Patent Application
20140087397
A method for adjusting therapy provided to a patient either diagnosed with or at risk for a DNA-repair defect or with cancer, such as a risk for cancer based on a diagnosis of Fanconi Anemia (FA). The method involves determining, in a biological sample of the patient, the level of at least one biomarkers or a panel of biomarkers that is/are altered compared to the level of the same biomarker(s) from a biological sample of a control individual such that the alteration is indicative of FA.
Early identification of biomarkers for cancer cells such as squamous cell carcinoma (SCC), particularly head and neck squamous cell carcinoma (HNSCC) subsequently analyzed, that identify defects in the FA pathway which under normal conditions result in DNA repair, permit altered or alternative patient treatments resulting in more favorable patient outcomes.
Traditional cancer therapies such as chemotherapy and radiation result in high morbidity and mortality in FA patients, due to their global hypersensitivity to DNA damage. It is presumed that FA tumors are exquisitely sensitive to conventional therapies such as chemotherapy and radiation, but the FA patient's hypersensitivity to DNA damage significantly limits their effectiveness, with cisplatin therapy being one example. The only curative therapy for hematologic FA abnormalities, a bone marrow transplant (BMT), causes significant morbidity and mortality in FA patients from the genotoxic conditioning regimens and the transplant itself.
The disclosed method thus involves detection of a biomarker or a biomarker panel or signature early in a patient's treatment that permits alternative and more tailored patient therapies, yielding a more favorable patient outcome. The method diagnoses FA using the disclosed biomarkers and/or biomarker signatures, and subsequently adjusts or modifies solid tumor treatment. In embodiments, modifying or adjusting treatment includes dosage, intensity, such as with radiation therapy, length of treatment, frequency of treatment, and inclusion or elimination of any additional treatments, e.g., decreasing treatment duration or intensity because of FA cells' hypersensitivity.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Patients with FA are at increased risk for cancer in general, and for certain types of cancers in particular. It is known that FA patient have accelerated transformation of hematopoietic cells resulting in leukemia, and have accelerated transformation of keratinocytes resulting in SCC. In one embodiment, the inventive method assess levels of biomarkers in patients at risk for or diagnosed with FA, compared to a control, and adjusts or alters the patient's existing cancer therapy based on the results. In one embodiment, the inventive method assess levels of biomarkers in patients at risk for or diagnosed with other DNA repair deficiencies, compared to a control, and adjusts or alters the patient's existing cancer therapy based on the results. In one embodiment, the method assesses an elevated risk of cancer susceptibility in a healthy individual, compared to the general population, and instructus preventative measures based on the results, e.g., frequent cancer screens. The method determines, in a sample of a patient's biological fluid, at least one biomarker that, individually or collectively, is indicative of FA. Such early identification of biomarkers for cancer cells, such as head and neck squamous cell carcinoma, subsequently analyzed, that identify FA pathway defects, can result in alternative chemotherapeutic and/or irradiation methods of treatment. Also as subsequently analyzed, because FA deficient cells are hypersensitive to DNA damage, low dose clastogenic treatments may be effective for sporadic SCC with FA mutations. Identification and diagnosis of FA in cancer patients is particularly useful where achieving tumor eradication, while minimizing toxicity, is a difficult balance.
A biomarker may be biochemical entities including precursors, breakdown proteins, derivatives, modified (e.g., glycosylated, phosphorylated, etc.) forms, metabolites, etc. of proteins including amino acids and peptides, lipids, vitamins, etc. The patent assayed may be in a normal unstressed condition, or may be in a stressed condition, e.g., having undergone a fast for at least twelve or eighteen hours, etc. Biomarkers include 3-hydroxyisovalerate, acetate, isopropanol, glycerol-3-phosphocholine, choline, histamine, glutamate, alanine, glycine, isoleucine, lysine, phenylalanine, threonine, tyrosine, fat-soluble vitamins A and D, cholesterol, ketone bodies, acetone, and/or acetoacetate/acetoacetaldehyde. Any biological sample may be assayed, e.g., cells, tissues, blood including plasma and/or serum, urine, feces, cerebrospinal fluid, saliva, lavage fluid, intrathecal fluid, etc. The term biological sample includes any or all of the above unless a specific source, e.g., urine, is stated. An altered level of the biomarker(s), compared to a control patient not having either cancer or FA i.e., a healthy control patient is indicative or suggestive of FA. In one embodiment, the confidence level of the diagnosis of FA is increased when the number of biomarkers having altered levels is increased.
