WO2013075110A2

WO2013075110A2 - A novel eml4-alk variant: primers, probes, methods, and kits for the detection thereof

Info

Publication number: WO2013075110A2
Application number: PCT/US2012/065877
Authority: WO
Inventors: Craig Stephens; Martin Maus
Original assignee: Response Genetics, Inc.
Priority date: 2011-11-18
Filing date: 2012-11-19
Publication date: 2013-05-23
Also published as: WO2013075110A3

Abstract

The present invention relates primers, probes, and kits for the detection of EML4-ALK variant sequences in a sample. The present invention also relates methods of detection EML4-ALK variants using the primer, probes, and kits disclosed herein.

Description

A NOVEL EML4-ALK VARIANT: PRIMERS, PROBES, METHODS, AND KITS FOR

THE DETECTION THEREOF

RELATED APPLICATIONS

This application claims priority to US provisional application 61/561 ,391 , filed on

November 18, 201 1 , which is herein incorporated by reference.

FIELD OF THE INVENTION

The invention relates to primers, probes and kits for the detection of variants in the EML4-AL gene sequence, and methods of use thereof.

DESCRIPTION OF RELATED ART

Cancer arises when a normal cell undergoes neoplastic transformation and becomes a malignant cell. Transformed (malignant) cells escape normal physiologic controls specifying cell phenotype and restraining cell proliferation. Transformed cells in an individual's body thus proliferate, forming a tumor. When a tumor is found, the clinical objective is to destroy malignant cells selectively while mitigating any harm caused to normal cells in the individual undergoing treatment.

Lung cancer is the leading cause of cancer-related death in the United States and worldwide. Despite recent advances in the treatment of the disease, the overall 5-year survival rate in the United States remains only 15%.

Historically, the histological appearance of lung cancer has been used to guide treatment decisions, primarily distinguishing patients with small-cell lung cancer (SCLC) from those with non-small-cell lung cancer (NSCLC). Three major subtypes of NSCLC -adenocarcinoma, squamous cell carcinoma, and large-cell carcinoma-were grouped together as a single entity for therapy and enrollment of patients onto clinical trials. With the recent regulatory approval of particular drugs for specific subtypes, the discrimination of specific NSCLC subtypes has grown in significance.

In recent years, it has also become evident that adenocarcinomas have distinct genomic changes that allow them to be further classified into clinically relevant molecular subsets. Each subset can be defined by a specific genomic alteration that is responsible for both the initiation and maintenance of the lung cancer (i.e., a driver mutation). Importantly, specific mutations induce differential sensitivity of tumors to targeted therapeutic agents. For example, tumors highly sensitive to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) often contain dominant somatic mutations in exons that encode a portion of the tyrosine kinase domain of EGFR. Conversely, tumors with somatic mutations in KRAS, which encodes a GTPase downstream of EGFR, display primary resistance to these drugs.

When knowledge of specific genetic changes is linked to a targeted therapy, dramatic improvements in response and clinical benefit can be observed without the need for initial treatment with cytotoxic chemotherapy.

Recently, another potential driver mutation in NSCLC was discovered, namely fusion of the N-terminal portion of the protein encoded by the echinoderm microtubule-associated proteinlike 4 (EML4) gene with the intracellular signaling portion of the receptor tyrosine kinase encoded by the anaplastic lymphoma kinase (ALK) gene. These fusions are the result of inversions within the short arm of chromosome 2 (involving 2p21 and 2p23, approximately 12 Mb apart). Whereas other genetic alterations involving ALK have been identified in anaplastic large-cell lymphomas, inflammatory myo fibroblastic tumors, and neuroblastomas, none have yet been found in lung cancers. The EML4-ALK fusion gene was believed to be novel to NSCLC. Reports have identified it in breast cancer and colorectal carcinoma. See Lin et al., Mol. Cancer. Res., 2009; 7: 1466-1476.

To date, multiple EML4-ALK variants have been identified in lung cancer. Although the fusions contain variable truncations of EML4 (occurring at exons 2, 6, 13, 14, 15, 18, and 20), the ALK fusion in all of them starts at a portion encoded by exon 20 of the kinase gene. To date, all EML4-ALK fusions tested biologically demonstrate gain of function properties.

The echinoderm microtubule-associated protein like protein (EML4) has a basic region at the amino terminus, and further has carboxyl-terminal WD (tryptophan-aspartic acid) domains. The physiological functions of EML4 are not clearly known.

Full-length ALK expression has been reported in some cancer cells of ectodermal origin, such as neuroblastoma, glioblastoma, breast cancer, and melanoma (the full-length ALK expression has not been observed in cancer cells of endodermal and mesodermal origins). Full- length ALK is expressed in many neuroblastoma cell lines. However, the autophosphorylation of ALK is not observed in these neuroblastoma cell lines. Moreover, ALK expression has been reported, from the cohort analysis of neuroblastoma patients, to be weakly associated with cancer. It has been suggested that ALK expression in neuroblastoma may reflect its expression in normal neural differentiation, rather than its association with cancer. On the other hand, in reported cases, ligands such as pleiotrophin and midkine as well as the gene amplification of ALK itself increase the autophosphorylation of ALK and mobilize intracellular signals. It has also been reported that ALK may contribute to cancer cell growth.

