Patent Description:
For example, thymidylate synthase (TS) is an integral enzyme in DNA biosynthesis where it catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) and provides a route for de novo synthesis of pyrimidine nucleotides within the cell (<NPL>). Thymidylate synthase is a target for chemotherapeutic drugs, most commonly the antifolate agent <NUM>-fluorouracil (<NUM>-FU). As an effective agent for the treatment of colon, head and neck and breast cancers, it is believed the primary action of <NUM>-FU is to inhibit TS activity, resulting in depletion of intracellular thymine levels and subsequently leading to cell death.

Thymidylate synthase is also known to have clinical importance in the development of tumor resistance, as demonstrated by studies that have shown acute induction of TS protein and an increase in TS enzyme levels in neoplastic cells after exposure to <NUM>-FU (<NPL>; <NPL>). The ability of a tumor to acutely overexpress TS in response to cytotoxic agents such as <NUM>-FU may play a role in the development of fluorouracil resistance. the levels of TS protein appear to directly correlate with the effectiveness of <NUM>-FU therapy, that there is a direct correlation between protein and RNA expression and TS expression is a powerful prognostic marker in colorectal and breast cancer (<NPL>. In advanced metastatic disease, both high TS mRNA, quantified by RT-PCR, and high TS protein expression, have been shown to predict a poor response to fluoropyrimidine-based therapy for colorectal (Johnston et al. (<NUM>) supra. ; <NPL>; <NPL>), gastric (<NPL>), and head and neck (<NPL>; <NPL>) cancers.

Similarly, mutation of the KRAS oncogene is predictive of a very poor response to panitumumab (VECTIBIX®) and cetuximab (ERBITUX®) therapy in colorectal cancer (<NPL>). Currently, one of the most reliable ways to predict whether a colorectal cancer patient will respond to one of the EGFR-inhibiting drugs is to test for certain "activating" mutations in the gene that encodes KRAS, which occur in <NUM>% of colorectal cancers. Studies show patients whose tumors express the mutated version of the KRAS gene will not respond to cetuximab or panitumumab.

One important source for this type of information comes in the form of formalin-fixed, paraffin-embedded tissue ("FFPET") samples, that are routinely created from biopsy specimens taken from patients undergoing a variety of diagnostic and/or therapeutic regimens for a variety of different diseases. These samples are usually associated with the corresponding clinical records and often play an important role in diagnosis and determination of treatment modality. For example, tumor biopsy FFPET samples are often linked with cancer stage classification, patient survival, and treatment regime, thereby providing a potential wealth of information that can be cross-referenced and correlated with gene expression patterns. However, the poor quality and quantity of nucleic acids isolated from FFPET samples has led to their underutilization in gene expression profiling studies.

It is known that RNA can be purified and analyzed from FFPET samples (<NPL>), however, RNA isolated from FFPET samples is often moderately to highly degraded and fragmented. In addition to being degraded and fragmented, chemical modification of RNA by formalin restricts the binding of oligo-dT primers to the polyadenylic acid tail and can impede the efficiency of reverse transcription.

In view of these difficulties, the analysis of nucleic acids from formalin fixed, paraffin embedded tissue (FFPET) has proven challenging due to the multiple steps required for generating PCR-amplifiable genetic material. The procedure to isolate nucleic acids from FFPET may include removal of paraffin (deparaffinization), lysis of preserved sample (protease digestion), reversal of cross-links acquired during the fixation process and solid phase-based purification of nucleic acids. These protocols are typically complex and labor intensive.

The invention is defined in the claims. Methods and reagents for the isolation of nucleic acids from cell or tissue samples (e.g., fine needle aspirates and/or fixed embedded tissue samples (e.g., FFPET samples, and/or cryosections) are provided. In some embodiments, the methods are simple, easily semi-automated or fully automated and typically require minimal hands-on time, while extracting nucleic acids of high yield and PCR-amplifiable quality.

The invention is defined in the claims. Formalin-fixed, paraffin-embedded tissue (FFPET) samples represent the most commonly collected and stored samples for use in the diagnosis and prognosis of diseases, including, but not limited to, cancer. Nevertheless, historically these samples have been underutilized for the purpose of gene expression profiling because of the poor quality and quantity of FFPET nucleic acids. The analysis of nucleic acids from formalin fixed, paraffin embedded tissue (FFPET) is challenging due to the multiple steps required for generating amplifiable (e.g., PCR-amplifiable) genetic material. The procedure to isolate nucleic acids from FFPET has typically involved removal of paraffin (deparaffinization), lysis of preserved sample (protease digestion), reversal of cross-links acquired during the fixation process, and solid phase-based purification of nucleic acids.

There are various sample-prep procedures for extracting PCR-ready DNA/RNA, but most are complex and labor intensive. The compositions and methods described herein overcome these and other problems and provide reagents and protocols that can be used to rapidly isolate amplifiable quality nucleic acid samples (e.g., DNA, RNA). The methods provided are simple, easily semi- or fully-automated, and require requiring minimal hands-on time. The nucleic acids are extracted at high yield and are of PCR-amplifiable quality.

In certain embodiments, a lysis solution is provided that can be used to extract nucleic acids from a paraffin embedded formalin fixed sample using a single solution and incubation at a single temperature. This is provides a significant improvement is simplicity, efficiency and cost over previous two buffer/two temperature systems used to isolate nucleic acids from tissue samples.

It will be noted that while the discussion below focuses on FFPE samples, the lysis reagents described herein and uses thereof are effective with essentially any cellular or tissue sample including, but not limited to, fresh tissue sections, frozen tissue sections, cell biopsies, needle aspirates, cell buttons, tissue microarrays, and the like.