This information is applied to increase, decrease, alter, combine, etc. dose, type, duration, extent, etc. of the patient's present cancer therapy based on the diagnosis of FA. The cancer therapy may be targeted or general, and may include surgical therapy, radiation therapy, chemotherapy, immunotherapy, etc. As subsequently analyzed and explained, one type of patient which the invention has particular application is a patient that has been diagnosed with or is at risk for squamous cell carcinoma (SCC). As subsequently analyzed and explained, one type of patient for which the invention has particular application is a patient that has or is at risk for bone marrow failure (BMF).
The group of diagnostic or suggestive biomarkers for FA may be termed an FA biomarker panel or FA biomarker signature. One panel includes biomarkers that participate in biochemical reactions or process involved in the metabolism of lipids. This may be further parsed as biomarkers involved in the metabolism of specific lipids, such as cholesterol. The patient may be required to endure a fast sufficient to generate a state of metabolic ketosis. One exemplary but not limiting biomarker panel includes at least two of 3-hydroxyisovalerate, acetate, isopropanol, glycerol-3-phosphocholine, phosphocholine, choline, and/or histamine. One exemplary but not limiting biomarker panel includes at least two of glutamate, alanine, glycine, isoleucine, lysine, phenylalanine, threonine, and/or tyrosine.
Biomarkers may be determined by any method. One method is nuclear magnetic resonance (NMR). A biological fluid sample from a control patient is assayed by NMR for a least one biomarker, and that level is compared to the level of the same biomarker(s) from a biological fluid in a FA patient. The biological samples assayed from the control and FA patient should be the same, e.g., control individual urine compared to FA patent urine, or control individual plasma compared to FA patient plasma. The results obtained by comparing the peak size of each biomarker from the control individual, versus the peak size of each biomarker from the FA patient, are than assessed. Treatment of the FA patient is altered, or not, as previously described, based on the difference. Depending on the degree of variation of the particular biomarker within the general population, even a small difference in a stable biomarker may be useful. The assessment can be incorporated into a kit using biochemical assays known in the art to determine the concentration of the biomarker(s). For example, known biochemical assays exist for a signature panel of, e.g., 3-hydroxyisovalerate, acetate, isopropanol, glycerol-3-phosphocholine, phosphocholine, choline, and histamine. The assays may be adapted or adaptable, as known in the art, depending upon the biological fluid, etc.
Fanconi anemia (FA), a rare inherited chromosomal-instability autosomal recessive disease, results from mutations in at least one of fifteen genes encoding proteins in the FA pathway shown in
The importance of the FA pathway, initially regarded as a specialized ICL repair system due to rare incidence of FA, was demonstrated by finding that important breast cancer susceptibility genes BRCA2, PALB2, and BRIP1 are identical to FANCD1, FANCN, and FANCJ. Correspondingly, the FA pathway is activated beyond ICL by forms of genotoxic stress including ultraviolet (UV) and ionizing radiation, and oxidative stress. FA deficient cells are used as a model system to study ATR signaling and BRCA functions in DNA repair.
Carriers of FANCD1/BRCA2 and RAD51C mutations are predisposed to breast and ovarian cancer. Carriers of FANCJ and FANCN mutations are predisposed to breast cancer at lower penetrance. Carriers of FANCD1/BRCA2, FANCC and other FA gene mutations are predisposed to pancreatic cancer. Defects in the FA/BRCA pathway exist in the general, non-FA population, so associated cancer susceptibility is not limited to the rare, inherited scenario.
FA is also characterized by cancer susceptibility, demonstrating that the FA pathway is also important in tumor suppression. FA patient are at risk for developing acute myeloid leukemia (AML). FA patients are at extreme risk for developing squamous cell carcinoma (SCC) of the head and neck (HNSCC), and gynecological tract. The incidence for developing SCC is even greater in bone marrow transplant individuals and individuals recovered from hematopoietic disease.
FA pathway tumor suppression in hematopoietic, versus epithelial, cells could be mechanistically similar or distinct. It is thus important to assess any link between BMF and subsequent solid tumor development, and to optimize therapies to decrease risk of malignant transformation in both hematopoietic and epidermal cells. In one embodiment, leukemia patients who recover poorly from chemotherapy, and young SCC patients, particularly those experiencing serious toxicity from chemotherapy and/or radiation, are candidates for biomarker assessment.
In one embodiment, the inventive method assesses stress responses, DNA repair, and SCC susceptibility.
