-expressed at least 2-fold in aggressive tumor cases compared to non-aggressive (Gleason pattern 3) tumor cases, with a p-value <0.0005. This comparison identified 122 probesets, with a 90th percentile FDR of 1.6%.
To determine the overlap between the VOG approach and analysis by the pFC method, results were compared at two stages in the biomarker discovery process. First, the ability of VOG and pFC to identify over-expressed genes in tumor cells (step 1) was examined. Analysis of the same expression data by both methods, comparing non-neoplastic tissues to all tumor tissue samples, identified similar numbers of candidates (
The VOG-Δp list of candidates included probesets representing the same gene as well as probe sets representing different genes with similar expression patterns in the tissues studied. Grouping probesets based on a Pearson correlation of 0.7 for the expression patterns (
Genes that had high Δp values or high expression levels in aggressive tumors were selected for validation. Gene expression was analyzed in formalin-fixed, paraffin-embedded (FFPE) tissues that were independent from the tissue samples used in nucleic acid expression profiling described above. The tissues were obtained from men who had a prostatectomy and at least five years of follow up information. Cases were defined as high-grade (Gleason score of 7 or higher) prostate cancer tissues from men who failed systemically within five years of prostatectomy. Controls were defined as high-grade prostate cancer tissues from men who did not have systemic progression seven years after their surgery. The case and control tissues were matched with respect to Gleason score, pre-operative PSA level, age, year of surgery, and margin, seminal vesicle and/or nodal invasion. One hundred pairs were randomly selected for validation experiments, and all samples for which tissue was available were used in the analysis (sixty-seven pairs). The laboratory personnel were blinded to case-control status, and processing of the samples was randomized to prevent experimental bias. With the exception of one sample, all of the remaining controls were free of systemic progression for at least eight years after surgery.
A section of each FFPE tissue was placed on a slide and stained with H&E. A pathologist circled the slide around the areas of the tumor tissue that had an aggressive phenotype. Subsequent sections (10 mm) of each tissue were prepared under RNase free conditions and deparaffinized with xylene. The identified tumor areas were scraped into 1.5 mL tubes containing digestion buffer from the RecoverAll kit (Ambion, Austin, Tex.). Total RNA was isolated according to the manufacturer's protocol. The isolated RNA was treated with DNase using the Turbo DNA free kit (Ambion) according to the manufacturer's instructions. The amount of nucleic acid in each sample was measured using the Quant-iT™ RiboGreen kit (Invitrogen Carlsbad, Calif.). Reverse transcription was performed using Superscript III First Strand Synthesis system (Invitrogen) and 500 ng of RNA from each sample in 40 μL reaction volumes.
Quantitative PCR was performed using 12.5 ng of cDNA (RNA equivalents) per reaction in a 20 μL reaction volume with SYBR green PCR Master Mix (Applied Biosystems, Foster City, Calif.). An ABI 7900HT instrument was used with the manufacturer's default cycling conditions. The concentration of each primer was 0.15 or 0.2 nM. The primers were designed to amplify a 70-85 base pair fragment corresponding to the Affymetrix target sequence for the nucleic acid of interest. Primer Express software (ABI) was used in the primer design. The primer pairs were validated by generating standard curves using dilutions of pooled prostate cDNA from frozen cancerous and non-cancerous tissues and analysis of endpoint dissociation curves. GAPDH was amplified in each sample to verify that an adequate amount of amplifiable cDNA was present as a test for RNA quality. To check for genomic DNA contamination, RNA samples lacking reverse transcriptase were tested in a quantitative PCR reaction. Samples with a cycle threshold (Ct) less than 35 per 12.5 ng RNA were considered contaminated with DNA and were re-processed. Ct values above 35 were called “undetermined” and were manually set to 40 cycles for data analysis. For each sample, the average cycle number was used in data analysis.