In certain embodiments the lysis solutions comprise a high concentration sodium salt (e.g., NaCl), a buffer sufficient to maintain the pH of the solution at a pH ranging from about pH <NUM> to about pH <NUM>, or from about pH <NUM> to about pH <NUM>, a chelating agent, a magnesium salt (e.g., MgCl<NUM>), and a detergent. In certain embodiments the lysis solution additionally contains an antifoaming agent, and/or a preservative/biocide, and/or a protease. In certain embodiments the lysis solution omits the protease which can then be added immediately prior to use.

One illustrative, but non-limiting embodiment of a lysis solution is shown in Table <NUM>.

This formulation is intended to be illustrative, but non-limiting. Using the teachings provided herein, other lysis solutions useful for a <NUM>-step, <NUM> temperature extraction of nucleic acids from a tissue sample will be available to one of skill in the art.

In various embodiments the lysis solution comprises a sodium salt (NaCl). In certain embodiments the sodium salt is at a concentration ranging from about <NUM> to about <NUM>, or from about <NUM> up to about <NUM>, or is about <NUM>. In certain embodiments a calcium salt (e.g., CaCl) may be used in addition to a sodium salt.

In some aspects of the disclosure, the lysis solution comprises a buffer that buffers the solution at a pH ranging from about pH <NUM> up to about pH <NUM>. In some embodiments the buffer buffers the solution at a pH ranging from about pH <NUM>, or about pH <NUM>, or about pH <NUM> up to about pH <NUM> or up to about pH <NUM>, or up to about pH <NUM>, or up to about pH <NUM>. In certain embodiments the pH is buffered at pH <NUM> (+/- <NUM>).

In certain embodiments, the concentration of the buffer ranges from about <NUM> up to about <NUM>, or from about <NUM> up to about <NUM>, or is about <NUM>.

In certain aspects of the disclosureany of a number of buffers used in biology are suitable. Such include, but are not limited to buffers such as citrate buffer, Tris, phosphate, PBS, citrate, TAPS, Bicine, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, Cacodylate, SSC, MES, and the like. An illustrative, but non-limiting list of buffer compounds is provided in Table <NUM>.

In one illustrative, but non-limiting embodiment, the buffer is a HEPES HEPES sodium salt (MW <NUM>) present at about <NUM>.

The various buffers described above are intended to be illustrative and not limiting. Using the teaching and examples provided herein, numerous other buffers for use in a lysis solution in accordance with the methods described herein will be available to one of skill in the art.

As indicated above, in some embodiments, the lysis solution comprises one or more chelating agents. Chelating agents are well known to those of skill in the art and include, but are not limited to N-acetyl-L-cysteine, ethylenediaminetetraacetic acid (EDTA), diethylene triamine pentaacetic acid (DTPA), ethylenediamine-N,N'-disuccinic acid (EDDS), <NUM>,<NUM>-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and phosphonate chelating agents (e.g., including, but not limited to nitrilotris(methylene)phosphonic acid (NTMP), ethylenediamine tetra(methylene phosphonic acid) (EDTMP), diethylenetriamine penta(methylene phosphonic acid (DTPMP), <NUM>-hydroxy ethylidene-<NUM>,<NUM>-diphosphonic acid (HEDP), and the like). In some embodiments the chelating agent comprises EDTA, or DTAP. In some embodiments, the chelating agent comprises EDTA.

In some embodiments, when present, the chelating agent is present in the lysis solution at a concentration ranging from about <NUM> up to about <NUM>, or from about <NUM> up to about <NUM>. In some embodiments, the chelating agent is present at a concentration ranging from about <NUM>, or from about <NUM>, or from about <NUM>, or from about <NUM> up to about <NUM>, or up to about <NUM>, or up to about <NUM>, or up to about <NUM>, or up to about <NUM>. In some embodiments, the chelating agent is present at a concentration of about <NUM>. In some embodiments, the chelating agent ranges from about <NUM> up to about <NUM>, or from about <NUM> up to about <NUM>, or from about <NUM> to about <NUM>, or is about <NUM>.

In certain embodiments the lysis solution contains a magnesium salt. In certain embodiments the magnesium salt is MgCl<NUM>. In certain embodiments the concentration of the magnesium salt in the lysis solution ranges from about <NUM> up to about <NUM>, or from about <NUM> up to about <NUM>, or is about <NUM>.

As indicated above, in some aspects of the disclosure, the lysis solution comprises one or more detergents. In some embodiments, the detergent comprises an ionic detergent or a non-ionic detergent. In some embodiments, the detergent includes one or more detergents shown in Table <NUM>.

In some embodiments the detergent comprises Tween <NUM>, full strength.

In some embodiments, when present, the detergent is present in the lysis solution at a concentration ranging from about <NUM> up to about <NUM>, or from about <NUM> up to about <NUM>, or from about <NUM> up to about <NUM>, or from about <NUM> up to about <NUM>. In some embodiments the detergent ranges from about <NUM>, or from about <NUM>, or from about <NUM> or from about <NUM> or from about <NUM> up to about <NUM> or up to about <NUM>, or up to about <NUM>, or up to about <NUM>, or up to about <NUM>, or up to about <NUM>. In some embodiments, the detergent is present at a concentration of about <NUM>. In some embodiments, the detergent is present at a percentage ranging from about <NUM>% (v/v) up to about <NUM>% (v/v), or from about <NUM>% (v/v) up to about <NUM>% (v/v) or from about <NUM>% up to about <NUM>% (v/v). In some embodiments the detergent constitutes about <NUM>% to about <NUM>% of said solution, or about <NUM>% to about <NUM>% of said solution, or about <NUM>% of the lysis solution.

In some embodiments, the detergents used in the lysis solutions described herein need not be limited to the detergents described above. Using the teaching and examples provided herein, other detergents will be available to one of skill in the art.

In some embodiments, the lysis solution additionally comprises one or more of the following: a second detergent, a chaotrope and/or reducing agent, calcium chloride or other salt, and/or a protease.