In some cases of human malignant lymphoma and inflammatory myo fibroblastic tumor, the ALK gene has been reported to be fused with other genes (NPM, CLTCL, TFG, CARS, SEC31 LI , etc.) as a result of chromosomal translocation or inversion and thereby form a fusion type of tyrosine kinase. Moreover, a method for identifying a protein as a fusion partner for ALK using ALK antibodies has been reported. Since most partner molecules have a complex formation domain, the fusion protein itself has been thought to form a complex. This complex formation has been considered to cause loss of control of the tyrosine kinase activity of ALK and induce carcinogenesis with abnormally activated intracellular signals

EML4-ALK is a fusion-type protein tyrosine kinase that is generated in human non- small-cell lung cancer (NSCLC) as a result of a recurrent chromosome inversion, inv

(2)(p21p23).

There is data suggesting that the documented presence of an EML4-ALK variant, which is observed in as many as 10% to 15% of patients with non-small-cell lung cancer, indicates that the malignancy will be unresponsive to tyrosine kinase inhibition of EGFR. (J Clin Oncol.

2009;27:4247-4253). Of particular interest is the finding that among the patients who were light smokers or never-smokers and did not possess an EGFR mutation, one third were found to have an EML4-ALK variant. These data add to growing evidence for the relevance of specific molecular testing in non-small-cell lung cancer such that, in the relatively near future, it might be possible to develop therapeutic paradigms optimized for the individual patient based on unique characteristics of each cancer. Such decisions will include not only the particular drugs that should be used, but also (as appears to be the situation with EML4-ALK) the agents that should be avoided due to a predicted lack of efficacy.

The discovery of the fusion of the anaplastic lymphoma kinase (ALK) with the echinoderm microtubule-associated protein like 4 (EML4) as an oncogene has lead to a validation as a clinical target in NSCLC in a short period of time. The ALK-Inhibitor crizotinib has demonstrated to prolong progression- free survival compared to the standard of care chemotherapy in patients with advanced NSCLC that are ALK positive.

SUMMARY OF THE INVENTION

The present invention provides primers, probes, and kits for the detection of EML4-ALK variants. The present invention also provides methods of detection EML4-ALK variants using the primer, probes, and kits of the present invention. Particular primer combinations as disclosed herein may be used in amplifying EML4-ALK variants and distinguishing between variants. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a table listing the sequences of the primers and probes used in the amplification and detection of EML4-ALK gene variants.

Figure 2 shows a table listing the specificity of primers and probes used in the amplification of EML4-ALK gene variants for particular variants.

Figure 3 shows a picture of a gel that was run for testing a sample for the presence of EML4- ALK gene variant 2. Figure 4 shows a picture of a gel that was run for testing a sample for the presence of EML4- ALK gene variant 1 and a picture of a gel run for variant 3a/3b.

Figure 5 provides the sequence of variant 3 a. The text in italics is from the EML4 gene. The text in regular font is from the ALK gene. The text in bold shows the location of the primers used to detect the variant. The forward primer indicated is SEQ ID NO:22. The reverse primer indicated is SEQ ID NO: 27. The text underlined is the location of the probe used to detect variant 3a, which is SEQ ID NO: 26.

Figure 6A provides provides the sequence of variant 3b. The text in italics is from the EML4 gene. The text in regular font is from the ALK gene. The text in bold shows the location of the primers used to detect the variant. The forward primer indicated is SEQ ID NO:22. The reverse primer indicated is SEQ ID NO:27. The text underlined is the location of the probe, which is SEQ ID NO: 26. The double underlined text is the additional insert found in variant 3b that is not present in variant 3a. Figure 6B provides sequence information regarding variant 3c. The novel variant, detected in two NSCLC specimens, is longer than variant 3a and shorter than variant 3b, representing a 18bp insertion of intron 19 of ALK in between exon 6 of EML4 and exon 20 of ALK. The 18 bp insert is shown italics. The bold text are sequences from EML4 exon 6 and the underlined text are sequences from ALK exon 20.

Figure 7 shows a picture of a gel that was run for testing a sample for the presence of EML4- ALK gene variant 3c. Lane 1 was for markers. Lane 2 are variant markers showing both variant 3a and 3b (labeled MS). Lane three is a negative control (labeled NTC). Lane 4 (labeled H- 2228) are synthetic variants 3a and 3b used as a control. Lane 5 is from a patient sample having variant 3c (labeled as RDX1 1-8167). Lane 6 is a control made for variant 3c.

DETAILED DESCRIPTION OF THE INVENTION

The term "first primer" is used interchangeably with "forward primer." Similarly, the term "second primer" is used interchangeably with "reverse primer."

The present invention provides a method for detecting the presence of an EML4-ALK variant in a sample by (a) isolating a nucleic acid from the sample wherein the sample comprises DNA sequences, (b) performing an amplification reaction of said sequences using a first primer capable of annealing specifically to an EML4-ALK variant sequence at a first position in a EML4-ALK DNA sequence and a second primer capable of annealing specifically at a second position in a EML4-ALK DNA sequence, and (c) visualizing or detecting amplification products produced by said amplification reaction, wherein detection of the EML4-ALK variant specific amplification product is a positive indicator of a EML4-ALK variant in the sample. The first and second primers anneal to different strands of double stranded EML4-ALK DNA sequence. In the methods disclosed below, not only can the listed primers be used but also primers having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% identity with the listed primers may be used, as long as they are able to specifically bind to and allow amplification of the EML4-ALK variants. The first primer is selected from the group consisting of SEQ ID NO: l , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25. The second primer is SEQ ID NO: 10, SEQ ID NO:27, SEQ ID NO: 30 or SEQ ID NO:31. The amplification reaction is capable of producing an EML4-ALK variant specific amplification product when the DNA sequences of the sample comprise an EML4-ALK DNA sequence comprising a variant sequence at said first position of said EML4-ALK DNA sequence.

