WO2019036632A1 - Compositions and methods for detection of cd30 mrna isoforms for use in drug selection - Google Patents

Abstract

Provided is a method for detecting CD30 mRNA expression in a biological sample from an individual with lymphoma by in situ hybridization using probes that can discriminate between different isoforms. The levels of overall expression and relative levels of the isoforms of CD30 mRNA can be used for identification of individuals who are likely to respond to a specific anti-cancer agent, such as brentuximab vedotin. A suitable individual can then be treated with the anti-cancer agent. Also provided are kits for detection of CD30 mRNA and its isoforms.

Description

COMPOSITIONS AND METHODS FOR DETECTION OF CD30 mRNA ISOFORMS

FOR USE IN DRUG SELECTION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.

62/547,179, filed on August 18, 2017, the disclosure of which is incorporated herein by reference.

FIELD OF THE DISCLOSURE

[0002] This disclosure relates generally to the field of CD30 expression, and more particularly to compositions and methods for detecting isoforms of CD30 expression, and using analysis of the isoforms to guide patient care.

BACKGROUND OF THE DISCLOSURE

[0003] Antibody-drug conjugate (ADC) is a type of immunoconjugate that consists of monoclonal antibodies and a cytotoxic component connected by a cleavable linker. Currently, only two ADCs have been approved by FDA for the eradication of malignant cells, namely the Brentuximab vedotin and ado-trastuzumab (Masuda S, et al. The Lancet

Oncology; 17:e371). Brentuximab vedotin is an anti-CD30-monomethyl auristatin E

(MMAE) immunoconjugate that was approved in 2011 for the treatment of refractory Hodgkin's lymphoma and anaplastic large-cell lymphoma with positive CD30 expression (Smaglo et al. Nat Rev Clin Oncol 2014; 11 :637-48). Upon binding to CD30 epitope on tumor cells, the immunoconjugate is endocytosed and fused with the lysosome, after which the linker gets cleaved via hydrolysis by Cathepsin B. Activated MMAE is then released into the cytoplasm to inhibit the formation of mitotic spindle, irreversibly resulting in cell cycle arrest.

[0004] Recently, a 5-year follow-up study reported that patients with

refractory/relapsed Hodgkin's disease achieved 41% overall survival and 22% progression- free survival after treatment with brentuximab vedotin, suggesting the immunoconjugate has good disease control and is an effective treatment strategy (Chen et al., Blood

2016; 128: 1562-6). Paradoxically, a recent phase 2, open-label, multicenter study suggested that CD30 expression does not predict patient response to brentuximab vedotin, as patient with low CD30 expression can also achieve great objective response to the therapy (Chen, et al., Blood 2016; 128: 1562-6). Moreover, some of the adverse effects related to the administration of brentuximab vedotin, such as neutropenia, neuropathy, fatigue, nausea, diarrhea, pyrexia, arthralgia, and headache and so on are similar to those observed with less specific chemotherapy regimens (Fanale MA, et al. Clin Cancer Res 2012; 18:248-55). Taken together, the pharmacodynamics of the drug is deemed questionable, and the selection of suitable patients for treatment is currently not possible. Since the expression of CD30 protein is not a predictive marker of clinical response to brentuximab vedotin treatment, the development of an alternative mechanism that can identify patients who respond to this agent is necessary. The present disclosure is pertinent to this need.

SUMMARY OF THE DISCLOSURE

[0005] The present disclosure provides compositions and methods for detecting CD30 mRNA expression in cells, and identifying CD30 splice variants using in situ hybridization techniques. The splice variants are identified by using probes that can hybridize to specific exon junctions and can discriminate between the different isoforms. For example, differential staining was observed in cells using probes directed to signal peptide, transmembrane portion, and/or cytoplasmic portion of CD30 protein.

[0006] In one aspect, this disclosure provides a method of deteting CD30 mRNA expression in a biological sample comprising cells, which are fixed and permeabilized by using in situ hybridization using one or more probes, wherein each probe is specific to CD30 mRNA isoform 1, CD30 mRNA isoform 2, both CD30 isoform 1 and isoform 2, or the mRNA encoding CD30 signal peptide. For example, the probes may be specific for a transmembrane portion of CD30 (specific to isoform 1), cytoplasmic portion of CD30 (specific to isoform 2), or a portion that contains both a transmembrane and a cytoplamic portion (that is common to both isforms 1 and 2). By comparing the relative levels of the isoforms with lymphoma patients who have responded or not responded to brentuximab vedotin treatment, it can be determined if an individual is a candidate for treatment with this anti-cancer drug. Based upon such determination, a suitable candidate may be then treated with brentuximab vedotin. A candidate who is not a suitable candidate for treatment with brentuximab vedotin, may be treated with a drug other than brentuximab vedotin. Examples of probes that can be used in the present disclosure are BA-Hs-TNFRSF8-tvl-ElE2 probe, the BA-Hs-TNFRSF8-tv3-ElE2 probe, the BA-Hs-TNFRSF8-E4E5 probe, or the BA-Hs- TNFRSF8-lzz-st probe - as further described herein. [0007] In one aspect, this disclosure provides a method of treating lymphoma in an indivdual. The method comprises determining in a biological sample obtained from an individual who has been diagnosed with or is suspected of having lymphoma, expression of CD30 mRNA by in situ hybridization, and based on a determination of the presence CD30 mRNA (compared to a reference level), administering brentuximab vedotin to the indivdiual. The reference level may be obtained from one or more individuals have responded positively to treatment with brentuximab vedotin. The CD30 mRNA determination may include determination of relative levels of isoform 1 and isoform 2, and comparison of those levels with a reference level. [0008] In one aspect, this disclosure provides a kit comprising one or more probes, wherein the one or more probes comprise the BA-Hs-TNFRSF8-tvl-ElE2 probe, the BA-Hs- TNFRSF8-tv3-ElE2 probe, the BA-Hs-TNFRSF8-E4E5 probe, or the BA-Hs-TNFRSF8- lzz-st, and optionally reagents for use in in situ hybridzation.