Suitable genes for normalization of quantitative PCR data were identified. The microarray data were searched for genes with minimum variation in expression across the 101 samples of cancerous and non-cancerous prostate epithelial cells analyzed as described in Example 1. The suitability of candidate genes was further assessed by analyzing the genes for minimum variation in expression across bulk kidney tissue samples. This was important because although candidate prostate cancer biomarkers were discovered using laser capture microdissected cells, further evaluation of the biomarkers was to be performed using bulk tissues. Candidate genes for normalization of quantitative PCR data were, therefore, analyzed for minimum variation in expression across various bulk kidney tissues that were profiled in another microarray study. This process identified NDUFA7 and RSP16 as the best genes for normalization. HPRT and GAPDH were also examined. These genes were ultimately assessed by quantitative RT-PCR of FFPE prostate samples in a case-control study. Among these genes, NDUFA7 produced the minimum variation across the cases and the controls (
The case-control tissue samples were analyzed for expression of genes with Δp values greater than 30 and genes with high Δp values and high expression levels in aggressive tumors. Four genes, RRM2, TOP2A, HSPC150, and CDC2, were identified as having Δp>30, and these genes were grouped in the same cluster (Table 2) by the data reduction step described in Example 2. RRM2 and Top2a were examined by quantitative PCR, and a significant correlation was observed between the expression levels of the two genes across the case-control samples (R2=0.59,
About 40% of the genes selected for quantitative RT-PCR validation had significantly higher expression levels in cases than controls (p<0.05;
It was determined whether the pFC method would have identified genes showing association with outcome in the case-control analysis. The 122 probesets identified by pFC comparison of non-aggressive versus aggressive tumors (
laevis)
The results described in Example 3 were obtained using case and control tissues that were matched with respect to clinical parameters, including margin, seminal vesicle and/or nodal invasion. When only non-invasive controls were included in the case/control study, additional genes with prognostic value were identified (Table 3). These genes can serve as prognostic biomarkers in biopsy tissues, where invasive status is not known.
Cohort Study for Endorsing High-Risk Population Marker Development
To confirm the need for novel biomarkers to predict outcome in high risk prostate cancer patients, an analysis of a cohort of prostate cancer patients was undertaken. This cohort analysis was performed independent of the case-control study used to evaluate the candidate biomarkers. This analysis, used the Mayo Radical Prostatectomy database cases from 1990-2004, where 441 systemic progressions were observed in 10,626 patients initially treated with RRP. From this data, a Cox proportional hazards model was computed using only existing clinical and pathologic parameters as predictors. Independent analyses were performed on the entire cohort and on the high risk subgroup (GS≥7).
Validation 1 Case-Control Design
The records of all men treated by RRP at the Mayo Clinic between 1990 and 1999, with archived paraffin-embedded material from the RRP specimen, were examined. Men with systemic progression within five years of RRP were identified, and matched with controls known to be free of systemic progression at seven years. Subjects were also matched on Gleason score, TNM stage, margin status, and preoperative serum PSA. Patients undergoing RRP at the Mayo Clinic were evaluated postoperatively at least quarterly for one year, semiannually for one year, and annually thereafter. Digital rectal examination and serum PSA were evaluated at each visit. If patients had an abnormal elevation in serum PSA postoperatively, a radioactive bone scan, plain radiograph in the presence of an abnormal bone scan, and/or computerized tomography were performed. Patients that did not return to the Mayo Clinic were mailed kits for blood submission and serum PSA testing, and additional medical information was obtained from the local physicians as needed (Blute et al., J. Urol., 165(1): 119-25 (2001)). Systemic progression was defined as the development of metastatic disease as determined by clinical, radiologic (bone or CT scan), or pathologic (biopsy) evaluation. PSA progression alone was not sufficient for declaration of a systemic progression. Based on this data, a set of 200 (initially) samples, comprising matched cases and controls, was defined. Tissues were acquired from the tissue bank and reviewed by two pathologists blinded to the case-control status. 76 cases and 81 controls passed the pathology review and had sufficient tissue sample available for experimental analysis. The clinical and pathologic features of the selected subjects were noted (Table 4). Due to the exclusion of several subjects following review by the pathologists, exact case-control matching was not preserved. However, the retained cases and controls remained balanced on age, preoperative serum PSA, TNM stage, Gleason score, margin status, and adjuvant (<90 days after surgery) therapy, and an unmatched analysis was used. Based on the final numbers, the study had about 80% power to detect (alpha=0.05, two-sided) a mean difference in gene expression between cases and controls equivalent to 0.45 standard deviations. For present/absent gene expression, a 22 percentage point difference (e.g., 40% vs. 62%) between cases and controls could be detected.