In the FA pathway, multi-protein interactions coordinate DNA repair. Fifteen complementation groups and corresponding FA genes are known; protein products function as either signal transducers and/or DNA processing factors within the larger FA-BRCA DNA damage response network, subsequently described. The FA/BRCA pathway is activated during DNA replication, and by DNA ICL and other lesion damage. Protein components assemble into at least three complexes within the cell nucleus: FA core complex (FACC), ID complex of (mono)ubiquitinated FANCD2 and FANCI proteins, and a complex of the FANCN and BRCA2 proteins associated with homologous recombination that binds chromatin near DNA lesions downstream from the ID complex.
FACC has eight FA proteins: FANCA, B, C, E, F, G, L, and M, and other FA-associated proteins such as FAAP100, FAAP20 and FAAP24. Each protein is a prerequisite for proper complex assembly and subsequent DNA repair, explaining why mutations in different FA genes yield similar but not necessarily identical clinical phenotypes. FANCM (blue) with MHF1/2 (dark grey) and FAAP24 (light grey) recruit a large multi-subunit ubiquitin E3 ligase, termed FACC (blue), to sites of DNA damage. FACC then mono-ubiquitinates FANCD2 and FANCI; mono-ubiquitinated FANCD2-FANCI (green) is recruited to sites of damage by FANCJ (yellow) and BRCA1 (yellow). These FA proteins colocalize with downstream FA proteins RAD51, FANCN, FANCD1/BRCA2 (yellow), and facilitate DNA ICL repair. Multiple FACC constituents and associated components of the pathway are phosphorylated, including FANCA, G, M, D1/BRCA2, D2, and I; these phosphorylation events are important for ICL repair. FACC forms a nuclear, high molecular weight E3 ubiquitin ligase complex, based on the sole E3 ubiquitin ligase domain of FANCL. FANCL contains additional domains responsible for directing substrate binding (DRWD domain) and four E2 protein interactions (RING domain) by using UBE2T. FANCM provides ICL resistance to cells. FANCM and FAAP24, with the histone-fold containing proteins MHF1 and MHF2, recognize both DNA lesions and stalled replication forks, and subsequently generate single stranded DNA, which is thought to activate both ATR and its downstream target Chk1.
ATR and ATM kinases, key upstream regulators of the FA pathway, phosphorylate several FA pathway components and specifically direct cellular response to DNA damage during S-phase. FANCI phosphorylation likely turns on the FA pathway and initiates interactions between FACC and ID complex (composed of FANCI and FANCD2). Following phosphorylation, FANCI at Lys523 and FANCD2 at Lys562 are monoubiquitinated by FACC ubiquitin ligase, important for downstream function of the FA pathway in DNA repair. FANCD2 mono-ubiquitination is thus widely used as a read-out for FA pathway activation. It is detectable by a shift from the nonubiquitinated short form (FANCD2-S) to a slower migrating, long form (FANCD2-L) in immunoblots. FANCD2-L relocalization to chromatin is visualized using immunofluorescence (IF) by formation of nuclear FANCD2 foci that colocalize with other nuclear DNA damage response proteins (γH2AX or Rad51). Mutation of core complex I components such as FANCA is thus reflected experimentally by the absence of FANCD2 monoubiquitination, and by the absence of FANCD2 and Rad51 foci following DNA damage induction. This has been described for multiple cell types including keratinocytes cultured from FA patient skin biopsies, where retroviral FANCA complementation successfully re-instated detection of FANCD2-L by immunoblot and IF.
ID complex activation initiates its localization to nuclear foci together with BRCA1 and Rad51, essential factors for proper homologous recombination. After ID complex loads onto chromatin, it binds the FAN1 protein, responsible for nuclease activity during repair. Although FANCI monoubiquitination is not required for proper repair, its phosphorylation may play a role in localizing the ID complex to chromatin foci, described as sites of repair because they harbor numerous repair factors such as BRCA1, BRCA2, PCNA, or Rad51. Subsequent DNA nuclease recruitment and activation “unhooks” the crosslink, followed by homologous recombination (HR), nucleotide excision repair (NER) on the opposing strand, and translesion synthesis (TLS) to repair the gap.
Post-DNA repair, the FA pathway is inactivated by de-ubiquitination of the ID complex. Inactivation occurs when USP1, a de-ubiquitinating enzyme, associates with FANCD2 within the nuclear foci and removes the monoubiquitin moiety. The absence of de-ubiquitination of FANCD2, like the absence of ubiquitination, leads to DNA sensitivity to crosslinking agents such as cisplatin or mitomycin C. Although the FA pathway has thus become a model system for ICL repair, its molecular components coordinate and integrate many DNA repair machineries including homologous recombination (HR), nucleotide excision repair (NER), and translesion synthesis (TLS).