[0009] In one aspect, this disclosure provides a substrate in contact with permebalized cells, wherein the cells comprise mRNA/DNA probe duplexes, wherein the DNA probe component of the duplex is at least one probe selected from the group consising of the BA- Hs-TNFRSF8-tvl-ElE2 probe, the BA-Hs-TNFRSF8-tv3-ElE2 probe, the BA-Hs- TNFRSF8-E4E5 probe, or the BA-Hs-TNFRSF8-lzz-st probe.

BRIEF DESCRIPTION OF THE FIGURES

[0010] Figure 1. Flow chart providing a representation of the steps of embodiments of the present method for cells in suspension and formalin fixed paraffin embedded (FFPE) sections.

[0011] Figure 2. Test run demonstration of use of the present method for detecting expression of different mRNA. Both positive and negative controls are shown. [0012] Figure 3. Identification of regions of CD30 protein detected by the different probes, and use of the probes to detect the expression of CD30 mRNA in two patient samples from HL patients. Both positive (PPIB) and negative (DapB) controls are included.

[0013] Figure 4. Representation of Reed Sternberg cell in Hodgkin's lymphoma staining with hematoxylin/eosin (left) and CD30 expression by immunohistochemistry (right). [0014] Figure 5. Representation of cells from classical Hodgkin's lymphoma (CHL) and anaplastic large cell lymphoma (ALCL) using hematoxylin/eosin;

immunohistochemistry; and by in situ hybridization using probes for signal peptide (SP), transmembrane portion specific to isoform 1 (TMP), cytoplasmic portion specific to isoform 2 (CP), or a probe that is specific to a transmembrane portion and a cytoplamic portion that is common to both isoforms 1 and 2 (TMP and CP). The transmembrane region that probe BA- Hs-T FRSF8-E4E5 hybridizes to is distinct from the transmembrane region that probe BA- Hs-T FRSF8-tvl-ElE2 hybridizes to, and the cytoplamic region that probe BA-Hs- T FRSF8-E4E5 hybridizes to is distinct from the cytoplamic region that probe BA-Hs- T FRSF8-tv3-ElE2 hybridizes to.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0015] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. [0016] Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein.

[0017] All nucleotide sequences described herein, their RNA and DNA equivalents, and complimentary sequences are included in this disclosure. All polynucleotide and amino acid sequences associated with GenBank accession numbers described in this disclsoure are incorporated herein by reference as those sequences are listed in the database as of the filing date of this application or patent. The disclosure includes all combinations of probes described herein, cells that are fixed to a substrate and which comprise probes that are directly and/or indirectly detectably labeled, and/or are hybridized to mRNA within fixed cells, and kits for performing assays as described herein.

[0018] In embodiments, this disclosure relates to detection of CD30 mRNA expression in neoplastic cells, as well as in background tumor infiltrating cells. The disclosure includes generating and analyzing expression patterns of CD30 mRNA, which can be used in, for example, clinical applications to direct personalized approach to target drug therapy application for individuals in need of treatment for cancer, and who may be, for example, candidates for treatment with brentuximab vedotin, or another agent that targets CD30 and or expression of CD30. Thus, it is expected that patterns of mRNA expression revealed using embodiments of the present invention may act as surrogates to determine and correlate drug specific interactions in tumor cells, and within their microenvironment cell population. The disclosure accordingly includes detecting, measuring, and/or quantifying one or more isoforms (i.e., splice variants) of CD30 mRNA, including but not necessarily limited to determining relative proportions of splice variants.

[0019] In particular, in view of the data presented in this disclosure, the invention provides new approaches for identification of individuals who may be, or may not be, candidates for treatment with brentuximab vedotin, or a similar agent, or another agent that targets CD30. In more detail, the incorporation of mRNA analysis into in situ hybridization (ISH) is a powerful tool that can be used to study gene expression in tissue samples without losing their morphological architectures. The disclosure therefore provides for using any suitable approach that involves probes described herein to determine the presence, absence, proportion, ratio, amount, relative amount, or other parameters that reflect CD30 mRNA expression in biological samples that are prepared in any manner that is suitable for ISH analysis. In embodiments ISH of this disclosure is performed using cells that are

permabilized. Those skilled in the art will recognize that permeabilized cells are those cells which have been exposed to organic reagents (commonly referred to in the art as 'fixatives') which can include but are not limited to organic solvents, such as acetone, alcohol, and aldehyde containing solutions, such as formaldehyde, paraformaldehyde, and the like. Cells exposed to such reagents are commonly referred to as "fixed" and the process of treating them with such reagents is referred to as "fixing" the cells. The disclosure thus includes samples of fixed cells (including fixed tissue samples), wherein the cells comprise CD30 mRNA that is hybridized to at least one probe described herein. In embodiments, the disclosure comprises use of paraffin embedded tissue samples, wherein the samples comprise CD30 mRNA that is hybridized to at least one probe described herein. In embodiments, the disclosure comprises permeablization of cells that are fixed to a substrate, hybridization of one or more probes that are described herein, and detection of the hybridization of the probes to determine in situ CD30 mRNA splice variant expression. Thus, the probes may be detectably labeled such that the CD30 mRNA splice variants can be detected via a signal produced by a detectable label attached to the probe, or the probes may comprise a substrate to which detection agents may be added, as described further below.