In some embodiments the lysis solution additionally includes one or more proteases. Suitable proteases include, but are not limited to serine proteases, threonine proteases, cysteine proteases, aspartate proteases, metalloproteases, glutamic acid proteases, metalloproteases, and combinations thereof. Illustrative suitable proteases include, but are not limited to proteinase k (a broad-spectrum serine protease), subtilysin trypsin, chymotrypsin, pepsin, papain, and the like.

In some embodiments, when present in the lysis solution the protease is present at an amount that provides an activity that ranges from 1U/ml up to about 200U/ml of lysis solution. In some embodiments, the amount provides an activity ranging from about <NUM> U/ml, or from about <NUM> U/ml, or from about <NUM> U/ml, or from about <NUM> U/ml, up to about <NUM> U/ml, or up to about <NUM> U/ml, or up to about <NUM> U/ml, or up to about <NUM> U/ml, or up to about <NUM> U/ml, or up to about <NUM> U/ml of lysis solution. In some embodiments, the amount of protease ranges from about <NUM> to about <NUM>/ml. In some embodiments, the amount of protease ranges from about <NUM>/mL, or about <NUM>/mL, or about <NUM>/mL, or about <NUM>/mL, or about <NUM>/mL, or about <NUM>/mL, or about <NUM>/mL, or about <NUM>/mL up to about <NUM>/mL, or up to about <NUM>/mL, or up to about <NUM>/mL, or up about <NUM>/Ml, or up to about <NUM>/mL.

In some embodiments, the lysis solutions in the methods described herein need not be limited to the use of the proteases described above. Using the teaching and examples provided herein, other proteases will be available to one of skill in the art.

In various embodiments methods of use of the lysis solutions described herein are provided. One embodiment of the methods is schematically illustrated in <FIG>. As shown therein, one or more sections of a fixed, paraffin-embedded, tissue sample, are incubated in a lysis solution at a temperature ranging from about <NUM> to about <NUM>, typically a single temperature of about <NUM>. In certain embodiments the lysis solution lacks a protease, however, more typically a protease (e.g., proteinase K) is included.

The nucleic acids can be recovered from the lysis solution, e.g., using an alcohol extraction (e.g., an alcohol precipitation). The procedure results in a relatively high yield extraction and produces a nucleic acid (e.g., DNA, RNA) of sufficient quality for PCR amplification, detection, and/or quantification of a target nucleic acid sequence. In some embodiments the incubating is for a period of time up to about <NUM> hours. However, in typical embodiments, the incubating can range from about <NUM>, <NUM>, or <NUM> minutes up to about <NUM> hour. As noted above, in some embodiments no protease is required. Similarly, in some embodiments, the method does not include further steps of deparaffinization and/or additional reagents for deparaffinization. In some embodiments the method does not utilize an organic solvent for deparaffinization and/or the incubating is not in the presence of an organic solvent. According, the method is rapid, simple, and easily amenable to automation and high throughput methodologies.

The nucleic acids extracted using the methods and reagents described herein are of good quality and can readily be amplified to detect and/or quantify one or more target nucleic acid sequences in the sample. The nucleic acids are compatible with any of a number of amplification methods including, but not limited to polymerase chain reaction (PCR) (see. e.g., <NPL>,) including RT-PCR, ligase chain reaction (LCR) (see, e.g., <NPL>; <NPL>; <NPL>), transcription amplification (see, e.g., <NPL>), self-sustained sequence replication (see, e.g., <NPL>), dot PCR, linker adapter PCR, and the like.

Moreover it was a surprising discovery that samples processed in accordance with the methods using the materials described herein, particularly using the lysis solution(s) described herein (see, e.g., Table <NUM>) give earlier Ct results, sometimes better than <NUM> Cts, or better than <NUM> Cts, or better than <NUM> Cts, as compared to various commercial lysis systems.

Additionally a lysate stability study was performed in which FFPE cell buttons and FPE patient samples were lysed, mixed with Ethanol and then stored at -20C with scheduled test dates (see, e.g., Example <NUM>, experiment G, on-going thru Day <NUM>, and <FIG> and <FIG>). In one experiment, presently out to <NUM> days consistent cycle thresholds were observed over the course of the <NUM> days for all targets. It is thus possible to measure multiple pulls from the original lysed scroll to perform either a repeat test (if needed) or reflex cartridge test(s).

While in some embodiments, the extracted nucleic acids are used in amplification reactions, other uses are also contemplated. Thus, for example, the extracted nucleic acids (or their amplification product(s)) can be used in various hybridization protocols including, but not limited to nucleic acid based microarrays. In some embodiments any nucleic acid-based microarray can be used with the methods described herein. Such microarrays include but are not limited to, commercially available microarrays, for example microarrays available from Affymetrix, Inc. (Santa Clara, CA), Agilent Technologies, Inc. (Santa Clara, CA), Illumina, Inc. (San Diego, Calif. ), GE Healthcare (Piscataway, N. ), NimbleGen Systems, Inc. (Madison, Wis. ), Invitrogen Corp. (Carlsbad, Calif. ), and the like.

The methods and reagents described herein are thus applicable to basic research aimed at the discovery of gene expression profiles relevant to the diagnosis and prognosis of disease. The methods are also applicable to the diagnosis and/or prognosis of disease, the determination particular treatment regiments, monitoring of treatment effectiveness and the like. In some embodiments the methods are also applicable to other fields where the quality of nucleic acid is poor, such as forensics, archeology, medical history, paleontology, and the like. In view of the teachings and protocols provided herein, these and other applications will readily be recognized by those of skill in the art.