The present invention provides a method for detecting the presence of an EML4-ALK variant in a sample by (a) isolating a nucleic acid from the sample wherein the sample comprises RNA sequences, (b) performing an amplification reaction of said sequences using a first primer capable of annealing specifically to EML4-ALK variant sequence at a first position in a EML4- ALK RNA sequence and a second primer capable of annealing specifically at a second position in a EML4-ALK RNA sequence, and (c) visualizing or detecting amplification products produced by said amplification reaction, wherein detection of a EML4-ALK variant specific amplification product is a positive indicator of a EML4-ALK variant in the sample. The first and second primers anneal to different strands of double stranded EML4-ALK RNA sequence. The first primer is selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25. The second primer is SEQ ID NO: 10, SEQ ID NO:27, SEQ ID NO: 30 or SEQ ID NO:31. The amplification reaction is capable of producing an EML4-ALK variant specific amplification product when the RNA sequences of the sample comprise an EML4-ALK RNA sequence comprising a variant sequence at said first position of said EML4-ALK RNA sequence.

The present invention provides a method for detecting the presence of an EML4-ALK variant 3c in a sample by (a) isolating a nucleic acid from the sample wherein the sample comprises RNA or DNA sequences, (b) performing an amplification reaction of said sequences using a first primer capable of annealing specifically to EML4-ALK variant sequence at a first position in a EML4-ALK RNA or DNA sequence and a second primer capable of annealing specifically at a second position in a EML4-ALK RNA or DNA sequence, and (c) visualizing or detecting amplification products produced by said amplification reaction, wherein detection of a EML4-ALK variant specific amplification product is a positive indicator of a EML4-ALK variant in the sample. The first and second primers anneal to different strands of double stranded EML4-ALK RNA or DNA sequence. The first primer is selected from the group consisting of SEQ ID NO:22 and SEQ ID NO:23. The first primer is preferably SEQ ID NO: 22. The second primer is selected from the group consisting of SEQ ID NO: 10, SEQ ID NO:27, SEQ ID NO: 30 or SEQ ID NO:31. The second primer is preferably SEQ ID NO:27. The amplification reaction is capable of producing an EML4-ALK variant specific amplification product when the RNA or DNA sequences of the sample comprise an EML4-AL RNA or DNA sequence comprising a variant sequence at said first position of said EML4-ALK RNA or DNA sequence. A probe used to identify the presence of a 3a, 3b, or 3c variant is the probe having SEQ ID NO:26. the novel variant 3c is discussed in more detail in figure 6B and Example 5.

The term variant includes but is not limited to, truncation variations, elongation variants, point mutations, deletions, etc.

Embodiments of the present invention comprise EML4-ALK variant specific forward and reverse primers (i.e. primers that are capable of binding to and amplifying EML4-AL variants as opposed to not binding to and amplifying EML4-ALK non-variants). Exemplary forward primers include SEQ ID NO: 1-9 and SEQ ID NO: 12-25. Exemplary reverse primers include SEQ ID NO: 10, SEQ ID NO:27, and SEQ ID NO:30-31. In addition, to the sequences set forth in SEQ ID NO: 1-9 and SEQ ID NO: 12-25, SEQ ID NO: 10, SEQ ID NO:27, and SEQ ID NO:30- 31 , the invention also provides primers having 80%, 85%, 90%, 95% and 99% identity to the sequences set forth in the SEQ ID NOs. One skilled in the art can appreciate that one may be able to take the enumerated sequence in the SEQ ID NO: and alter one or a few bases and the primer may still work in the methods of the present invention. In addition, one may shift the primers upstream or downstream, and/or add a few or subtract a few bases and still achieve a desired amplicon of suitable length that includes the variant and allow for later identification of the presence of the various EML4-ALK variants. One can easily test these primer variants by methods known in the art, for example by performing amplification of a control sample that has the variant present and then determining if the new primer was indeed able to amplify and pick up this variant. Embodiments of the present invention comprise oligonucleotide probe sequences, wherein the oligonucleotide is used as a probe for the detection of EML4-ALK variant sequences. This probe was designed to avoid any known EML4-ALK polymorphisms.

Optionally, the oligonucleotide is detectably labeled. Exemplary probes include SEQ ID NO: l 1 , SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:32. In addition, to the probe sequences set forth in SEQ ID NO:l 1 , SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:32, the invention also provides probes having 80%, 85%, 90%, 95% and 99% identity to the sequences set forth in the SEQ ID NO: l 1 , SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:32. One skilled in the art can appreciate that one may be able to take the enumerated sequence in the SEQ ID NO and alter one or a few bases, and the probe may still work in the methods of the present invention. In addition, one may shift the probe upstream or downstream, and/or add a few or subtract a few bases, and it may still be able to bind to and identify the various EML4-ALK variants. One can easily test these new probe by methods known in the art, for example by taking a sample known to contain a variant, labeling the probe and allowing the probe to bind to the variant, washing away any unbound probe and then looking to see if there is label present bound to the variant.