[0020] In embodiments, the disclosure comprises analyzing any biological sample from an individual using ISH as described herein. In embodiments, the sample is a biological sample that comprises or is suspected of comprising abnormal cells, wherein the abnormal cells express CD30 mRNA. In embodiments, the sample comprises or is suspected of comprising cancer cells that express CD30 mRNA. In embodiments, the sample can be any suitable tissue sample, and may comprise for example, a biopsy or a portion of a biopsy. In embodiments, the tissue sample comprises a sample of a solid tumor. In embodiments, sample comprises or is suspected of comprising at least one of: any lymphoma cells, including but not limited to cells of anaplastic large cell lymphoma, classic Hodgkin's lymphoma, or Plasmablastic lymphoma. In embodiments the sample may comprise cells of embryonal carcinoma or mesothelioma, and other lymphoproliferative disorders such as cutaneous T cell lymphoproliferative disorders, a subset of diffuse large B-cell lymphoma and Epstein Bar virus derived lymphoproliferative neoplasms.

[0021] In certain approaches the invention includes determining CD30 splice variants using ISH and the probes described herein, and comparing the splice variant analysis to any suitable reference. In embodiments the reference comprises a value for CD30 mRNA splice variant expression. In embodiments the reference comprises an established normal range, a standardized curve, positive, negative, or matched controls, etc. The reference can comprise a splice variant value obtained from analysis of a non-diseased tissue from the same individual, or samples from one or more individuals who have been determined to be normal with respect to CD30+ expression. In embodiments, the reference is a CD30 value obtained from one or more individuals who responded to brentuximab vedotin, and/or individual(s) who did not respond to brentuximab vedotin.

[0022] The probes of this disclosure can comprise or consist of any nucleotide sequence described herein. In general the probes can comprise or consist of from 10-40 nucleotides. The probes may be directly labeled, or they may serve as a substrate for labeling reagents, as described further herein. In embodiments, any assay used in the disclosure can include from one to six probes. In embodiments, the probes comprise segments, wherein at least one segment comprises a polynucleotide sequence that is complimentary to CD30 mRNA, and can be complimentary to a segment of CD30 mRNA that spans an exon junction. In embodiments the probes are DNA probes. In an embodiment, the probe comprises a segment that is complimentary to an exon junction that is unique to a CD30 mRNA splice variant, and/or exon junctions that are present in a subset of splice variants. In embodiments the probes further comprise a linker segment, and a segment that is complimentary to another sequence that is used, for instance, for amplification and detectable labeling, whereby the detectable label produces a detectable signal, including but not limited to visually detectable signals that can be detected by any suitable type of microscopy used for ISH analysis. Kits comprising the probes, and reagents suitable for use in detecting hybridized probes, are included in the disclosure. The present disclosure thus provides for use of CD30 mRNA as a surrogate marker to predict patient's response to brentuximab vedotin treatment. To demonstrate feasibility of this approach, the disclosure provides for establishing a direct correlation between selected Formalin-fixed, paraffin-embedded (FFPE) samples and patient responses to brentuximab vedotin and their CD30 mRNA expression.

[0023] In a non-limiting approach, the disclosure utilizes a BaseScope™ assay as an ISH technique to enable to detection of specific exon junctions with morphological context. Using this method. The BaseScope™ assay is known in the art and reagents for its performance are commercially available, such as from Advanced Cell Diagnostics, Inc. In embodiments, probes of this disclosure are used in ISH BaseScope™ assay that is described in any of U.S. patents 7,709, 198; 8,604,182; 8,658,361; and 8,951,726, the disclosures of which are incorporated by reference. A representative description of the BaseScope™ assay, which comprises the steps of cell permeablization, hybridization, amplification, visualization, and quantification is available at acdbio.com/basescope%E2%84%A2-manual-assays, the description from which is incorporated by reference. In embodiments, probes of this disclosure comprise paired zz sequences to enhance specificity. [0024] Embodiments of this disclosure permit discrimination between the different variants of CD30 (e.g. transmembrane CD30 and cytoplasmic CD30), the translational patterns of CD30 mRNA and resulting protein molecules can also be analyzed and included in aiding with patient treatment decisions.

[0025] In embodiments, the disclosure comprises determining whether or not an individual is a candidate for brentuximab vedotin treatment (or treatment with another agent that targets CD30). In embodiments, this approach comprises performing an assay as described herein and comparing the determination of the splice variants of mRNA encoding CD30 to a reference value obtained or derived from individuals who responded to

brentuximab vedotin treatment. If the determination of the splice variants of mRNA encoding CD30 for individual are the same or similar to the reference value, it is indicative that the individual is a candidate for treatment with the brentuximab vedotin, in which case the disclosure may further comprised administering the brentuximab vedotin to the individual. But if the determination of the splice variants of mRNA encoding CD30 for individual are different from the reference value, it may be indicative that the individual is not a candidate for treatment with the brentuximab vedotin. In another embodiment, the reference value can be from an individual who did not response to brentuximab vedotin. In that case, a determination of the splice variants of mRNA encoding CD30 for individual being the same or similar to the reference value will be indicative that the individual is not a candidate for treatment with the brentuximab vedotin

[0026] Those skilled in the art will recognize that certain diagnostic threshold values for CD30 mRNA splice variant expression can be developed, given the benefit of the present disclosure, to aid in the determination of whether or not an individual is a candidate for treatment with brentuximab vedotin.