Using the methods described herein DNA and/or RNA can be isolated from any biological sample. Such samples include, but are not limited to fresh samples or cell/tissue aspirates, frozen sections, needle biopsies, cell cultures, fixed tissue samples, cell buttons, tissue microarrays, and the like. The methods are particularly well suited for use with fixed paraffin-embedded tissue (e.g., FFPET) samples. While histological samples are typically fixed with an aldehyde fixative such as formalin (formaldehyde) and glutaraldehyde, it is believed the methods described herein additionally work with tissues fixed using other fixation techniques such as alcohol immersion, and the like.

Illustrative samples include, but are not limited to, FFPET samples from human tissues, laboratory animal tissues, companion animal tissues, or livestock animal tissues. Thus, for example, the samples include tissue samples from humans including, but not limited to samples from healthy humans (e.g., healthy human tissue samples), samples from a diseased subject and/or diseased tissue, samples used for diagnostic and/or prognostic assays and the like. Suitable samples also include samples from non-human animals. FFPET samples from, for example, a non-human primate, such as a chimpanzee, gorilla, orangutan, gibbon, monkey, macaque, baboon, mangabey, colobus, langur, marmoset, lemur, a mouse, rat, rabbit, guinea pig, hamster, cat dog, ferret, fish, cow, pig, sheep, goat, horse, donkey, chicken, goose, duck, turkey, amphibian, or reptile can be used in the methods described herein.

In addition, FFPET samples of any age can be used with the methods described herein including, but not limited to, FFPET samples that are fresh, less than one week old, less than two weeks old, less than one month old, less than two months old, less than three months old, less than six months old, less than <NUM> months old, less than one year old, at least one year old, at least two years old, at least three years old, at least four years old, at least five years old, at least six years old, at least seven years old, at least eight years old, at least nine years old, at least ten years old, at least fifteen years old, at least twenty years old, or older.

In some embodiments the methods described herein are performed on one or more sections taken from a fixed, embedded tissue sample (e.g., an FFPET sample). The sections can be of any desired thickness. Thus, in some embodiments, both thin sections or thick sections are contemplated, including, but not limited to, sections that are less than <NUM> micron thick, about <NUM> micron thick, about <NUM> microns thick, about <NUM> microns thick, about <NUM> microns thick, about <NUM> microns thick, about <NUM> microns thick, about <NUM> microns thick, about <NUM> microns thick, about <NUM> microns thick, about <NUM> microns thick, about <NUM> microns thick, or about <NUM> microns thick, depending upon the desired application. In certain applications, the sections can be, for example, up to about <NUM> micron thick, up to about <NUM> microns thick, up to about <NUM> microns thick, up to about <NUM> microns thick, up to about <NUM> microns thick, up to about <NUM> microns thick, up to about <NUM> microns thick, up to about <NUM> microns thick, up to about <NUM> microns thick, up to about <NUM> microns thick, up to about <NUM> microns thick, up to about <NUM> microns thick, or up to about <NUM> or <NUM> microns thick. In some embodiments, the sections can be defined by a range of sizes, including, but not limited to, between about <NUM> and about <NUM> microns thick, between about <NUM> and about <NUM> microns thick, between about <NUM> and about <NUM> microns thick, or between about <NUM> and about <NUM> microns thick.

In many cases, the fixed embedded tissue samples (e.g., FFPET samples) comprise an area of diseased tissue, for example a tumor or other cancerous tissue. While such FFPET samples find utility in the methods described herein, FFPET samples that do not comprise an area of diseased tissue, for example FFPET samples from normal, untreated, placebo-treated, or healthy tissues, also can be used in the methods described herein. In some embodiments of the methods described herein, a desired diseased area or tissue, or an area containing a particular region, feature or structure within a particular tissue, is identified in a FFPET sample, or a section or sections thereof, prior to isolation of nucleic acids as described herein, in order to increase the percentage of nucleic acids obtained from the desired region. Such regions or areas can be identified using any method known to those of skill in the art, including, but not limited to, visual identification, staining, for example hematoxylin and eosin staining, immunohistochemical labeling, and the like. In any event, in some embodiments, the desired area of the tissue sample, or sections thereof, can be dissected, either by macrodissection or microdissection, to obtain the starting material for the isolation of a nucleic acid sample using the methods described herein.

While, in certain embodiments, the lysis reagents and methods described herein are particularly well suited for use with formalin-fixed paraffin embedded (FFPE) samples, it will be appreciated that the reagents and methods need to be limited to use with such samples. For example, in certain embodiments the lysis reagent(s) and methods described herein can be used on whole cells that are, for example, applied onto a glass slide as a smear. In certain embodiments the smears are derived from a fine needle aspirate. Smears can be a vehicle that has been associated with FNAs where the drawn sample is applied to a slide as a smear. The cells can be stained for visual observations but they can also be left unstained and simply allowed to air dry. In certain embodiments using these unstained smears cells can be scrapped off the slide and utilized with the lysis reagent and methods described herein.

In another illustrative, but non-limiting approach the fine needle aspirate cells can be injected directly into the lysis reagent. The sample can continue with the lysing procedure. In certain embodiments it is possible to transport the sample (in the lysis reagent) to a different site where the analysis procedure can be completed. In certain embodiments the fine needle aspirate sample can also be made into an FFPE cell button.

The use of fine needle aspirates provides a method of avoiding the tedious process of preparing formalin fixed paraffin embedded samples and can significantly speed up the testing process. This method may be quite useful in developing areas of the world.

In addition, to fine needle aspirates, it will also be appreciated that the reagent(s) (e.g., lysis solution) and methods of use thereof are amendable to use with essentially any method of cell collection. Such methods include, but are not limited to scrapes (e.g., buccal scrapes, gynecological scrapes, throat scrapes, scrapes during surgical procedures, etc.), wipes (obtained, for example, using a cotton swab), and aspirates including, but not limited to vacuum assisted biopsies.