The present invention provides primers comprising, consisting of, or consisting essentially of SEQ ID NO: 1 -9, SEQ ID NO: 12-25, SEQ ID NO: 10, SEQ ID NO:27, and SEQ ID NO:30-31 or a primer that is 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1-9, SEQ ID NO: 12-25, SEQ ID NO: 10, SEQ ID NO:27, or SEQ ID NO:30-31.

The present invention provides probes comprising, consisting of, or consisting essentially of SEQ ID NO: 1 1 , SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:32 or a probe that is 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: l 1 , SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:32.

Accordingly, methods of the present invention may use primers comprising, consisting of, or consisting essentially of SEQ ID NO: 1-9, SEQ ID NO: 12-25, SEQ ID NO: 10, SEQ ID NO:27, and SEQ ID NO:30-31 or a primer that is 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: l -9, SEQ ID NO: 12-25, SEQ ID NO: 10, SEQ ID NO:27, or SEQ ID NO:30-31.

Methods of the present invention may use probes comprising, consisting of, or consisting essentially of SEQ ID NO: 1 1 , SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:32 or a probe that is 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: l 1, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:32.

The present invention also provides a kit comprising at least one of SEQ ID NO: 1-32 or primer or probe 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1-32.

In performing a method of the present invention, EML4-ALK variant expression levels are assayed in patient tumor samples to prognosticate the efficacy of a treatment regimen. For example, if the tumor shows the presence of an EML4-ALK variant, then the patient may be unresponsive to tyrosine kinase inhibition of EGFR and as such perhaps should not be treated with trysoine kinase inhibitors of EGFR such as Erlotinib, BIBW-2992, Brivanib, WZ3146, or gefitinib. Accordingly, the present invention provides a method of determining a

chemotherapeutic regimen for treating a tumor in a patient comprising: (a) obtaining a tissue sample of the tumor (b) isolating DNA or RNA from the tumor sample; (c) subjecting the DNA or RNA to amplification using a pair of oligonucleotide primers capable of amplifying a region of the EML4-ALK gene, to obtain a EML4-ALK amplified sample; (d) determining the presence of a EML4-ALK variant in the amplified sample; (e) determining a chemotherapeutic regime based on the presence of an EML4-ALK variant in the sample.

In performing an embodiment of the present invention, tumor cells are preferably isolated from the patient. Solid or lymphoid tumors or portions thereof are surgically resected from the patient or obtained by routine biopsy. DNA or RNA isolated from frozen or fresh samples is extracted from the cells by any of the methods typical in the art, for example, Sambrook, Fischer and Maniatis, Molecular Cloning, a laboratory manual, (2nd ed.), Cold Spring Harbor

Laboratory Press, New York, (1989). Preferably, care is taken to avoid degradation of the DNA or RNA during the extraction process. Samples

Methods of the present invention comprise obtaining a sample of a tissue or a body fluid from the subject (e.g., a mammal), wherein the sample contains nucleic acid. Non-limiting examples of tissue or body fluids that can be used include blood, plasma, lymph, tumor biopsies, and body tissue. In one embodiment, the tissue sample comprises paraffin embedded (PE) tissue specimens or formalin fixed paraffin embedded (FFPE) specimens. In certain embodiments, the sample is, but is not limited to, a fresh sample, frozen sample, a product of a needle biopsy, or from a pleural effusion. In some embodiments, the nucleic acid is deoxyribonucleic acid (DNA). In some embodiments, the nucleic acid is ribonucleic acid (RNA).

Methods of the invention can be applied to any type of tissue from a patient. Sources of such tissue include but are not limited to nervous system, thyroid, skin, gastrointestinal tract, large intestine, biliary tract, ovary, eye, prostate, central nervous system, liver, small intestine, breast, pancreas, soft tissue, upper, aerodigestive tract, adrenal gland, autonomic ganglia, haematopoietic and lymphoid tissue, lung, esophagus, pituitary, and stomach. For examination of resistance of tumor tissue to a certain treatment, it is preferable to examine the tumor tissue. In one embodiment, a portion of normal tissue from the patient from which the tumor is obtained is also examined to allow a comparison between the normal/healthy and the tumor tissue.

Isolating nucleic acid

Embodiments of the present invention utilize methods of DNA isolation known to those skilled in the art. In general, the aim is to separate DNA present in the nucleus of the cell from other cellular components. The isolation of DNA usually begins with lysis, or breakdown, of tissue or cells. This process is essential for the destruction of protein structures and allows for release of nucleic acids from the nucleus. Lysis is carried out in a salt solution, containing detergents to denature proteins or proteases (enzymes digesting proteins), such as Proteinase K, or in some cases both. It results in the breakdown of cells and dissolving of membranes.

Methods of DNA isolation include, but are not limited to, phenol: chloroform extraction, high salt precipitation, alkaline denaturation, ion exchange column chromatography, resin binding, and paramagnetic bead binding.