In FA patients, clonal evolution plays a key role in progression to leukemia and most FA patients experience progressive BMF and often leukemia. Marrow dysfunction occurs at an early age and is associated with stem cell loss in the hematopoietic compartment, responsible for most FA childhood mortality. The risk of BMF in FA patients is 90% by age 50. Rapid hematopoietic cell loss then forces compensatory chronic proliferation, which likely results in clonal evolution and leukemogenesis. Clonal selection and resistance to cytokine-induced cell death has been reported. In an environment of genomic instability, loss of function of tumor suppressors, as well as oncogenic translocations, can be acquired and selected for rapidly. Chromosomes 1, 3 and 7 were more frequently involved in FA AML than in de novo AML. The specific involved clone may thus distinguish FA AML from de novo cases. The only curative therapy for hematologic FA abnormalities is a bone marrow transplant (BMT). However, genotoxic conditioning regimens and the transplant itself are a significant cause of morbidity and mortality in this patient population, which tolerates chemotherapy and radiation poorly.
FA patients that survive BMF through treatment remain susceptible to cancers, typically myeloid leukemias and SCC. FA patients with biallelic mutations in BRCA2 have an exceptionally high risk of developing AML by age 5, and the overall prevalence of AML in FA patients is 33% by age 40. Sporadic AML may also carry FA defects, but only a small number of mutations have been identified, e.g., FANCF silencing was demonstrated in an AML cell line, and hypermethylation of the promoter regions of FANCC and FANCL was identified in sporadic acute leukemia. The high risk of AML and myelodysplastic syndrome (MDS) in pediatric FA patients, and the similarities between chromosomal abnormalities in FA and AML/MDS suggest molecular link(s) between FA pathway irregularities and AML/MDS in the general population.
Many FA patients develop AML and/or MDS, thus, the FA genotype is viewed as preleukemia. Stabilization and prevention of pre-leukemic phenotypes is the expressed goal, ideally without increasing the risk of solid tumor development. However, a thorough understanding of the role of FA proteins in squamous epithelium is lacking, impeding the rational design of strategies that prevent both hematopoietic and epithelial transformation. Molecular and phenotypic aspects of FA that might differ substantially across tissue and cancer types are presented, and care must be taken to ensure that leukemia prevention and treatment in FA patients does not further exacerbate development of solid tumors.
Mutations in the FA pathway lead to increased risk of SCC, with anogenital region and head and neck SCC (HNSCC) the most common. HNSCC is the sixth leading cancer worldwide, with prolonged tobacco and alcohol use as principal risk factors. In the general population, about 25% of HNSCC are caused by human papillomavirus (HPV) infection, particularly the HPV16 genotype. HPV status determines significant biological differences; HPV positive tumors exhibit improved treatment response and prognosis versus HPV negative tumors. The prevalence of HPV in FA SCC remains controversial.
Because only a few number of individuals exposed to tobacco, alcohol, or HPV ultimately develop cancer, one who does must have an inherent predisposition that collaborates with these genotoxic exposures for cancer to occur. Within the general population there are different levels of DNA safeguarding capability, which play a role in cancer development. Unfortunately, overall treatment outcomes for HNC have not improved, and conventional clastogenic therapies have substantial side effects on normal physiological functions, e.g., swallowing, speech, and physical appearance. These responses to therapy, together with tumor development, are also modified by genetic predispositions, and appear to be greatly amplified in FA patients. Thus, while FA tumors are predicted to be exquisitely sensitive to conventional chemotherapy and radiation, the FA patient's global hypersensitivity to DNA damage limits their effectiveness, particularly radiation therapy.
Array based comparative genome hybridization of 21 sporadic oral SCC revealed deregulation of FA and FA-associated genes, including BRCA1, BRCA2 (FANCD1), FANCG and FANCD2. Table 1 lists in either FA genes themselves, or in a select gene subset associated with DNA repair. Table 1 shows somatic mutations of HNSCC patient tumors in a select subset of FA- and DNA-repair associated genes, each letter in parentheses represents each patient. Mutations were identified by exome sequencing of tumors isolated from sporadic HNSCC patients. Table 1 represents new analyses previous data that revealed 38/74 (51%) sequenced tumors harbored at least one somatic mutations in the indicated gene subset. A majority of these tumors (18/38=47%) harbored multiple mutations, ranging from 2-7. Among these mutated genes, BRCA2, FANCM (5 mutations reported), ATR, UBE4A (4 mutations reported), BRCA1, USP43 and USP44 (3 mutations reported) are most frequent. These findings indicate a subset of primary, therapy-naïve HNSCC harbor mutations in important DNA repair pathways including FA. Such mutations may contribute functionally to increased tumor growth and/or may modify sensitivity to conventional drug therapies.