In certain illustrative, but non-limiting embodiments, the sample comprises a diseased area or tissue comprising cells from a cancer. In some embodiments the cancer comprises a cancer selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), Adrenocortical carcinoma, AIDS-related cancers (e.g., kaposi sarcoma, lymphoma), anal cancer, appendix cancer, astrocytomas, atypical teratoid/rhabdoid tumor, bile duct cancer, extrahepatic cancer, bladder cancer, bone cancer (e.g., Ewing sarcoma, osteosarcoma, malignant fibrous histiocytoma), brain stem glioma, brain tumors (e.g., astrocytomas, brain and spinal cord tumors, brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, breast cancer, bronchial tumors, burkitt lymphoma, carcinoid tumors (e.g., childhood, gastrointestinal), cardiac tumors, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous t-cell lymphoma, duct cancers e.g. (bile, extrahepatic), ductal carcinoma in situ (DCIS), embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer (e.g., intraocular melanoma, retinoblastoma), fibrous histiocytoma of bone, malignant, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., ovarian cancer, testicular cancer, extracranial cancers, extragonadal cancers, central nervous system), gestational trophoblastic tumor, brain stem cancer, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, langerhans cell cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kaposi sarcoma, kidney cancer (e.g., renal cell, Wilm's tumor, and other kidney tumors), langerhans cell histiocytosis, laryngeal cancer, leukemia, acute lymphoblastic (ALL), acute myeloid (AML), chronic lymphocytic (CLL), chronic myelogenous (CML), hairy cell, lip and oral cavity cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer (e.g., childhood, non-small cell, small cell), lymphoma (e.g., AIDS-related, Burkitt (e.g., non-Hodgkin lymphoma), cutaneous T-Cell (e.g., mycosis fungoides, Sézary syndrome), Hodgkin, non-Hodgkin, primary central nervous system (CNS)), macroglobulinemia, Waldenström, male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, melanoma (e.g., childhood, intraocular (eye)), merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, mouth cancer, multiple endocrine neoplasia syndromes, multiple myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, Myelogenous Leukemia, Chronic (CML), multiple myeloma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity cancer, lip and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter, transitional cell cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma (e.g., Ewing, Kaposi, osteosarcoma, rhadomyosarcoma, soft tissue, uterine), Sézary syndrome, skin cancer (e.g., melanoma, merkel cell carcinoma, basal cell carcinoma, nonmelanoma), small intestine cancer, squamous cell carcinoma, squamous neck cancer with occult primary, stomach (gastric) cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, trophoblastic tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, Wilm's tumor, and the like.

It will be recognized that the methods described herein are believed to be compatible with essentially any fixed (e.g., formalin fixed, glutaraldehyde fixed, etc.) paraffin embedded tissue sample. Such samples include, but are not limited to biopsies and fine needle aspirates and archived samples (e.g. tissue microarrays), and the like.

In some embodiments, one or more tissue sections are heated in the lysis solution. In this regard, it is noted that where thinner sections are used it is possible and can be desirable to utilize a plurality of sections (e.g., at least <NUM> sections, or at least <NUM> sections, or at least <NUM> sections, or at least <NUM> sections, or at least <NUM> sections, or at least <NUM> sections, or at least <NUM> sections, or at least <NUM> sections, or at least <NUM> sections). Particularly where the section is <NUM> thick or smaller multiple sections can be desirable.

In some aspects of the disclosure, the sections are heated in the lysis solution at a temperature of about <NUM> up to about <NUM>. In some embodiments the sections are heated at a temperature ranging from about <NUM>, or from about <NUM>, or from about <NUM>, or from about <NUM>, or from about <NUM>, or from about <NUM> up to about <NUM>, or up to about <NUM>, or up to about <NUM>. In some embodiments, the sections are heated at a temperature ranging from about <NUM> to about <NUM>. In certain embodiments the heating is at <NUM>.

In some embodiments, the incubation time ranges from about <NUM> minutes up to about <NUM> hours. In some embodiments, the incubation time ranges from about <NUM> minutes, or from about <NUM> minutes, or from about <NUM> minutes, or from about <NUM> minutes, or from about <NUM> minutes up to about <NUM> hours, or up to about <NUM> hours, or up to about <NUM> hours, or up to about <NUM> hours, or up to about <NUM> hours, or up to about <NUM> hours, or up to about <NUM> hours, or up to about <NUM> hours, or up to about <NUM> hours, or up to about <NUM> hour. In some embodiments, the incubation time ranges from about <NUM> minutes up to about <NUM> hour.

In one illustrative, but non-limiting, embodiment the one or more sections are incubated (heated) in the lysis solution (e.g., a solution as shown in Table <NUM>) for about <NUM> minutes at a temperature of about <NUM>. In another illustrative, but non-limiting, embodiment the one or more sections are incubated (heated) in the lysis solution (e.g., a solution as shown in Table <NUM>) for about <NUM> minutes at a temperature of about <NUM>.

These heating temperatures and periods are illustrative and not intended to be limiting. Using the teaching provided herein, one of skill may optimized the protocol for a particular sample type at a particular time and temperature.

After the tissue section(s) are heated in the lysis solution the extracted nucleic acid (e.g., DNA, RNA) can be recovered. Numerous methods for DNA and/or RNA recovery are known to those of skill in the art.

In some embodiments, the nucleic acid is precipitated and/or bound to a solid substrate. Precipitation and/or binding to a substrate is readily accomplished by use of an alcohol, for example a lower alcohol (e.g., a C<NUM>-C<NUM> alcohol). In some embodiments the alcohol is ethanol or isopropanol. In some embodiments the alcohol is ethanol. It will be recognized that in some embodiments, dry alcohols can be used.