Embodiments of the present invention utilize methods of RNA isolation known to those skilled in the art. RNA may be isolated and prepared for hybridization by a variety of methods including, but not limited to, Trizol^® and Guanidinium thiocyanate-phenol-chloroform

extraction. The principle of RNA isolation is based on cell/tissue lysis, followed by extraction, precipitation, and washing. It will be understood by those skilled in the art the selection of RNA isolation will depend on sample type. Incorporated by reference is US 12/144,388 directed to a method of RNA isolation from paraffin embedded tissue, a common source for oncogene marker testing. Amplification

Embodiments of the present invention utilize thermal and isothermal amplification methods including, but not limited to, polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), ligase chain reaction (LCR), helicase dependent amplification (HDA) and Nucleic Acid Sequence Based Amplification (NASBA) and

Amplification Refractory Mutation System (ARMS). In certain embodiments, primers and probes of the invention are used in ARMS.

Detection/Visualization

Embodiments of the present invention utilize detection/visualization methods including, but not limited to, labeling primers used during the amplification step such that the amplification products are labeled with a detectable marker and hybridizing the amplification product to oligonucleotide probes labeled with a detectable marker. Detectable markers include, but are not limited to, chemiluminescent tags, fluorescent tags, and radioactive tags. Labeled amplification product can be directly measured using methods corresponding to the type of label used according to methods known to one skilled in the art. Labeled probes can be hybridized to the amplification product according to methods known to one skilled in the art.

Using primers and probes of the present invention, the invention provides methods of assaying for the presence of particular EML4-ALK variants. In one embodiment, the assay of the invention detects the presence of variant 1 in exon 13 of the EML4 fused with exon 20 of the ALK gene. Forward primers having SEQ ID NO: 1 or 12, reverse primers having SEQ ID NO: 27, 30 or 31 and probes having SEQ ID NO: 26 or 32 are used in methods of the present invention for the detection of variant 1.

In another embodiment, the assay of the invention detects the presence of variant 2 in exon 20 of the EML4 fused with exon 20 of the ALK gene. Forward primers having SEQ ID

NO: 2, 13 or 14, reverse primer having SEQ ID NO: 27, 30 or 31 and probe having SEQ ID NO: 26 or 32 are used in methods of the present invention for the detection of variant 2.

In yet another embodiment, the assay of the invention detects the presence of variant 3a in exon 6a of the EML4 fused with exon 20 of the ALK gene. Forward primers having SEQ ID NO: 3, 15, 22 or 23, reverse primers having SEQ ID NO: 27, 30 or 31 and probes having SEQ ID NO: 26 or 32 are used in methods of the present invention for the detection of variant 3a. In yet another embodiment, the assay of the invention detects the presence of variant 3b in exon 6b of the EML4 fused with exon 20 of the ALK gene. Forward primers having SEQ ID NO: 4, 16, 22 or 23, reverse primers having SEQ ID NO: 27, 30 or 31 and probes having SEQ ID NO: 26 or 32 are used in methods of the present invention for the detection of variant 3b.

In yet another embodiment, the assay of the invention detects the presence of variant 3c in exon 6b of the EML4 fused with exon 20 of the ALK gene. Forward primers having SEQ ID NO: 4. 16, 22 or 23, reverse primers having SEQ ID NO: 27, 30 or 31 and probes having SEQ ID NO: 26 or 32 are used in methods of the present invention for the detection of variant 3b. In certain preferred embodiments, the forward primer is SEQ ID NO: 22 and the reverse primer is SEQ ID NO: 27 and the probe is SEQ ID NO: 26.

The assay of the invention can also detect the presence of variant 4 in exon 14 with insertion of 1 1 nucleotides of unknown origin fused with ALK in exon 20 starting at nucleotide 50. Forward primers having SEQ ID NO: 5, 17, or 25, reverse primers having SEQ ID NO: 10, and probes having SEQ ID NO: 1 1 or 28 are used in methods of the present invention for the detection of variant 4.

In another embodiment, the assay of the invention detects the presence of variant 5a in exon 2 of the EML4 fused with exon 20 of the ALK. Forward primers having SEQ ID NO: 6 or 18, reverse primers having SEQ ID NO: 27, 30 or 31 and probes having SEQ ID NO: 26 or 32 are used in methods of the present invention for the detection of variant 5a.

In another embodiment, the assay of the invention detects the presence of variant 5b in exon 2 of the EML4 fused with exon 20 of the ALK gene with 5b containing an additional 1 17 nucleotides from intron 19. Forward primers having SEQ ID NO: 7 or 19, reverse primers having SEQ ID NO: 24 and probes having SEQ ID NO: 25 or 29 are used in methods of the present invention for the detection of variant 5a.

In yet another embodiment, the assay of the invention detects the presence of variant 6 in exon 13 of the EML4 fused with exon 20 of the ALK gene with an additional 69 nucleotides from intron 19. Forward primers having SEQ ID NO: 8 or 20, reverse primers having SEQ ID NO: 27, 30 or 31 and probes having SEQ ID NO: 26 or 32 are used in methods of the present invention for the detection of variant 6.

In still another embodiment, the assay of the invention detects the presence of variant 7 in exon 14 of the EML4 fused with exon 20 of the ALK gene starting at nucleotide 13. Forward primers having SEQ ID NO: 9, 21 or 25, reverse primer having SEQ ID NO: 10 and probes having SEQ ID NO: 1 1 or 28 are used in methods of the present invention for the detection of variant 7.