[0027] In embodiments, the disclosure comprises determining CD30 mRNA splice variant expression, and fixing the determination of the CD30 mRNA splice variant expression as disclosed herein in a tangible medium of expression, such as a compact disk, a DVD, or any other form of electronic file. Thus, tangible forms of media comprising a CD30 mRNA splice variant expression determination as set forth herein are included in the present disclosure. In embodiments, the invention includes communicating or otherwise transferring the tangible medium comprising a determination of CD30 mRNA splice variant expression to a health care provider, such as by electronically transmitting a file containing the

determination to the health care provider. [0028] In one embodiment, the disclosure includes simultaneously analyzing the expression of CD30 mRNA and protein at the single cell level in FFPE samples, and relating this analysis to patient responses to brentuximab vedotin.

[0029] In certain implementations probes used in this disclosure are pertinent to discriminating splice variants of mRNA encoding CD30 and thus, in embodiments, at least one of the probes may span an exon junction. Probes of this disclosure may comprise cDNA segments of CD30 mRNA, or complementary sequences. In embodiments, the terms "isoform" and "transcript variant" are used herein interchangeably. Transcript variant may be abbreviated herein as "tv".

[0030] In embodiments the disclosure uses a target probe set specific to CD30 isoform 1 (NM_001243.4, SEQ ID NO: 1, the probe targeting nucleotides 278-317). This is a 40nt long probe and is referred to herein as BA-Hs-TNFRSF8-tvl-ElE2). In embodiments the disclosure uses a target probe set specific to CD30 isoform 2 (targeting M_001281430.2, SEQ ID NO:2, nucleotides 278-316) which is a 39nt long probe, referred to herein as BA-Hs-T FRSF8-tv3-ElE2). In embodiments, the disclosure uses a target probe set that targets both CD30 isoforms (targeting M_001243.4, nucleotides 617-651). This is a 35nt long probe and is referred to herein as BA-Hs-TNFRSF8-E4E5. In an embodiment, to detect the presence of the CD30 signal peptide (which does not include an exon junction probe) a target probe set specific to mRNA encoding the signal peptide can be used. This target probe set will detect both CD30 Isoform 1 and 2 (targeting NM_001243.4, nts 224-272). This probe is referred to herein as BA-Hs-TNFRSF8-lzz-st). Use of some of these probes in a non-limiting demonstration is shown in Figure 2 and 3.

[0031] In an embodiment, the BA-Hs-TNFRSF8-tvl-ElE2 probe can be used to detect the mRNA sequence of CD30 transcript variant ("tv") 1; the mRNA equivalent of the cDNA sequence of tvl is under NCBI Reference Sequence NM_001243.4 and is:

1 auacgggaga acuaaggcug aaaccucgga ggaacaacca cuuuugaagu gacuucgcgg 61 cgugcguugg gugcggacua gguggccgcg gcgggagugu gcuggagccu gaaguccacg 121 cgcgcggcug agaaccgccg ggaccgcacg ugggcgccgc gcgcuucccc cgcuucccag 181 gugggcgccg gccgccaggc caccucacgu ccggccccgg ggaugcgcgu ccuccucgcc 241 gcgcugggac ugcuguuccu gggggcgcua cgagccuucc cacaggaucg acccuucgag 301 gacaccuguc auggaaaccc cagccacuac uaugacaagg cugucaggag gugcuguuac 361 cgcugcccca uggggcuguu cccgacacag cagugcccac agaggccuac ugacugcagg 421 aagcagugug agccugacua cuaccuggau gaggccgacc gcuguacagc cugcgugacu 481 uguucucgag acgaccucgu ggagaagacg ccgugugcau ggaacuccuc ccgugucugc 541 gaaugucgac ccggcauguu cuguuccacg ucugccguca acuccugugc ccgcugcuuc 601 uuccauucug ucuguccggc agggaugauu gucaaguucc caggcacggc gcagaagaac 661 acggucugug agccggcuuc cccagggguc agcccugccu gugccagccc agagaacugc 721 aaggaacccu ccaguggcac caucccccag gccaagccca ccccgguguc cccagcaacc 781 uccagugcca gcaccaugcc uguaagaggg ggcacccgcc ucgcccagga agcugcuucu 841 aaacugacga gggcucccga cucucccucc ucugugggaa ggccuaguuc agauccaggu 901 cuguccccaa cacagccaug cccagagggg ucuggugauu gcagaaagca gugugagccc 961 gacuacuacc uggacgaggc cggccgcugc acggccugcg ugagcuguuc ucgagaugac 1021 cuuguggaga agacgccaug ugcauggaac uccucccgca ccugcgaaug ucgaccuggc 1081 augaucugug ccacaucagc caccaacucc ugugcccgcu guguccccua cccaaucugu 1141 gcagcagaga cggucaccaa gccccaggau auggcugaga aggacaccac cuuugaggcg 1201 ccaccccugg ggacccagcc ggacugcaac cccaccccag agaauggcga ggcgccugcc 1261 agcaccagcc ccacucagag cuugcuggug gacucccagg ccaguaagac gcugcccauc 1321 ccaaccagcg cucccgucgc ucucuccucc acggggaagc ccguucugga ugcagggcca 1381 gugcucuucu gggugauccu gguguugguu gugguggucg gcuccagcgc cuuccuccug 1441 ugccaccgga gggccugcag gaagcgaauu cggcagaagc uccaccugug cuacccgguc 1501 cagaccuccc agcccaagcu agagcuugug gauuccagac ccaggaggag cucaacgcag 1561 cugaggagug gugcgucggu gacagaaccc gucgcggaag agcgaggguu aaugagccag 1621 ccacugaugg agaccugcca cagcgugggg gcagccuacc uggagagccu gccgcugcag 1681 gaugccagcc cggccggggg ccccucgucc cccagggacc uuccugagcc ccgggugucc 1741 acggagcaca ccaauaacaa gauugagaaa aucuacauca ugaaggcuga caccgugauc 1801 guggggaccg ugaaggcuga gcugccggag ggccggggcc uggcggggcc agcagagccc