In some embodiments the alcohol is used to simply precipitate the nucleic acid(s). In some embodiments, the alcohol is used to precipitate the nucleic acids in the present of a compatible solid phase that results in binding of the nucleic acid to that solid phase.

For example, in some embodiments, the alcohol treatment is performed in the present of a glass or cellulose substrate resulting in the binding of the nuclei acid(s) to that substrate. Remaining contaminants can be washed away while retaining the recovered nucleic acids that are then ready for amplification or other uses.

In some embodiments the solid phase comprises glass, silica, or cellulose. The solid phase can be provided by the walls of a container, as a fiber (e.g., glass fiber), as a membrane (e.g., cellulose membrane), in the form of beads (e.g., microparticles, or nanoparticles, etc.), and the like.

In certain embodiments, the nucleic acid recovery can be performed in a GENEXPERT® cartridge, e.g., as described below.

The nucleic acids extracted using the methods and reagents described herein are of good quality and can readily be amplified to detect and/or quantify one or more target nucleic acid sequences in the sample. The nucleic acids are particular well suited to PCR amplification reactions including, but not limited to RT-PCR. While in some embodiments, the extracted nucleic acids are used in amplification reactions, other uses are also contemplated. Thus, for example, the extracted nucleic acids (or their amplification product(s)) can be used in various hybridization protocols including, but not limited to nucleic acid based microarrays.

The nucleic extraction methods and reagents described herein are applicable to basic research aimed at the discovery of gene expression profiles relevant to the diagnosis and prognosis of disease. The methods are also applicable to the diagnosis and/or prognosis of disease, the determination particular treatment regiments, monitoring of treatment effectiveness and the like.

The methods described herein simply and efficiently produce extracted nucleic acids well suited for use in RT-PCR systems. While they can be used in any such system, in some embodiments, as illustrated herein in the Examples, the nucleic acids are particularly well suited for use in the GENEXPERT® cartridge and systems (Cepheid Systems Inc.

The GENEXPERT® system is a closed, self-contained, fully-integrated and automated platform that represents a paradigm shift in the automation of molecular analysis, producing accurate results in a timely manner with minimal risk of contamination. The GENEXPERT® system combines on-board (in cartridge) sample preparation with real-time PCR (polymerase chain reaction) amplification and detection functions for fully integrated and automated nucleic acid analysis in a cartridge (GENEXPERT® cartridge). The system is designed to purify, concentrate, detect and identify targeted nucleic acid sequences thereby delivering answers directly from samples (see, e.g., <CIT>, <CIT>, <CIT>, <CIT>, and <CIT>). In various embodiments, components of the cartridge can include, but are not limited to, processing chambers containing reagents, filters, and capture technologies useful to extract, purify, and amplify target nucleic acids. A valve enables fluid transfer from chamber to chamber and contains nucleic acids lysis and filtration components. An optical window enables real-time optical detection (e.g., of PCR amplification products). A reaction tube can be provided that permits very rapid heating and/or thermal cycling.

In certain embodiments an illustrative GENEXPERT® cartridge comprises a plurality of chambers disposed around a central valve assembly and selectively in fluid communication with the central valve assembly where the central valve assembly is configured to accommodate a plunger that is capable of drawing fluid into or out of a chamber in fluid communication with the central valve. Rotation of the valve assembly determines which chamber are in fluid communication with the central valve.

Accordingly, in some embodiments, methods are provided for identification and/or quantitative measurement of a target nucleic acid sequence in a fixed paraffin embedded tissue sample (optionally utilizing a GENEXPERT® cartridge and system). In some embodiments the methods comprise extracting a nucleic acid (e.g., a DNA, an RNA) from a fixed paraffin embedded biological tissue sample according any of the extraction methods described herein, subjecting the extracted nucleic acid to amplification using a pair of oligonucleotide primers capable of amplifying a region of a target nucleic acid, to obtain an amplified sample; and determining the presence and/or quantity of the target nucleic acid. In some embodiments, the target nucleic acid is a DNA (e.g., a gene). In some embodiments, the target nucleic acid is an RNA (e.g., an mRNA, a non-coding RNA, and the like).

In some embodiments, the nucleic acids extracted using the methods described herein are well suited for use in diagnostic methods, prognostic methods, methods of monitoring treatments (e.g., cancer treatment), and the like. Accordingly, in some illustrative, but non-limiting embodiments, the nucleic acids extracted from fixed paraffin-embedded samples (e.g., from FFPET samples) can be used to identify the presence and/or the expression level of a gene, and/or the mutational status of a gene.

Such methods are particular well suited to identification of the presence, and/or expression level, and/or mutational status of one or more cancer markers. Accordingly, in some embodiments, the nucleic acids extracted using the methods described herein are utilized to detect the presence, and/or copy number, and/or expression level, and/or mutational status of one or more cancer markers. Illustrative, but non-limiting cancer markers are shown in Table <NUM>.

In some embodiments, the target nucleic acid comprises a microRNA described in <CIT> and <CIT>. In some embodiments the target nucleic acid comprises a nucleic acid marker for the presence and/or severity and/or prognosis of lung cancer. In some embodiments the target nuclei acid comprises a target nucleic acid marker for lung cancer (e.g., non-small cell lung cancer) described in in <CIT>. In some embodiments the target nucleic acid comprises a nucleic acid marker for the presence and/or severity and/or prognosis of cervical cancer and/or cervical dysplasia. In some embodiments the target nuclei acid comprises a target nucleic acid marker for cervical dysplasia and/or cervical cancer described in in <CIT>.

The foregoing target nucleic acids are illustrative and non-limiting. Using the teaching provided herein, numerous other target nucleic acid sequences will be available to one of skill in the art.