Validation 2 Case-Control Design
An independent nested-case control validation study was conducted using 21 systemic progression patients (cases) from the fresh frozen prostatectomy cohort from 2000-2005 inclusive. Controls were selected from those at risk at the time of the case's event and matched on Gleason score, pathologic stage, age, and year of surgery. The risk-set sampling design was preferred since the follow-up time was not sufficient to select 7-year systemic-progression-free matched controls. Four potential controls were identified with the first one or two that qualified selected for further experimental processing. The total number of controls with sufficient RNA was 36.
Processing of Formalin Fixed Paraffin Embedded (FFPE) Samples
In all experiments, the processing of samples was randomized to prevent processing biases. Each case was reviewed by a pathologist, and tumor was identified on H-E stained sections. Subsequent sections (10 (μm) from each case were prepared under RNase free condition and de-paraffinized with Xylene. Identified tumor areas were scraped into 2 mL tubes containing digestion buffer (RecoverAll kit, Ambion, Austin, Tex.). Total RNA was isolated according to the RecoverAll RNA isolation procedure. The isolated RNA was treated with DNase using Ambion Turbo DNA free kit, according to the manufacturer's instructions (Ambion). The amount of RNA in each case was measured by the Quant-iT™ RiboGreen kit (Invitrogen Carlsbad, Calif.). Reverse transcription was performed using Superscript III First Strand Synthesis system (Invitrogen) and 500 ng of RNA from each case in a 40 μL reaction volume.
Processing of Frozen Samples for Validation
Tissue was cut by the tissue-processing core facility and not allowed to thaw. Sections were stored on slides at −80° C. for less than one week. Slides were placed directly from −80° C. to 75% ethanol (ETOH) for 30 seconds×2 to remove optimum cutting temperature (OCT) compound (Tissue-Tek), then placed in 95% ETOH for 30 seconds and 100% ETOH for 30 seconds and air dried. Tissue was scraped directly into lysis solution from the RNeasy mini or midi kit (Qiagen, Valencia, Calif.) and processed immediately according to manufacturer's instructions. Reverse transcription and qPCR were performed as described for FFPE tissue except 200 ng of RNA was used in the RT reaction and 1 μL cDNA was used in subsequent qPCR (5 ng RNA equivalent).
Quantitative PCR (qPCR) was performed on each sample by adding 12.5 ng total RNA equivalent of cDNA to a 20-μL reaction volume for each gene of interest using SYBR green PCR Master Mix (Applied Biosystems ABI, Foster City, Calif.) on the ABI 7900HT real time PCR machine using the manufacturer's default cycling conditions. Primers for qPCR were designed using Primer Express software (ABI) to amplify a 70-85 base pair fragment from the Affymetrix target sequence for the gene of interest. The primer pair concentrations (0.15 or 0.2 μM final) were optimized by generating standard curves using a pool of prostate cDNA from normal and tumor tissue. To check for genomic DNA, No-RT samples were run in a qPCR reaction and those with cycle threshold (Ct) less than 35 for GapDH were considered contaminated with DNA and were re-processed. In the analysis of data, “undetermined” values for Cts were replaced with a Ct of 40, which was the maximum cycle number in the qPCR experiments. All samples were analyzed in duplicate, and all studies were carried out under approved protocols. Primers used for the quantitative RT-PCR expression analysis of the genes in the final model are:
Data Analysis
All quantitative PCR measurements were normalized by subtracting the number of cycles measured for a candidate gene from the average number of cycles measured for GAPDH and RPS28 in the same tissue sample. This normalization method inverts the amplification values, such that higher values correspond to higher expression. The full set of un-normalized data was used to assess the variance of the assay using a Bland-Altman plot of the average of a pair of replicates versus their difference. Assay variability was close to constant for values below 33, from which point it increased linearly. The inverse of the estimated variance was used to create a weighted average for each replicate pair. In samples where one replicate measure returned no value, the other replicate value was used. In samples where both replicate measures returned no value, the measurement was treated as missing.