1861 gaguuggagg aggagcugga ggcggaccau accccccacu accccgagca ggagacagaa

1921 ccgccucugg gcagcugcag cgaugucaug cucucagugg aagaggaagg gaaagaagac

1981 cccuugccca cagcugccuc uggaaaguga ggccugggcu gggcuggggc uaggagggca

2041 gcaggguggc cucugggagg ccaggauggc acuguuggca ccgagguugg gggcagaggc

2101 ccaucuggcc ugaacugagg cuccagcauc uaguggugga ccggccgguc acugcagggg

2161 ucuggugguc ucugcuugca uccccaacuu agcugucccc ugacccagag ccuaggggau

2221 ccggggcuug uacagaagag acaguccaag gggacuggau cccagcagug auguugguug

2281 aggcagcaaa cagauggcag gaugggcacu gccgagaaca gcauuggucc cagagcccug

2341 ggcaucagac cuuaaccacc aggcccacag cccagcgagg gagaggucgu gaggccagcu

2401 cccggggccc cuguaacccu acucuccucu cucccuggac cucagaggug acacccauug

2461 ggcccuuccg gcaugccccc aguuacugua aauguggccc ccagugggca uggagccagu

2521 gccugugguu guuucuccag agucaaaagg gaagucgagg gauggggcgu cgucagcugg

2581 cacugucucu gcugcagcgg ccacacugua cucugcacug gugugagggc cccugccugg

2641 acugugggac ccuccuggug cugcccaccu ucccuguccu guagcccccu cggugggccc

2701 agggccuagg gcccaggauc aagucacuca ucucagaaug uccccaccaa uccccgccac

2761 agcaggcgcc ucggguccca gaugucugca gcccucagca gcugcagacc gccccucacc

2821 aacccagaga accugcuuua cuuugcccag ggacuuccuc cccaugugaa cauggggaac

2881 uucgggcccu gccuggaguc cuugaccgcu cucugugggc cccacccacu cuguccuggg

2941 aaaugaagaa gcaucuuccu uaggucugcc cugcuugcaa auccacuagc accgacccca

3001 ccaccugguu ccggcucugc acgcuuuggg guguggaugu cgagaggcac cacggccuca

3061 cccaggcauc ugcuuuacuc uggaccauag gaaacaagac cguuuggagg uuucaucagg

3121 auuuuggguu uuucacauuu cacgcuaagg aguaguggcc cugacuuccg gucggcuggc

3181 cagcugacuc ccuagggccu ucagacgugu augcaaauga gugauggaua aggaugaguc

3241 uuggaguugc gggcagccug gagacucgug gacuuaccgc cuggaggcag gcccgggaag

3301 gcugcuguuu acucaucggg cagccacgug cucucuggag gaagugauag uuucugaaac

3361 cgcucagaug uuuuggggaa aguuggagaa gccguggccu ugcgagaggu gguuacacca

3421 gaaccuggac auuggccaga agaagcuuaa gugggcagac acuguuugcc caguguuugu

3481 gcaaggaugg aguggguguc ucugcaucac ccacagccgc agcuguaagg cacgcuggaa

3541 ggcacacgcc ugccaggcag ggcagucugg cgcccaugau gggagggauu gacauguuuc

3601 aacaaaauaa ugcacuuccu uaccuagugg cccuucacac aacuuuugaa ucucuaaaaa

3661 uccauaaaau ccuuaaagaa cuguaaaaaa aaaaaaaaaa aaaaaa (SEQ ID NO: !)

[0032] In an embodiment, the BA-Hs-TNFRSF8-tv3-ElE2 probe can be used to detect the tv3 mRNA equivalent of the cDNA sequence under NCBI Reference Sequence: NM_001281430.2); the mRNA sequence is:

1 auacgggaga acuaaggcug aaaccucgga ggaacaacca cuuuugaagu gacuucgcgg 61 cgugcguugg gugcggacua gguggccgcg gcgggagugu gcuggagccu gaaguccacg 121 cgcgcggcug agaaccgccg ggaccgcacg ugggcgccgc gcgcuucccc cgcuucccag 181 gugggcgccg gccgccaggc caccucacgu ccggccccgg ggaugcgcgu ccuccucgcc 241 gcgcugggac ugcuguuccu gggggcgcua cgagccuucc cacagggcug uucccgacac 301 agcagugccc acagaggccu acugacugca ggaagcagug ugagccugac uacuaccugg 361 augaggccga ccgcuguaca gccugcguga cuuguucucg agacgaccuc guggagaaga 421 cgccgugugc auggaacucc ucccgugucu gcgaaugucg acccggcaug uucuguucca 481 cgucugccgu caacuccugu gcccgcugcu ucuuccauuc ugucuguccg gcagggauga 541 uugucaaguu cccaggcacg gcgcagaaga acacggucug ugagccggcu uccccagggg