In some, a normal level (a "control") for each target nucleic acid (e.g., RNA) can be determined as an average (or median) level or range that is characteristic of normal cells or other reference material, against which the level measured in the sample can be compared. The determined average (or median) or range of target nucleic acid (e.g., RNA) in normal subjects can be used as a benchmark for detecting above-normal levels of target RNA indicative of a disease state (e.g., the presence of or predilection for a cancer). In some embodiments, normal levels of target nucleic acid can be determined using individual or pooled RNA-containing samples from one or more individuals, such as, in the case of cervical cancer, from patients undergoing hysterectomy for benign gynecologic disease.

In some embodiments, determining a normal level of expression of a target nucleic acid (e.g., RNA) comprises detecting a complex comprising a probe hybridized to a nucleic acid selected from a target RNA, a DNA amplicon of the target RNA, and a complement of the target RNA. That is, in some embodiments, a normal level of expression can be determined by detecting a DNA amplicon of the target RNA, or a complement of the target RNA rather than the target RNA itself. In some embodiments, a normal level of such a complex is determined and used as a control. The normal level of the complex, in some embodiments, correlates to the normal level of the target RNA.

In some embodiments, a control comprises RNA from cells of a single individual, cells known to be healthy from the same subject. In some embodiments, a control comprises RNA from a pool of cells from multiple individuals. In some embodiments, a control is drawn from anatomically and/or cytologically normal areas of the of the individual from whom the test sample was obtained. In some embodiments, a control comprises commercially-available human RNA, such as, for example in the case of cervical cancer, human cervix total RNA (Ambion; AM6992). In some embodiments, a normal level or normal range has already been predetermined prior to testing a sample for an elevated level.

In some embodiments, the normal level of target RNA can be determined from one or more continuous cell lines, typically cell lines previously shown to have expression levels of the at least one target RNA that approximate the level of expression in normal cells.

In some embodiments, a method comprises detecting the level of expression of at least one target RNA. In some embodiments, a method further comprises comparing the level of expression of at least one target RNA to a normal level of expression of the at least one target RNA. In some embodiments, a method further comprises comparing the level of expression of at least one target RNA to a control level of expression of the at least one target RNA. A control level of expression of the at least one target RNA is, in some embodiments, the level of expression of the at least one target RNA in a normal cell. In some such embodiments, a control level may be referred to as a normal level. In some embodiments, a greater level of expression of the at least one target RNA relative to the level of expression of the at least one target RNA in a normal cell indicates cervical dysplasia.

In some embodiments, the level of expression of the at least one target RNA is compared to a reference level of expression, e.g., from a confirmed neoplasia. In some such embodiments, a similar level of expression of the at least one target RNA relative to the reference sample indicates the presence of a neoplasia.

In some embodiments, a level of expression of at least one target RNA that is at least about two-fold greater than a normal level of expression of the respective at least one target RNA indicates the presence of a disease state (e.g., a cancer). In some embodiments, a level of expression of at least one target RNA that is at least about two-fold greater than the level of the respective at least one target RNA in a control sample comprised of normal cells indicates the presence of a cancer. In some embodiments, a level of expression of at least one target RNA that is at least about <NUM>-fold, at least about <NUM>-fold, at least about <NUM>-fold, at least about <NUM>-fold, at least about <NUM>-fold, at least about <NUM>-fold, at least about <NUM>-fold, or at least about <NUM>-fold greater than the level of expression of the respective at least one target RNA in a control sample comprised of normal cells indicates the presence of a cancer. In some embodiments, a level of expression of at least one target RNA that is at least about <NUM>-fold, at least about <NUM>-fold, at least about <NUM>-fold, at least about <NUM>-fold, at least about <NUM>-fold, at least about <NUM>-fold, at least about <NUM>-fold, or at least about <NUM>-fold greater than a normal level of expression of the at least one target RNA indicates the presence of a cancer.

In some embodiments, a control level of expression of a target RNA is determined contemporaneously, such as in the same assay or batch of assays, as the level of expression of the target RNA in a sample. In some embodiments, a control level of expression of a target RNA is not determined contemporaneously as the level of expression of the target RNA in a sample. In some such embodiments, the control level of expression has been determined previously.

In some embodiments, the level of expression of a target RNA is not compared to a control level of expression, for example, when it is known that the target RNA is expressed at very low levels, or not at all, in normal cells. In such embodiments, detection of a high level of the target RNA in a sample is indicative of a cancer.

In certain embodiments kits are provided for the extraction of a nucleic acid from a cell and/or tissue sample. In certain embodiments the kit will typically comprises a container containing a lysis solution as described herein. In certain embodiments the kit further comprises a container containing a protease (e.g., proteinase K, trypsin, chymotrypsin, papain, etc.). In certain embodiments the protease and the lysis solution are mixed together. In certain embodiments the protease and the lysis solution are provided in separate containers.

In certain embodiments the kit can further comprise a device for the collection of a cell or tissue sample. Illustrative devices include, but are not limited to a device selected from the group consisting of a device or device tip for performing a scrape, a wipe, a device or device tip for obtaining an aspirate, a punch biopsy device, and a blade for obtaining a skin biopsy. For example, in certain embodiments, the kit comprises a device or device tip for obtaining a fine needle aspirate and/or for obtaining a vacuum assisted aspirate. In certain embodiments the kit comprises a device for performing a buccal scrape, or a gynecological scrape. Illustrative devices include, but are not limited to a multispatula, an extended tip spatula, a cytobrush, a cytopick, a cervexbrush, swab, a baynebrush. , a profilebrush, a bulb aspirator, an Ayre spatula, an Aylesbury device, and the like. In typical embodiments the device for collection of a cell or tissue sample is provided in packaging that preserves sterility of the sample collecting device before use.

In certain embodiments the kit can comprise a container configured to receive a cell or tissue sample and to store that sample in said lysis solution or in a buffer. In certain embodiments the container configured to receive a cell or tissue sample is configured for storage and/or shipping. Thus, in certain embodiments, the container configured to receive a cell or tissue sample, is provided with a label to identify the sample, and, in certain embodiments sealable packaging to hold the container during storage and/or shipping and/or a shipping container.