Fusion Status Analysis
The presence of TMPRS S2-ERG, TMPRS S2-ETV1, and TMPRS S2-ETV4 fusions was assigned based the expression values of ERG, ETV1, and ETV4 genes. The thresholds for status assignments were determined for each gene by analyzing the normalized CT expression values of these genes. Samples where the expression was greater than the upper threshold in any of the three genes (ERG>−4.8, ETV1>−5.5, ETV4>−6) were designated fusion positive. Samples where the expression was less than the lower threshold for all three genes (ERG<−5.4, ETV1<−6.8, ETV4<−5.9) were assigned fusion negative. All other samples were assigned an undetermined status. A discrete variable was then used to denote the predicted fusion status of the cases and controls by assigning a “1” to fusion+samples, a “−1” to fusion-samples, and a “0” to the samples with undetermined status.
Ploidy Analysis
Flow cytometry was performed as described elsewhere (Zanetta et al., Am. J. Obstet. Gynecol., 175(5): 1217-25 (1996)). The nuclear content of 10,000 nuclei was measured with a FACScan (Becton Dickinson, Sunnyvale, Calif.) flow cytometer. Cell cycle evaluation of the DNA histogram was performed with a Modfit 5.2 (Verity Software, Topsham, Me.) computerized software program. Tumors with only one identifiable gap0-gap1 peak were classified as DNA diploid (2n). Tumor samples that contained a significant increase in the 4n peak (more than 9% of nuclei) and an identifiable 8n population were categorized as DNA tetraploid. Tumor DNA content was classified as DNA aneuploid if a separate, identifiable gap0-gap1 peak was present. All DNA histograms were analyzed and classified without knowledge of the clinicopathologic features or patient outcome. The ploidy status parameter was defined as 0 for diploid tumors, 1 for tetraploid tumors, and 2 for anauploid.
Statistical Methods
In the cohort study, the Cox proportional hazards model was used to evaluate predictors of time to systemic progression. The concordance statistic (C), which has an interpretation similar to ROC curve area, was used to compare models. In the case-control study, the association of gene expression with systemic progression was assessed using univariate and multivariate logistic regression with the dependent variable (0=control, 1=case) and the normalized genes as the independent predictors. Computations were done using the S-Plus statistical package (Insightful Corp, Seattle Wash.) and the open-source R statistical package. The clustering analysis was performed using clustering methods in R.