601 ucagcccugc cugugccagc ccagagaacu gcaaggaacc cuccaguggc accauccccc

661 aggccaagcc caccccggug uccccagcaa ccuccagugc cagcaccaug ccuguaagag

721 ggggcacccg ccucgcccag gaagcugcuu cuaaacugac gagggcuccc gacucucccu

781 ccucuguggg aaggccuagu ucagauccag gucugucccc aacacagcca ugcccagagg

841 ggucugguga uugcagaaag cagugugagc ccgacuacua ccuggacgag gccggccgcu

901 gcacggccug cgugagcugu ucucgagaug accuugugga gaagacgcca ugugcaugga

961 acuccucccg caccugcgaa ugucgaccug gcaugaucug ugccacauca gccaccaacu

1021 ccugugcccg cugugucccc uacccaaucu gugcagcaga gacggucacc aagccccagg

1081 auauggcuga gaaggacacc accuuugagg cgccaccccu ggggacccag ccggacugca

1141 accccacccc agagaauggc gaggcgccug ccagcaccag ccccacucag agcuugcugg

1201 uggacuccca ggccaguaag acgcugccca ucccaaccag cgcucccguc gcucucuccu

1261 ccacggggaa gcccguucug gaugcagggc cagugcucuu cugggugauc cugguguugg

1321 uugugguggu cggcuccagc gccuuccucc ugugccaccg gagggccugc aggaagcgaa

1381 uucggcagaa gcuccaccug ugcuacccgg uccagaccuc ccagcccaag cuagagcuug

1441 uggauuccag acccaggagg agcucaacgc ugaggagugg ugcgucggug acagaacccg

1501 ucgcggaaga gcgaggguua augagccagc cacugaugga gaccugccac agcguggggg

1561 cagccuaccu ggagagccug ccgcugcagg augccagccc ggccgggggc cccucguccc

1621 ccagggaccu uccugagccc cgggugucca cggagcacac caauaacaag auugagaaaa

1681 ucuacaucau gaaggcugac accgugaucg uggggaccgu gaaggcugag cugccggagg

1741 gccggggccu ggcggggcca gcagagcccg aguuggagga ggagcuggag gcggaccaua

1801 ccccccacua ccccgagcag gagacagaac cgccucuggg cagcugcagc gaugucaugc

1861 ucucagugga agaggaaggg aaagaagacc ccuugcccac agcugccucu ggaaagugag

1921 gccugggcug ggcuggggcu aggagggcag caggguggcc ucugggaggc caggauggca

1981 cuguuggcac cgagguuggg ggcagaggcc caucuggccu gaacugaggc uccagcaucu

2041 agugguggac cggccgguca cugcaggggu cugguggucu cugcuugcau ccccaacuua

2101 gcuguccccu gacccagagc cuaggggauc cggggcuugu acagaagaga caguccaagg

2161 ggacuggauc ccagcaguga uguugguuga ggcagcaaac agauggcagg augggcacug

2221 ccgagaacag cauugguccc agagcccugg gcaucagacc uuaaccacca ggcccacagc

2281 ccagcgaggg agaggucgug aggccagcuc ccggggcccc uguaacccua cucuccucuc

2341 ucccuggacc ucagagguga cacccauugg gcccuuccgg caugccccca guuacuguaa

2401 auguggcccc cagugggcau ggagccagug ccugugguug uuucuccaga gucaaaaggg

2461 aagucgaggg auggggcguc gucagcuggc acugucucug cugcagcggc cacacuguac

2521 ucugcacugg ugugagggcc ccugccugga cugugggacc cuccuggugc ugcccaccuu

2581 cccuguccug uagcccccuc ggugggccca gggccuaggg cccaggauca agucacucau

2641 cucagaaugu ccccaccaau ccccgccaca gcaggcgccu cgggucccag augucugcag

2701 cccucagcag cugcagaccg ccccucacca acccagagaa ccugcuuuac uuugcccagg

2761 gacuuccucc ccaugugaac auggggaacu ucgggcccug ccuggagucc uugaccgcuc

2821 ucugugggcc ccacccacuc uguccuggga aaugaagaag caucuuccuu aggucugccc

2881 ugcuugcaaa uccacuagca ccgaccccac caccugguuc cggcucugca cgcuuugggg

2941 uguggauguc gagaggcacc acggccucac ccaggcaucu gcuuuacucu ggaccauagg

3001 aaacaagacc guuuggaggu uucaucagga uuuuggguuu uucacauuuc acgcuaagga

3061 guaguggccc ugacuuccgg ucggcuggcc agcugacucc cuagggccuu cagacgugua

3121 ugcaaaugag ugauggauaa ggaugagucu uggaguugcg ggcagccugg agacucgugg 3181 acuuaccgcc uggaggcagg cccgggaagg cugcuguuua cucaucgggc agccacgugc

3241 ucucuggagg aagugauagu uucugaaacc gcucagaugu uuuggggaaa guuggagaag

3301 ccguggccuu gcgagaggug guuacaccag aaccuggaca uuggccagaa gaagcuuaag

3361 ugggcagaca cuguuugccc aguguuugug caaggaugga gugggugucu cugcaucacc

3421 cacagccgca gcuguaaggc acgcuggaag gcacacgccu gccaggcagg gcagucuggc

3481 gcccaugaug ggagggauug acauguuuca acaaaauaau gcacuuccuu accuaguggc

3541 ccuucacaca acuuuugaau cucuaaaaau ccauaaaauc cuuaaagaac uguaaaaaaa

3601 aaaaaaaaaa aaaaaa (SEQ ID NO:2) [0033] In an embodiment, the BA-Hs-TNFRSF8-E4E5 probe can detect a common exon junction shared by tvl and tv3; it detects the mRNA given above for NM_001243.4 and is specific for nucleotides 617-651 of the mRNA sequence.