(f)
(l)
In contrast to leukemogenesis, solid tumor development, and particularly HNSCC and anogenital SCC, occur early and with striking aggressiveness in FA patient. Surgical treatment remains the mainstay, but relapse-free, two-year survival rates are <50%. Chemotherapy and radiation treatments are associated with high mortality and morbidity due to FA patients' global hypersensitivity to DNA damage; complications from radiotherapy include mucositis and pancytopenia.
New therapies for clinical management of FA associated SCC are thus needed. Early intervention, the best line of defense, is key for increasing the chance of complete surgical resection and survival. The previous absence, until now, of relevant biomarkers impeded early detection of tumor cells in FA patients.
Hematopoietic stem cells (HSC) in FA patient, unlike normal stem cells, are hypersensitive to apoptosis-inducing cytokines such as tumor necrosis factor-alpha (TNF-alpha). Exposure to these cytokines reduces the number of FA HSC by increasing the apoptosis rate in these cells. These events lead to rapid bone marrow failure with decreased production of all differentiated cell types. FA HSC adapt to this hostile environment by developing mutations, which confer resistance to TNF-alpha but retain the characteristic sensitivity to crosslinkers. Reducing the selection pressure in FA patients in order to decrease the transformation of FA hematopoietic cells towards leukemia is important.
Patients with sporadic HNSCC frequently present at advanced disease stages because of inferior diagnostic approaches. This necessitates aggressive chemo- and/or radiation therapy, leading to extreme toxicity and sometimes death. Over 20% of FA patients diagnosed with a solid tumor had a FA diagnosis made only after cancer was detected, thus treatment toxicity was not predictable. There is thus an urgent need for improved, early diagnosis of HNSCC in FA patients, and also for a FA diagnosis prior to cancer development and treatment. Cancer prevention in this patient population should be the primary goal.
Previous approaches to identifying markers of sporadic HNSCC are restricted to global genomic and proteomic approaches. Metagenomics, a specific type of genomic analysis, isolates nucleic acids and performs unprejudiced DNA sequencing to categorize viruses and microorganisms present in a particular sample. Mass spectrometry (MS) determined changes in proteins M2BP, MRP14, and CD59, validated by immunoassays. Proteome/peptidome analysis and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was used, but requires optimization to establish practicality in early stage detection in a clinical setting, with time and cost also being factors. Such “omics” techniques have identified several potential biomarkers of head and neck cancer, but additional techniques will be beneficial. For example, results from these “omic” methods may not fully correlate to one another because regulation of certain genes does not always result in abundance in the corresponding proteins. This is not surprising, since the production of a protein may occur sometime after its gene is transcriptionally activated and indicates increased emphasis on protein expression levels within a given system. Proteins reflect biological processes that take place at a defined time in a certain system, and metabonomics is the next most accurate technology for observing intermediates and final products relating to both gene transcription and protein regulation.
Metabonomics is the study of an organism's metabolome: quantitative measurements of multiparametric metabolic response to pathophysiological stimuli or genetic modifications. It has minimal use to date in cancer biology, but is potentially useful for biomarker identification in disease because the metabolome is downstream of both the genome and proteome and is complementary to other “omic” methods. Metabonomics research in HNSCC should address normal metabolic function in epidermal compartments to define how changes in this metabolic fingerprint can function as early diagnostic tools. Defining changes in the metabolome related to disease onset and progression in the FA patient HNSCC provides information about regulatory pathways in cancer in general, and will drive biomarkers identification and therapeutic targets
Thus, methods for diagnosing FA and for adjusting or modifying cancer treatment in a FA patient, based on the diagnosis of FA, is needed and biomarkers for FA were identified.
In one embodiment, biomarkers were identified using NMR-based metabonomics in biological samples from FA patients and FA knockout mice. FA knockout mice exist for various components of the FA pathway (Bakker et al. Learning from a paradox: recent insights into Fanconi anaemia through studying mouse models. Dis Model Meh. 6 (2013) 40). However, these mice do not spontaneously mimic characteristic FA manifestations, e.g., anemia, leukemia, and solid tumor development. Based on variable expressivity of recognized symptoms, and also on lack of biomarkers, the true incidence of FA is likely grossly underestimated. The disclosed biomarkers will assist screening of susceptible patients such as leukemia patients who recover poorly from chemotherapy, and young patients with SCC or other solid tumors, particularly those who experience serious toxicity from chemotherapy and/or radiation.