Results
Cohort Study for Current High-Risk Prostate Cancer Marker Status
The study objective was to generate a multivariate molecular model that would add value to contemporary prognostic factors in predicting adverse outcomes. Using patients treated from 1990-2004 in the Mayo Radical Prostatectomy database, the Cox proportional hazards model was used to identify significant clinical predictors of time to systemic progression. The strongest predictors were RRP Gleason score, pathologic stage components (extra-capsular cancer, seminal vesicle involvement, lymph node metastases), and the use of adjuvant (within 90 days after RRP) hormonal therapy (Table 5). The model had a ROC area under the curve (as measured by the concordance statistic) of 0.82. Gleason score was the strongest predictor with ten year rates of systemic progression for GS 2-6, 7, and 8-10 of 1.9%, 10.6% and 23.6%, respectively. When the patient cohort was restricted to GS 7-10, the strongest significant clinical predictors of time to systemic progression were RRP Gleason score, seminal vesicle involvement, and lymph node involvement (Table 5), and the concordance statistic dropped to 0.69. Guided by these results, it was concluded that a high-risk prostate cancer RRP population, with GS≥7, would strongly benefit from molecular marker panels to improve prognostic predictions. To address this need, a case-control study was designed to identify molecular markers using GS≥7 patients with and without systemic progression. Cases were defined as men that developed systemic progression or died of prostate cancer within five years of RRP matched for Gleason score, TNM stage, margin status and preoperative serum PSA with controls that did not develop systemic progression or die of prostate cancer.
Case-Control Study
The cases and controls were very similar in their clinical and pathologic features with the exception of DNA ploidy. The gene expression pattern between the two groups was evaluated using univariate analysis (Table 6) for all 38 normalized genes and the predicted fusion parameter. Additionally, the odds ratios and p-values were reported with adjustment for ploidy status of the sample (diploid vs. tetraploid vs. aneuploid). As part of the Mayo Clinic standard practice, DNA ploidy was performed on all RRP cases. Therefore, since ploidy status has been previously indicated (Bostwick et al., Arch. Pathol. Lab. Med., 124(7):995-1000 (2000) and Epstein et al., Scand. J. Urol. Nephrol. Suppl., 216:34-63 (2005)) as potentially prognostic, it was evaluated and found to be significantly associated with case/control status. The odds ratio reflected the increased risk for each one unit increase in the assay, i.e., doubling of gene expression. Based on this analysis, the best univariate predictors were BIRC5, RRM2, and TOP2A. CDH10, also a significant predictor, had a negative coefficient suggesting a protective role. The other genes with a significant (p 0.05) prognostic univariate effect were GRIN3A, COL2A1, NRP1, and SSTR1. When hierarchical clustering was performed for the normalized nucleic acids based on absolute correlation (
Multivariate Analysis Finds an Optimal Model
A forward stepwise selection process was used to define a three variable model for the prediction of prostate cancer outcome. The first variable included in the model was TOP2A as it possessed a significant p-value. With TOP2A in the model, BIRC5 and RRM2 were not significant predictors. The predictive performance of the model did not change significantly if either BIRC5 or RRM2 were selected as the first variable of the model. When any one of TOP2A, BIRC5, and RRM2 was selected as the first variable in the model, the other two nucleic acids lost significance, demonstrating these nucleic acids are interchangeable and suggesting they are related. The next most significant variable added to the model was CDH10. After adding CDH10 and TOP2A to the model, the significance of the fusion status (predicted fusion derived from high expression of ERG, ETV1, and ETV4) increased and became the most significant remaining parameter. Adding fusion status to TOP2A and CDH10 rendered the remaining nucleic acids insignificant. Each successive step of this modeling improved the corresponding Area Under the Curve (AUC) in the Receiver Operating Characteristic (ROC) plot. The AUC with TOP2A alone was 0.71. It was increased to 0.74 with CDH10, and increased to 0.79 with the predicted fusion parameter (
Using the predicted risk score from the regression model (Table 7), an optimal threshold for the identification of men with systemic progression was identified resulting in 65% sensitivity and 86% specificity. The score derived from the regression model was displayed, revealing the overall separation of the cases and the controls (
The results provided herein demonstrate that models can be used to distinguish men treated by RRP who develop systemic progression from men treated by RRP who do not develop systemic progression. A three-variable model, which includes TOP2A expression, CDH10 expression, and predicted TMPRSS2-(ERG, ETV1, or ETV4) fusion status, exhibits a sensitivity of 65% and specificity of 86%. The model was developed in a case control study of men matched on Gleason score, TNM stage, margin status, and preoperative serum PSA who did and did not develop systemic progression or die of prostate cancer. Survival curves by score group could not be generated due to the case/control design, wherein the event rate is pre-determined to be 50%. If the logistic score were applied prospectively, an increase in score of 0.7 (which is quite plausible) would imply a doubling in risk. For Gleason score 7, where the cumulative systemic progression rate at 10 years was 11%, this change in score could imply an overall increase in risk to around 20%.