[0034] In an embodiment, the BA-Hs-TNFRSF8-lzz-st is specific for nts 224-272 of the mRNA sequence given above for NM_001243.4, and is specific for a transcript that encodes the signal peptide.

[0035] In an embodiment, this disclosure provides a method of detecting CD30 mRNA expression in cells using the probes provided herein. In an embodiment, this disclosure provides a method of deteting CD30 mRNA expression in a biological sample (which may be fixed) comprising cells (such as tissue section, smears comrpising cells, cell suspensions and the like) comprising hybridizing one or more probes, wherein the one or more probes comprises at least one probe that is specific to CD30 mRNA isoform 1, CD30 mRNA isoform 2, both CD30 isoform 1 and isoform 2, or the mRNA encoding CD30 signal peptide, and and detecting hybridization of the probes to determine CD30 expression in the sample. The one or more probes may be selected from BA-Hs-TNFRSF8-tvl-ElE2 probe, the BA-Hs-TNFRSF8-tv3-ElE2 probe, the BA-Hs-TNFRSF8-E4E5 probe, or the BA-Hs- TNFRSF8-lzz-st probe. In an embodiment, the method comprises determining expression of CD30 mRNA portions that represent the transmembrane CD30 and/or cytoplasmic CD30 in portions. The relative levels of the different mRNA portions can be compared to levels from reference controls (such as those that positively responded to a therapy, or did not respond to the therapy). Based on these comparisons, decisions regarding treatment with a particular treatment approach (such as brentuximab vedotin) can be made. For example, once an individual is identified as being suitable for treatment with brentuximab vedotin,

administration of brentuximab vedotin can be initiated. [0036] In an aspect, this disclosure provides a method of treating lymphoma comprising determining in a biological sample obtained from an individual who has been diagnosed with or is suspected of having lymphoma, expression of CD30 mRNA (total or relative levels of different isoforms, such as isoform 1 and 2) by in situ hybridization, and based on a determination of the presence CD30 mRNA compared to a reference level, administering brentuximab vedotin to the indivdiual.

[0037] In as aspect, this disclosure provides kit comprising one or more probes, wherein the one or more probes comprise the BA-Hs-TNFRSF8-tvl-ElE2 probe, the BA-Hs- TNFRSF8-tv3-ElE2 probe, the BA-Hs-TNFRSF8-E4E5 probe, or the BA-Hs-TNFRSF8- lzz-st, and optionally reagents for use in in situ hybridzation. In one embodiemt, this disclosure provides a kit comprising at least BA-Hs-TNFRSF8-tvl-ElE2 probe, and the BA- Hs-TNFRSF8-tv3-ElE2 probe, and optionally comprising the BA-Hs-TNFRSF8-E4E5 probe, or the BA-Hs-TNFRSF8-lzz-st, and also optionally comprising reagents for use in in situ hybridization, and also optionally comprising information regarding relative levels of CD30 mRNA isforms 1 and 2 from responders and non-responders of lymphoma patients treated with brentuximab vedotin, and optionally instructions for use of the kit and interpretation of results.

[0038] In one aspect, this disclosure provides a substrate in contact with permebalized cells, wherein the cells comprise mRNA/DNA probe duplexes, wherein the DNA probe component of the duplex is at least one probe selected from the group consising of the BA- Hs-TNFRSF8-tvl-ElE2 probe, the BA-Hs-TNFRSF8-tv3-ElE2 probe, the BA-Hs- TNFRSF8-E4E5 probe, or the BA-Hs-TNFRSF8-lzz-st probe.

[0039] The invention is further demonstrated by way of the Figures and data presented herein. EXAMPLE 1

[0040] In order to demonstrate the feasibility of the method, SKMEL7 and U937 cell lines were used as test run specimens to detect the range of mRNA expression for 4-hydroxy- tetrahydrodipicolinate reductase (DapB) and Peptidyl-prolyl cis-trans isomerase B (PPIB). Subsequently, formalin fixed paraffin embedded tissue sections from classical Hodgkin's Lymphoma (HL) (two cases) were tested.

[0041] A representative flowchart of non-limiting steps by which the present invention can be carried out is provided in Figure 1. Results are presented in Figures 2 and 3. Figure 4 demonstrates a comparison of Classical Reed Sternberg cell in HL, stained with hematoxylin and eosin (left panel) with classical Reed Sternberg cells in HL showing CD30 expression by immunohistochemistry (right panel).

[0042] The particular probes used for CD30 mRNA FFPE tissue sections are summarized in the table of Figure 3. In situ hybridization images are shown for two patient samples with Hodgkin's lymphoma (HL). Hybridization is viewed as discrete dots for probes specific for SP, TMP, CP and TMP+CP regions of CD30. Positive control (PPIB) and negative control (DapB) are also shown. The data demonstrate that using the probes described herein can detect a range of distribution of CD30 mRNA expression in FFPE classical HL tissue sections.

EXAMPLE 2

[0043] In this example, we identified detection of specific CD30 mRNA isoforms that correspond the different CD30 protein segments within the neoplastic cells and tumor microenvironment immune cells. Using this method, the CD30 mRNA expression patterns in classical Hodgkin lymphoma (CHL) and anaplastic large cell lymphoma (ALCL) were evaluated.