In certain embodiments the kit can optionally further include a sterile swab (e.g., an alcohol swab) for cleaning the sample site, and/or a drying pad (e.g., a gauze pad) for drying the site, and/or a dressing (e.g. bandage) for dressing the site after obtaining the sample.

In certain embodiments, the components for a single collection operation are packaged together in a packet. Such packets can include, for example, a single use disposable sample device, optionally a sterile swab, optionally a drying pad, and optionally a dressing. In certain embodiments the kit includes at least <NUM> packets, or at least <NUM> packets, or at least <NUM> packets, or at least <NUM> packets, or at least <NUM> packets, or at least <NUM> packets, or at least <NUM> packets.

In certain embodiments the kit can further contain instructional materials teaching collection methods utilizing the kit components and, optionally, providing guidance to overcome problems that may occur during collection. The instructional materials can also include information and/or instructions regarding the use of the lysis reagent and/or instructions for the collection, and/or storage, and/or shipping of a cell or tissue sample. In certain embodiments the kits additionally contain reagents and/or instructions teaching the use of the lysis buffer for isolation and recovery of a nucleic acid.

Often and typically the instructional materials are provided in written form and can be printed on the kit components themselves (e.g. on the cover of a box, container, or on an envelope, or can be provided as an insert/instructional page or booklet. While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.

The following examples are offered to illustrate, but not to limit the claimed invention.

Goals: Perform a Proteinase K treatment using a test buffer, followed by modified FFPE lysis on patient 015445T2.

Samples: FFPE patient slides (<NUM>), 015445T2. <NUM> thick and applied to glass slides.

IHC/FISH: ER: rich, PR: rich, HER2: amplified.

Goals: Test FFPE lysis reagent with <NUM>% emulsion instead of Anti-foam on Pt 015445T2 slide.

Samples FFPE patient slide, 015445T2. <NUM> thick and applied to glass slides.

Goals: Optimize amount of PK added during off board lysis (before heat step).

Samples Alamak FFPE cell buttons, BT474.

<FIG> shows cycle threshold as a function of NaCl concentration for ESR and PGR. <FIG> shows cycle threshold as a function of NaCl concentration for ERBB2 and CYFIP1. <FIG> shows cycle threshold as a function of NaCl concentration for MKi67.

Goals: Process BT474 cell button samples with m3FFPE, m3cFFPE, m3dFFPE and m3eFFPE lysis formulations.

Samples: BT474 FFPE cell buttons, cut and stored at <NUM>. <NUM> thick and applied to glass slides.

Each slide was transferred to a labeled <NUM> tube.

<NUM> of each designated lysis reagent was added to its tube.

20uL of Proteinase K was added to each sample.

The samples were vortexed for <NUM> seconds, then incubated at 80C for <NUM> minutes.

The samples were vortexed for <NUM> seconds and pulse spun.

Each sample was transferred to a labeled <NUM> vial containing <NUM> <NUM>% Ethanol.

The samples were each vortexed for at least <NUM> seconds.

Cartridge A's, NGB, were prepared with reaction beads in chamber <NUM> and liquid reagents in chambers <NUM> and <NUM>.

Four 520uL aliquots from each sample was transferred to chamber <NUM> in their designated cartridges.

All carts were run using the <NUM> Strat + 2X sonicate ADF.

Goals: Process BT474 FFPE cell buttons with m3f FFPE lysis reagents with varying antifoam concentrations.

Samples: BT474 FFPE cell buttons. Slides are <NUM> thick and applied to glass slides.

The samples were vortexed for <NUM> seconds, then incubated at <NUM> for <NUM> minutes.

Cart A's, NGB, were prepared with reaction beads in chamber <NUM> and liquid reagents in chambers <NUM> and <NUM>.

Four 520uL aliquots per test condition were transferred to chamber <NUM> in their designated cartridges.

Additionally a lysate stability study was performed in which FFPE cell buttons and FPE patient samples were lysed, mixed with Ethanol and then stored at -20C with scheduled test dates (see, e.g., Table <NUM>, below and <FIG> and <FIG>).

<FIG> shows the stability (repeatable of cycle threshold) for ESR, and PGR for samples stored over <NUM> days. <FIG> shows the stability (repeatable of cycle threshold) for ESR, and PGR for samples stored over <NUM> days.

Goal: Test the cores from slide <NUM>, (TMA30 block from Yale), in the Stratifier assay.

Test Samples: <NUM> cores on a single slide, TMA block from Yale. Slide YTMA <NUM>-<NUM>, Breast ER, <NUM>-<NUM>-<NUM>, slide <NUM>. Slide was cut <NUM> thick.

Claim 1:
A lysis solution for the extraction of a nucleic acid from a cell or tissue sample, said lysis solution comprising:
NaCl<NUM> at a concentration in the range of <NUM> to <NUM>;
a HEPES sodium salt buffer sufficient to maintain the pH of said solution at a pH ranging from pH <NUM> to pH <NUM>, wherein the concentration of said buffer is in the range of from <NUM> up to <NUM>;
a chelating agent selected from the group consisting of N-acetyl-L-cysteine, ethylenediaminetetraacetic acid (EDTA), diethylene triamine pentaacetic acid (DTPA), ethylenediamine-N,N'-disuccinic acid (EDDS), <NUM>,<NUM>-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and a phosphonate chelating agent, wherein the concentration of said chelating agent in said solution is in the range of from <NUM> to <NUM>;
MgCl<NUM> at a concentration less than <NUM>; and
an ionic detergent or a non-ionic detergent, wherein said detergent constitutes <NUM>% to <NUM>% of said solution.