Certain primers and probes of the present invention can be used to pick up (amplify and then visualize/detect) multiple variants. For example, Figure 2 shows the specificity of primers and probes that be used to detect more than one variant. For instance, forward primer having SEQ ID NO:22 and 23 can use used to detect either the 3a and/or the 3b variant. Forward primer having SEQ ID NO:25 can use used to detect either the 4 and/or 7 variant. The reverse primer having SEQ ID NO: 10 can be used to detect either the 4 and/or 7 variant. The probe having SEQ ID NO: 1 1 can be used to detect either the 4 and/or 7 variant. A probe having SEQ ID NO: 26 can be used to detect either the 1 , 2, 3a, 3b, 3c, 5a and/or 6 variant. A reverse primer having the SEQ ID NO: 27 can be used to detect either the 1, 2, 3a, 3b, 3c, 5a and/or 6 variant. Reverse primers having SEQ ID NO: 30 or 31 and probe having SEQ ID NO:32 can be used to detect the 1 , 2, 3 (a and b), 5a and/or 6 variants.

The present invention also provides a nucleic acid probe specific for the EML4-ALK variant 3c wherein the probe specifically binds to the following sequence (or its compelement): CTGACCACCCACCTGCAG (SEQ ID NO:33). The nucleic acid probe has the sequence of CTGCAGGTGGGTGGTCAG (SEQ ID NO: 34) or its complement.

The assay can be an EML4-ALK fusion assay, which is a specific RT-PCR based assay containing specific primers and probe that detect the fusion of the EML4 gene and the ALK gene at different splice sites in the two genes.

Assay Formats

Embodiments of the present invention can be utilized in a variety of formats including single-plex, multiplex assays and sequencing. Single-plex assays refer to those that measure a single target or analyte. A multiplex assay is a type of laboratory procedure that simultaneously measures multiple analytes (dozens or more) in a single assay. It is distinguished from procedures that measure one or a few analytes at a time. Multiplex assays are widely used in functional genomics experiments that endeavor to detect or to assay the state of all biomolecules of a given class (e.g., mRNAs, proteins) within a biological sample, to determine the effect of an experimental treatment or the effect of a DNA mutation over all of the biomolecules or pathways in the sample. An example of a multiplex assay is the DNA microarray for gene expression or single nucleotide polymorphism (SNP) detection assays. For example, the assay formats can be FISH (fluorescence in situ hybridization), RT-PCR (reverse transcription polymerase chain reaction), PCR (polymerase chain reaction), gene arrays, or protein arrays. Alternatively, embodiments of the present invention can be utilized with sequencing methods in order to detect target mutations.

Embodiments of the present invention can be used in assays wherein EML4-ALK gene variants are assessed along with other gene defects or anomalies. There are a number of genes screened in a variety of cancer assays. Embodiments of the present invention can be used to detect EML4-ALK singly, in pair- wise fashion with any other gene, or in multi-plex fashion for screening for gene mutations or variants in a variety of other genes. Other genes that can be screened included housekeeping genes and cancer associated genes. Examples of cancer associated genes that can be evaluated for mutations include but are not limited to EGFR, KRAS, TP53, K-RAS, p53, PIK3CA, and NKX2-1 (TTF1).

In addition, the invention provides the ability to screen for multiple EML4-ALK variants or to screen for just one EML4-ALK. For example, seven assays of the present invention (vl , v2, v3a, v3b, v3ab, v3c, v5a and v6) all can use a common FAM labeled probe (SEQ ID NO:32) and reverse primer (SEQ ID NO: 30 or 31). They can each contain one forward primer for discrimination between the different mutations. For example, by using reverse primer SEQ ID NO: 30 or 31 , and probe SEQ ID NO:32, variant 1 forward primers (SEQ ID NO: 1 or 12) can be used to identify variant 1 ; variant 2 forward primers (SEQ ID NO: 2 or 13) can be used to identify variant 2; variant 3a primer (SEQ ID NO: 3 or 15) can be used to identify variant 3a; variant 3b primers (SEQ ID NO: 4 or 16) can be used to identify variant 3b; variant 5a primers (SEQ ID NO: 6 or 18) can be used to identify variant 5a; and variant 6 primers (SEQ ID NO: 8 or 20) can be used to identify variant 6. In another embodiment, using reverse primer SEQ ID NO: 30 or 31, and probe SEQ ID NO:32, forward primers can be used that detect both variants 3a and 3b (SEQ ID NO: 22 or 23).

The present invention also provides three assays for identifying variant 4 and 7. For example, by using reverse primer SEQ ID NO: 10, and probe SEQ ID NO: 1 1 or 28, variant 4 primer SEQ ID NO: 17 can be used to identify variant 4. By using reverse primer SEQ ID NO: 10, and probe SEQ ID NO: 1 1 , variant 7 primers (SEQ ID NO: 9 or 21) can be used to identify variant 7. In another embodiment, by using reverse primer SEQ ID NO: 10, and probe SEQ ID NO: 1 1 , forward primer (SEQ ID NO:25) can be used to detect both variants 4 and 7.

The present invention also provides an assay for identifying variant 5b. Using reverse primer SEQ ID NO:24 and probe SEQ ID NO:29, with forward primers SEQ ID NO:7 or 19, one can detect EML4-ALK variant 5b.