Current assays used for clinical diagnosis of FA are highly specialized, time consuming, and expensive. Further, there are no biomarkers available for the diagnosis of many other DNA repair deficiencies.
One embodiment is a method for diagnosing individuals in the FA population and on a therapy regimen, especially cancer patients and especially HNSCC patients and patients after BMF, at the earliest possible stage, and then modifying the current therapy as needed, based on the biomarker and or biomarker signature level, to improve outcomes. Improved outcomes include, but are not limited to, improved treatment response, increased or decreased treatment dose, increased or decreased treatment frequency, different treatment types, removal of one treatment type, substitution of another treatment type, addition of another treatment type, etc.
One embodiment used nuclear magnetic resonance (NMR) metabonomics to identify a unique metabolic biomarker signature for FA in both mice and human biological samples. The method is innovative and clinically relevant, because the results provide an otherwise poorly understood link between metabolic response and DNA repair and provide for development of NMR spectroscopy as a diagnostic and surveillance tool in both children and adult FA patients. These data advance a mechanistic understanding of how metabolism marks or is functionally associated with DNA deficiency syndromes including FA.
One embodiment uses NMR with or without more routine clinical assays to detect and quantitate the biomarker or biomarker signature. Clinical chemistry assays for the disclosed biomarkers are known in the art, e.g., Burtis, Ashwood, and Bruns, Tietz Fundamentals of Clinical Chemistry (2007) 6th Ed., Saunders for assaying 3-hydroxyisovalerate, acetate, isopropanol, glycerol-3-phosphocholine, choline, histamine, glutamate, alanine, glycine, isoleucine, lysine, phenylalanine, threonine, tyrosine, vitamin A, vitamin D, cholesterol, ketone bodies, acetone, and/or acetoacetate/acetoacetaldehyde.
The methods include assessment whether an individual has or is predisposed to developing FA for making a change in treatment. For example, and without limitation, any form of fluid assay capable of detecting biomarkers may be used. The assay may be performed in a clinical laboratory or may be performed at a point of care and includes kit assays. A kit includes instructions for use and reagents for performing the assay. It may include a test substrate, e.g., test strip, dip stick, cassette, cartridge, panel, chip-based or bead-based array, multi-well plate, or series of containers, etc. The assay may be qualitative (present or absent), quantitative (concentration), or semi-quantitative (high or low relative to a control or to a predetermined threshold quantity). One or more reagents is provided in or on the test substrate to detect the presence (qualitative), concentration (quantitative), and/or relative amount compared to a control (semi-quantities) of one or more of the disclosed biomarkers. The kit may include assay panels for a number of biomarkers, with reagents provided at predetermined locations configured on separate test strips, or sequentially or in tandem on one test strip, and may include positive and/or negative controls on the same or different test strips. The biological sample may be dispensed directly or indirectly to contact the reagent(s), and the sample may be directly from the patient or previously obtained. The results may read by any of chromogenic, fluorogenic, electrochemiluminescent, etc. The assay may be an enzyme immunoassay (EIA), e.g., an enzyme-linked immunoassay (ELISA).
As an illustrative example only, a chip contains reagents immobilized at discrete, predetermined locations for detecting and quantitating in a fluid sample the concentration of the biomarkers 3-hydroxyisovalerate, acetate, isopropanol, glycerol-3-phosphocholine, choline, and/or histamine. The chip may be configured such that a detectable output, e.g. color change, is provided only if the concentration of one or more of these biomarkers exceeds a threshold value selected to distinguish between a biomarker concentration indicative of a healthy individual versus an individual having or predisposed to developing FA. Thus, the presence of a detectable output such as a color change provides an immediate indication that the sample contains significantly different concentrations of one or more biomarkers compared to a control, indicating that the subject has or is predisposed to developing FA and indicating that therapy should be altered.
Biological fluids and tissues, and cell culture extracts, were collected from mice and humans, and processed prior to analyses. Biological samples composed of cells obtained from culture plates, blood plasma, and urine were either concentrated using a SpeedVac centrifuge or a lyophilizer. The dried samples were then resuspended in a phosphate buffer containing both D2O and trimethylsylilpropionate (TMSP), an internal standard used for quantification of metabolite concentrations. Biological samples were then transferred to an NMR tube prior to analysis. All samples were analyzed on a 600 MHz NMR spectrometer. Spectral data were processed by data reduction software and analyzed by multivariate statistical analysis software that identified unique metabolic signatures compared to either isogenic FA-proficient cells or to healthy control subjects. These results may be further analyzed for biological relevance and linked to metabolic disease states, signaling pathways and other “omics” data. The results demonstrated the feasibility of using NMR-based metabonomics to identify clinical disease marker, assess drug or other therapy of a patient being treated for a disease, provide modifications to existing therapy and/or identify alternate drug or other therapies, in addition to addressing basic research questions.