[0044] Nineteen CHL and 11 ALCL cases were selected from the Roswell Park

Comprehensive Cancer Institute department of pathology archives. CD30 expression by immunohistochemistry (IHC) and CD30 mRNA isoform expression by BaseScope™ assay were measured on formalin fixed paraffin embedded ( FFPE ) tissue specimens. Four probe sets spanning the exon junctions for increased specificity were used to detect domains on the protein corresponding to signal peptide (SP), transmembrane portion (TMP), cytoplasmic portion (CP), and transmembrane and cytoplasmic portion (TMP &CP) of full length CD30 protein (Figure 3). The hybridization, amplification, and staining of probes are based on the manufacturer's protocol. The BaseScope™ assay probe signal is detected in the form of red dots, which are localized to the neoplastic cells and tumor microenvironment immune cells. Results are shown in Figure 5. Semi-quantitative analysis of both positive and negative signals using 100X magnification was performed on 100 neoplastic cells for each probe for each case, by two hematopathologists. For tumor microenvironment immune cells, an average number of positive and negative cells were obtained in five 100X magnification fields. [0045] In both CHL and ALCL, 100% of the neoplastic cells were positive for CD30 protein by immunohistochemistry. Table 1 illustrates relative expression of CD30 mRNA isoforms in CHL and ALCL neoplastic cells. Expression of CD30 mRNA in CHL the tumor microenvironment cells is also tabulated. [0046] Detection of CD30 mRNA isoforms using in situ hybridization as described herein offers a reliable and reproducible signal detection platform, as an alternative to CD30 protein expression by immunohistochemistry. The present assay provides a high-contrast low noise signal for easy visualization, accurate quantification, and precise positive signal localization. Different CD30 isoform patterns and signal frequency were observed within the neoplastic cells and immune cells in the tumor microenvironment. The CD30 mRNA isoform corresponding to transmembrane domain was a dominant expresser in both CHL and ALCL neoplastic cells and bystander cells in CHL. Thus this mRNA- based method provides a more sensitive method for antigen expression detection. Results are provided in Table 1 below. Table 1 : Relative expression of CD30 mRNA isoforms in CHL and ALCL

average negat ve average negat ve

[0047] Data shows that percent of cells which had 5 or more dots was higher for TMP or TMP + CP probes than for CP alone or SP probes for both ALCL and HL cells. [0048] It will be apparent from the foregoing description and the figures that this disclosure provides a demonstration of determining a range of CD30 mRNA expression in neoplastic lymphocytes, bystander non-neoplastic cells, and comparison of the same to the range of protein expression as detected by immunohistochemistry. It reveals the utility by way of a non-limiting implementation (the BaseScope™ assay), to detect the range of CD30 mRNA expression in routine formalin fixed paraffin embedded (FFPE) lymphoma tissue specimens.

[0049] While the invention has been described through illustrative examples, routine modifications will be apparent to those skilled in the art, which modifications are intended to be within the scope of the invention.

Claims

What is claimed is:

1. A method of deteting CD30 mRNA expression in a fixed biological sample comprising cells comprising hybridizing one or more probes, wherein the one or more probes comprises at least one probe that is specific to CD30 mRNA isoform 1, CD30 mRNA isoform 2, both CD30 isoform 1 and isoform 2, or the mRNA encoding CD30 signal peptide, and and detecting hybridization of the probes to determine CD30 expression in the sample.

2. The method of claim 1, wherin the one or more probes comprise the BA-Hs- TNFRSF8-tvl-ElE2 probe, the BA-Hs-TNFRSF8-tv3-ElE2 probe, the BA-Hs-TNFRSF8- E4E5 probe, or the BA-Hs-TNFRSF8-lzz-st probe.

3. The method of claim 1, wherein the detecting the hybridization of the probes is performed using an assay that is marked under the trademane BaseScope.

4. The method of claim 1, further comprising determining expression of transmembrane CD30 and/or cytoplasmic CD30 in the sample based upon CD30 mRNA isoform expression.

5. The method of any one of claims 1-4, further comprising determiming whether or not the individual from whom the sample was obtained is a candidate to be treated with brentuximab vedotin.

6. The method of claim 5, identifying the individual to be a candidate to be treated with brentuximab vedotin, and optionally administering the brentuximab vedotin to the indivdiual.

7. A method of treating lymphoma comprising determining in a biological sample obtained from an individual who has been diagnosed with or is suspected of having lymphoma, expression of CD30 mRNA by in situ hybridization, and based on a

determination of the presence CD30 mRNA compared to a reference level, administering brentuximab vedotin to the indivdiual.

8. The method of claim 7, wherein determination of CD30 also comprises determination of relative levels of isoform 1 and isoform 2.

9. A kit comprising one or more probes, wherein the one or more probes comprise the BA-Hs-T FRSF8-tvl-ElE2 probe, the BA-Hs-T FRSF8-tv3-ElE2 probe, the BA-Hs-

T FRSF8-E4E5 probe, or the BA-Hs-TNFRSF8-lzz-st, and optionally reagents for use in in situ hybridzation.

10. A substrate in contact with permebalized cells, wherein the cells comprise

mRNA/DNA probe duplexes, wherein the DNA probe component of the duplex is at least one probe selected from the group consising of the BA-Hs-TNFRSF8-tvl-ElE2 probe, the BA- Hs-TNFRSF8-tv3-ElE2 probe, the BA-Hs-TNFRSF8-E4E5 probe, or the BA-Hs-TNFRSF8- lzz-st probe.