EXAMPLES

EML4-ALK fusion single-plex assays for variants 1, 2, 3a, 3b, 3c, 4, 5a, 5b, 6, and 7 were developed. In addition, combination assays for the detection of variants 4 or 7 and 3a or 3b were developed, which did not distinguish between the pairs of variants. The assays used primers or probes selected from SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:30.

A control assay was used to assess the total cDNA in a sample. The control assay amplifies a region of exon 4 of the beta-actin gene using primers and 6FAM-labeled probe that were designed to avoid any known polymorphisms. "6FAM" is 6 - carboxyfluorescein.

Example 1

The assays for the variants 1 , 2, 3a, 3b, 3a/3b, 3c, 5a, and 6 use a common 6FAM labeled probe (SEQ ID NO:26) and reverse primer (SEQ ID NO:27). Each of the variants was amplified by hybridizing to a different and specific forward primer that discriminates among the different variants. An exception is the forward primer for 3a/3b (SEQ ID NO:22), which was designed to detect both variants 3a and 3b.

The assays for variants 4, 7, and the combination 4/7 used a common 6FAM-labeled probe (SEQ ID NO: l 1 ) and reverse primer (SEQ ID NO: 10). The forward primers were SEQ ID NO: 17 (for variant 4), SEQ ID NO:21 (variant 7) or SEQ ID NO:25 (combination of 4 and 7).

The assay for variant 5b used SEQ ID NO: 19 as the forward primer, SEQ ID NO:24 as the reverse primer, and SEQ ID NO:30 as the 6FAM-labeled probe. The assays had excellent linearity, PCR efficiency, accuracy, and reproducibility. For example, PCR efficiency, as measured by relation to beta-actin PCR, was 97.2 to 105.8% for the several assays.

The assays also detected the following truncations of EML4 using a real-time PCR assay based on a TaqMan assay:

Variant 1 in exon 13 of the EML4 fused with exon 20 of the ALK gene;

Variant 2 in exon 20 of the EML4 fused with exon 20 of the ALK gene;

Variant 3a/b in exon 6a/b of the EML4 fused with exon 20 of the ALK gene;

Variant 3c in exon 6 of the EML4 fused with exon 20 of the ALK gene;

Variant 4 in exon 14 having insertion of 1 1 nucleotides fused with ALK in exon 20 starting at nucleotide 50;

Variant 5a/b in exon 2 of the EML4 fused with exon 20 of the ALK gene with 5b containing an additional 1 17 nucleotides from intron 19;

Variant 6 in exon 14 of the EML4 fused with exon 20 of the ALK gene with an additional 69 nucleotides from intron 19; and

Variant 7 in exon 14 of the EML4 fused with exon 20 of the ALK gene starting at nucleotide 13.

The sensitivity of the EML4-ALK assays for variants was determined using serial dilutions of synthetic construct expressing the fusion transcripts in a background of U-87MG cDNA. The measured sensitivities are provided in the following table.

Example 2: Variant 2

FFPE tumor samples were obtained and the DNA was isolated and screened for the presence of variant 2. See figure 3. A positive control ("MxStd") was used along with a negative control ("NTC"). The forward primer as SEQ ID NO: 13, the reverse primer was SEQ ID NO:27 and the probe was SEQ ID NO:26. A control assay, labeled with FAM, was used to assess the total cDNA in a sample. This assay amplifies a region of exon 4 of the beta-Actin gene. The primers and probe have been designed to avoid any known b-Actin polymorphisms.

The sample labeled FFPE+ shows the presence of variant 2. Samples labeled FFPE- do not have the variant 2.

Example 3 : Variant 1

FFPE tumor samples were obtained and the DNA was isolated and screened for the presence of variant 1. See figure 4. A positive control ("MxStd") was used along with a negative control ("NTC"). The forward primer as SEQ ID NO: 12, the reverse primer was SEQ ID NO:27 and the probe was SEQ ID NO:26. A control assay, labeled with FAM, was used to assess the total cDNA in a sample. This assay amplifies a region of exon 4 of the beta-Actin gene. The primers and probe have been designed to avoid any known b-Actin polymorphisms.

The sample labeled FFPE+ shows the presence of variant 1. Samples labeled FFPE- do not have the variant 1.

Example 4: Variant 3a/b

FFPE tumor samples were obtained and the DNA was isolated and screened for the presence of variant 3a/b. See figure 4. A positive control ("MxStd") was used along with a negative control ("NTC"). The forward primer as SEQ ID NO:22, the reverse primer was SEQ ID NO:27 and the probe was SEQ ID NO:26 A control assay, labeled with FAM, was used to assess the total cDNA in a sample. This assay amplifies a region of exon 4 of the beta-Actin gene. The primers and probe have been designed to avoid any known b-Actin polymorphisms.

The sample labeled FFPE+ shows the presence of variant 3a or 3b . Samples labeled FFPE- do not have the variant 3a or 3b. Example 5: Variant 3C

RNA extracted from NSCLC specimens (FFPE)was amplified using primers and probes designed to detect specific EML4-ALK fusion gene fragments with a maximum amplicon of 170 bases by RT-PCR. The different assays can detect and identify each of the 9 variants (1, 2, 3a, 3b, 4, 5a, 5b, 6, 7). For the detection of variant 3, two PCR products of 136 bp (corresponding to variant 3 b) and 103 bp (corresponding to variant 3 a), representing the linking of exon 6 of EML4 and exon 20 of ALK, were detected by agarose gel electrophoresis. Any detected discrepancies to the known bands of variant 3a and variant 3b in gel electrophoresis were further investigated by sanger-sequencing.