In one embodiment, an FA-specific metabolic signature was identified in biological samples from a FA patient and from FA-knockout mice. The metabolic signature or pattern differs from that of family members for patients, and from littermates for mice.
Epithelial tissues, hematopoietic stem cells, serum, urine, and feces from FANCA, FANCC and FANCD2 knockout mice, and from their wild type and heterozygous littermates, are analyzed by NMR spectroscopy. The resulting NMR profiles provide the metabolic fingerprint of each murine specimen, and metabolites that are specific to FA knockout mice are identified using multivariate statistical significance analysis and existing small metabolite databases. Metabolic profiles of FANCA, FANCC and FANCD2 knockout mice are also compared to one another and unique biomarkers specific to each complementation group are identified. This is significant because distinct complementation groups in humans vary in disease severity and risk of cancer, but a diagnosis of complementation group is currently expensive and inconclusive in 20% of cases at the FA Comprehensive Care Center at Cincinnati Children's Hospital.
Urine, skin and blood from FA patients and their family members were analyzed by NMR spectroscopy. Unique metabolic signatures for both FA and specific complementation groups, where known, were identified. Over fifty urine samples from FA patients and their family members were collected, and urine samples from normal, age-matched controls, i.e., individuals who did not have FA, were obtained. Levels of acetone and acetaldehyde in the urine were a distinguishing feature of FA.
Metabonomic and RNA sequencing studies were performed with (i) immortalized keratinocytes or SCC cells isolated from FA patients, either control transduced or corrected for the appropriate FA gene using retroviral transduction, i.e., the gene mutation was identified and the non-mutated functional gene was added to restore FA pathway activity (data not shown), and with (ii) SCC cells cultured from tumors of individuals with sporadic HNSCC that were knocked down for a key component and central regulator of the pathway FANCD2. Results are shown in
Functional complementation and knockdown were verified in each case using methods known in the art. The FA status did not affect cellular proliferation, as shown in
Metabolic profiling analysis was conducted in seven cell lines, each either expressing or with a knockdown of FANCA. The results are shown in
The results are summarized below:
The Mahalanobis Distance (MD) is the distance between the centroids of each of the study groups. The distance is then determined to be statistically significant by determination of the T2 value and F-value. The experimental F-value is then compared to the Critical F-value and deemed significant if the experimental F-value is larger than the Critical F-value.
As described above, a unique metabolic signature distinguishes FA-proficient, meaning a FA pathway with no mutations or silencing events, and FA-deficient, meaning at least one gene in the FA pathway is mutated or silenced, cancer cells (
Thus, FA deficiency in the germline, or somatically in the general population, may stimulate tumor cell invasion and metastasis in vivo, as will be assessed by results of the growth of isogenic, FA-proficient versus FA-deficient cells in the flanks and tongues of immunodeficient mice.
Non-tumorigenic FANCD2 positive and FANCD2 negative tongues of mice were used for NMR. The results, shown in
The method permits identification of carriers of FA gene mutations who are themselves unaffected may be indentified using the inventive method, as shown by results in
First morning urine was collected from individuals with FA, their siblings, and unrelated, healthy controls. Aliquots of the specimens were frozen on dry ice, then thawed on ice. A 630 μL volume of urine was added to 70 μL phosphate buffer containing 100% D2O and trimethylsilylpropionate (TMSP), resulting in a final mixture of urine, buffer, 10% D2O and 0.58 mM TMSP. A 600 μL aliquot of each mixture was analyzed on a 600 MHz Varian NMR. Results are shown in
Analysis of urine from 33 FA patients, 20 unaffected siblings which are often heterozygous for FA, and two healthy control subjects revealed elevated levels of acetone and acetoacetate in FA patients compared to their siblings and healthy unrelated controls.
A subset of the FA-proficient and FA-deficient tumors was subjected to RNASeq analysis to determine transcriptional patterns that correlate with the observed metabolic and invasive changes. The results indicated that FA de-regulated cellular metabolism and decreased expression of enzymes involved with lipid metabolism.