In this case-control study, cases and controls were not matched for aneuploidy as this is not a standard pathologic assessment of prostate cancer in RRP specimens, and its association with prostate cancer outcome is debated. Ploidy generally correlates with other prognostic factors, such as Gleason score, tumor stage, and tumor volume: low-stage tumors are usually diploid, and high-stage tumors are usually non-diploid, and therefore some investigators have shown that there is no association of tumor ploidy with outcome in a multivariate model (Humphrey et al., Am. J. Surg. Pathol., 15(12): 1165-70 (1991)). However, in a Mayo Clinic study, when patients with tumor that had spread beyond the prostate were examined, tumor ploidy was found to be significantly associated with progression (Robertson and Paulson, Acta Oncol., 30(2):205-7 (1991) and Winkler et al., Mayo Clin. Proc., 63(2): 103-12 (1988)). In the Mayo Clinic practice, cancer ploidy is assessed on every RRP specimen, and therefore the significance of ploidy in the molecular model was investigated. It was found the ploidy was more weakly associated with cancer-specific outcome than the molecular markers but when the case-control was adjusted for ploidy, the coefficients of the model did not change significantly.
The samples evaluated in this study primarily consisted of patients of Caucasian descent. To obtain a more accurate assessment of prognostic performance in the population at large, a cohort study that includes proper minority population content can be used. Also, in the absence of an established assay to determine fusion status of the tumors, expression levels of the ETS gene family were used. This surrogate measure of fusion status has been applied in other studies (Demichelis et al., Oncogene, 26(31):4596-9 (2007)) and predicts the fusion status accurately.
The VOG-Δp and the pFC methods described in Example 3 were used to identify additional candidate nucleic acids for association with aggressiveness of prostate cancer. These nucleic acids were also evaluated with the same validation method that was described in Example 3 using the case-control group of patients that were matched with respect to clinical parameters, including margin, seminal vesicle, and/or nodal invasion. Both two-sided T-test and Wilcox tests were performed for these nucleic acids. These tests allow for protective nucleic acids with significant p-values also to pass threshold limits as well as nucleic acids with binary characteristics that would not look significant with the traditional t-test. Several nucleic acids passed at least one of the tests (Table 8). CDH10 is shown in bold because it is a protective gene (lower expression in cases than in controls). Also, TDO2 had a significant p-value with the Wilcox test and not with the t-test. This is attributed to the binary nature of its expression.
CDH10
0.0124
0.0071
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application is a continuation application of U.S. application Ser. No. 13/691,264 filed Nov. 30, 2012, now issued as U.S. Pat. No. 9,534,249; which is a continuation application of U.S. application Ser. No. 12/513,329 filed Jan. 28, 2010, now issued as U.S. Pat. No. 8,338,109; which is a 35 USC § 371 National Stage application of International Application No. PCT/US2007/083504 filed Nov. 2, 2007, now expired; which claims the benefit under 35 USC § 119 (e) to U.S. Application Ser. No. 60/856,536 filed Nov. 2, 2006, now expired. The disclosure of each of the prior applications is considered part of and is incorporated by reference in the disclosure of this application.
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| Number | Date | Country | |
|---|---|---|---|
| 20170159131 A1 | Jun 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60856536 | Nov 2006 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13691264 | Nov 2012 | US |
| Child | 15379237 | US | |
| Parent | 12513329 | US | |
| Child | 13691264 | US |