The novel variant, detected in two NSCLC specimens, is longer than variant 3a and shorter than variant 3b, (see figure 7) representing a 18bp insertion of intron 19 of ALK in between exon 6 of EML4 and exon 20 of ALK (see figure 6B). These finding have been confirmed by sanger-sequencing.

Compared to FISH technology, RT-PCR enables the detection of different isoforms of the EML4-ALK transforming gene, which can be validated by sequencing. This novel isoform contains the amino-terminal coiled-coil domain within EML4 and therefore is likely to show transforming activity of EML4-ALK.

Claims

A method for detecting the presence of an EML4-ALK variant 3c in a sample, said method comprising: (a) isolating a nucleic acid from said sample wherein the sample comprises DNA sequences; (b) performing an amplification reaction of said DNA sequences of said sample, wherein said amplification reaction comprises a first primer capable of annealing specifically to EML4-ALK variant sequence at a first position in a EML4-ALK DNA sequence wherein said first primer is selected from the group consisting of SEQ ID NO:22, and SEQ ID NO:23, and a second primer capable of annealing specifically at a second position in a EML4-ALK DNA sequence, wherein said first and second primers anneal to different strands of double stranded EML4-ALK DNA sequence, wherein the amplification reaction is capable of producing a EML4-ALK variant specific amplification product when the DNA sequences of the sample comprise a EML4-ALK DNA sequence comprising a variant sequence at said first position of said EML4-ALK DNA sequence; and (c) visualizing and/or detecting amplification products produced by said amplification reaction, wherein detection of a EML4-ALK variant specific amplification product is a positive indicator of a EML4-AL variant in said sample.

A method for detecting the presence of an EML4-ALK variant 3 c in a sample, said method comprising: (a) isolating a nucleic acid from said sample wherein the sample comprises RNA sequences; (b) performing an amplification reaction of said RNA sequences of said sample, wherein said amplification reaction comprises a first primer capable of annealing specifically to EML4-ALK variant sequence at a first position in a EML4-ALK RNA sequence wherein said first primer is selected from the group consisting of SEQ ID NO:22, and SEQ ID NO:23, and a second primer capable of annealing specifically at a second position in a EML4-ALK RNA sequence, wherein said first and second primers anneal to different strands of double stranded EML4-ALK RNA sequence, wherein the amplification reaction is capable of producing a EML4-ALK variant specific amplification product when the RNA sequences of the sample comprise a EML4-ALK RNA sequence comprising a variant sequence at said first position of said EML4-ALK RNA sequence; and (c) visualizing and/or detecting amplification products produced by said amplification reaction, wherein detection of a EML4-ALK variant specific amplification product is a positive indicator of a EML4-ALK variant in said sample.

3. The method of claim 1 or 2 wherein the second primer is selected from the group consisting of SEQ ID NO:26 and SEQ ID NO:27.

4. The method of claim 1 or 2 wherein the presence of the EML4-ALK variant is a positive indicator of non small cell lung carcinoma in said sample.

5. The method of claim 1 or 2, wherein the sample is selected from the group consisting of blood, tissue, or cells.

6. The method of claim 1 or 2, wherein the tissue sample is formalin fixed paraffin embedded tissue.

7. A method for determining a chemotherapeutic regimen for treating a tumor in a patient comprising: (a) obtaining a tissue sample of the tumor (b) isolating DNA or RNA from the tumor sample; (c) subjecting the DNA or RNA to amplification using a pair of

oligonucleotide primers capable of amplifying a region of the EML4-ALK gene, to obtain a EML4-ALK amplified sample; (d) determining the presence of a EML4-ALK variant 3c in the amplified sample; (e) determining a chemotherapeutic regime based on the presence of the EML4-ALK variant 3c in the sample.

8. The method of claim 7, wherein the oligonucleotide primers consist of a forward and reverse primer wherein the forward primer is SEQ ID NO:22 or an oligonucleotide primer at least 90

% identical thereto, and wherein the reverse primer is SEQ ID NO:27, or an oligonucleotide primer at least 90 % identical thereto.

9. A kit for detecting expression of a EML4-ALK variant 3c comprising forward and reverse oligonucleotide primers wherein the forward primer is selected from the group consisting of

SEQ ID NO:22, and SEQ ID NO:23 and wherein the reverse primer is selected from the group consisting of SEQ ID NO:26 and SEQ ID NO:27 or oligonucleotide primers at least 90% identical thereto.

0. A nucleic acid probe specific for the EML4-ALK variant 3c wherein the probe specifically binds to the following sequence: CTGACCACCCACCTGCAG (SEQ ID NO:33).

1. The method of claim 1 or 2, or wherein determining the presence of variant EML4-AL nucleic acid sequences comprises adding a probe wherein said probe comprises the oligonucleotide sequence of SEQ ID NO:26 or SEQ ID NO: 33, or a probe at least 90% identical thereto.