The results demonstrated the utility of the disclosed metabolites as biomarkers of FA, FA-deficient tumors, and/or BMF. The method is minimally invasive and provides results within a short time from sample collection to biomarker level determination in the patient compared to controls. Biological samples are those routinely clinically assayed, e.g., urine, plasma, saliva. In one embodiment, biomarker levels in patient saliva samples are compared to biomarker levels in patient related family member saliva samples. Saliva is relatively easily obtained, and salivary metabolic data may be related to urine data, and may be ultimately integrated with the FA SCC metabolome to provide clinical information on the presence of pre-malignant and malignant lesions in those same patients.
The disclosed NMR-based metabonomics revealed unique small metabolite signatures in FA cells, e.g., as shown in
Biological samples are obtained from patients and control by known protocols. In embodiments, the controls are matched by at least one characteristic including gender, age, genetic relative, etc. Samples may be obtained during routine screening or in connection with a regular check-up or physician visit, or together with administration of treatment such chemotherapy, radiation therapy, surgical therapy, etc.
In one embodiment, the patient is stressed prior to obtaining the biological sample for analysis. For example, one biomarker panel includes components involved with lipid metabolism, such as cholesterol metabolism. The patient thus may be subjected to dietary restrictions, e.g., instructed to fast for 12-18 hours, prior to sample collection to induce a state of ketosis or decreased cholesterol. Decreased cholesterol levels, i.e., hypocholesterolemia, is seen in Smith-Lemli-Opitz (SLO) syndrome, which is the result of a mutation in 7-dehydrocholesterol reductase (DHCR7). DHCR7 mRNA is decreased in FA cells and produces FA-like signs and symptoms such as developmental disorder, organ malformation including genitalia, distinctive facial features, microcephaly, intellectual disability, failure to thrive. Cellular and animal models of SLO demonstrate oxidative stress. Lack of cholesterol is associated with insufficient bile acid, taurine, in the gut and fewer micelles for vitamin and fat absorption leading to measurable vitamin deficiency. Thus, in one embodiment, FA biomarkers include cholesterol, vitamin A, and vitamin D. Because steroid hormones are generally synthesized from cholesterol, the reduction in cholesterol will likely result in decreased steroid hormone levels. Thus, in one embodiment, FA biomarkers include steroid hormones. Examples of steroid hormones are known in the art and include glucocorticoids, mineralocorticoids, androgens, estrogens, progestogens, and the closely related Vitamin D compounds.
Samples can be collected one or more times for a separate or combined analysis. The collected samples may be assayed immediately or stored under appropriate conditions for subsequent analysis. Analytical methods for bio marker level determination can include one or a combination of mass spectrometry (MS) coupled with gas chromatography (GCMS) or liquid chromatography (LCMS), HPLC, NMR spectroscopy, TLC, electrochemical analysis, refractive index spectroscopy, ultra-violet spectroscopy, fluorescent analysis, radiochemical analysis, near-infrared spectroscopy and light scattering analysis. In some embodiments, fluid samples may be processed prior to analysis. Biomarker quantitation may be performed using tools that compare the integral of a known reference signal with signals derived from a biomarker to determine concentration relative to the reference signal. Levels of the specific biomarker over or below a determined critical value, either in concentration or in amount, can indicate the presence of FA. The biomarker(s) concentrations or levels can be interpreted independently using an individual cut-off for each biomarker, interpreted collectively. For example, an altered concentration in at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, etc. more biomarkers may be used to diagnose FA. In one embodiment, altered concentrations of more biomarkers increases diagnosis certainty.
The disclosed method integrated metabolic information resulting in one or a panel of biomarkers for FA as a genetic syndrome and FA-associated cancer, to adjust and better target therapies for FA patients. Based on the known DNA repair defects in FA cells, tumors in FA patients are relatively more sensitive to conventional clastogenic treatments, permitting altered treatment such as dose de-escalation of chemotherapeutic agents. The results are applicable to FA patients with SCC and other tumors, and also to a significant proportion of patients with sporadic SCC and other tumors in the general population with mutations in FA or decreased expression of FA, and other DNA repair pathways.
Each of the following are expressly incorporated by reference herein in its entirety:
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The embodiments shown and described in the specification are only specific embodiments of inventors who are skilled in the art and are not limiting in any way. Therefore, various changes, modifications, or alterations to those embodiments may be made without departing from the spirit of the invention in the scope of the following claims. The references cited are expressly incorporated by reference herein in their entirety.
This application claims priority from U.S. application Ser. No. 61/705,396, filed on Sep. 25, 2012, which is incorporated by reference in its entirety.
This invention was made with government support under Grant CA102357 awarded by the Public Health Service. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61705396 | Sep 2012 | US |
