A METHOD FOR THE DIAGNOSIS AND TREATMENT OF CANCER AND AGENTS USEFUL THEREFOR
FIELD OF THE INVENTION
The present invention relates generally to a method for the diagnosis and treatment of cancer including cancer of pharyngeal tissue and in particular nasopharyngeal and related and/or surrounding tissue as well as cancer of cervical and related tissue. More particularly, the present invention provides a method for diagnosing nasopharyngeal cancer (NPC) and cervical cancer including distinguishing between different forms of cancers and to a method for the prophylaxis and treatment of cancer in a mammal and in particular a human. The present invention further provides agents useful in the prophylaxis and treatment of cancer in mammals and in particular humans.
BACKGROUND OF THE INVENTION
Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description. Nucleotide and amino acid sequences are referred to by a sequence identifier, i.e. <400>1, <400>2, etc. A sequence listing is provided after the claims.
The designation of nucleotide residues referred to herein are those recommended by the IUPAC- IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.
The increasing sophistication of recombinant DNA technology is greatly facilitating research and development in the medical and allied health fields. This is particularly the case in the field
of cancer research. Cancer is a major cause of mortality and morbidity in mammals and in particular humans. On purely economic terms, cancer is a major contributor to health costs in all communities. The genetic tools of recombinant DNA technology provide the means to investigate the molecular bases for cancer development and progression. Once these have been elucidated, a more rational approach can be adopted to design anti-cancer agents.
One particularly import cancer is nasopharyngeal cancer (NPC) which arises in the surface epithelium of the posterior nasopharynx. The World Health Organization (WHO) classifies NPC into three categories according to the degree of differentiation.
Type 1 NPC comprises squamous cell carcinomas which are highly differentiated with characteristic epithelial growth patterns and intra- and extra-cellular keratin filaments. Type II NPC comprises non-keratinizing carcinomas which retain epithelial cell shape and growth patterns. Type III NPC comprises undifferentiated carcinomas which produce no keratin and have no distinctive growth pattern. NPC is one of the most common malignancies in regions of south-eastern China, south-eastern Asia, Taiwan, eastern and northern parts of Africa and Alaska.
Viruses have been implicated in cell transformation and cancer development.
Epstein-Barr virus (EBV) has been demonstrated to be closely associated with Type II and III NPC. Type II and III NPC patients have elevated IgA and IgG antibody levels to the EBV viral capsid antigen (VCA) as well as the diffuse component of the early antigen. In contrast, patients with Type I highly-differentiated carcinomas have similar EBV serologic prolifes as that of the control populations and do not appear to have a special association with EBV infection. However, molecular studies have shown that EBV DNA exists in all three types of NPC. Northern blot analysis has also demonstrated the presence of EBV RNA in tissue biopsies from NPC patients. Nevertheless, direct evidence to show that EBV is the etiological agent for NPC has been difficult to obtain and such etiology has yet to be established. Consequently, detection and treatment protocols based on EBV are unlikely to be effective.
Genetic alterations detected through karyotyping studies have also been implicated in the development of NPC. However, this approach has also not resulted in clinically useful detection or treatment strategies.
In work leading up to the present invention, the inventors employed differential display and cloning techniques to isolate genes which might play a role in the tumor promotion and/or progression. In accordance with the present invention, the inventors have identified an association between altered gene expression and different forms of NPC. In addition, a similar pattern of expression is noted for other forms of cancer such as but not limited to cervical cancer. The recognition of this genetic phenomenon provides a means for the rational design of anti-cancer agents and diagnostic strategies.
SUMMARY OF THE INVENTION
Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
One aspect of the present invention is directed to an isolated nucleic acid molecule comprising a sequence of nucleotides which is expressed post-natally in poorly differentiated tumor cells but is substantially not expressed post-natally in either normal tissue corresponding to said tumor cells or in highly differentiated forms of said tumor cells.
Another aspect of the present invention is directed to an isolated nucleic acid molecule comprising a sequence of nucleotides which is substantially expressed in tumor cells when the nucleotide sequences are subjected to a hypomethylation.
A further aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides which is expressed post-natally in poorly differentiated NPC cells but is substantially not expressed post-natally in normal epithelial cells or in highly differentiated NPC cells.
Yet a further aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides which is expressed in cervical cancer cells when the nucleotide sequences are subject to hypomethylation.
Yet another aspect of the present invention is directed to an isolated nucleic acid molecule comprising a nucleotide sequence substantially as set forth in <400>1 or a nucleotide sequence having at least about 50% similarity to at least 30 contiguous nucleotides of <400>1 or a nucleotide sequence capable of hybridizing to <400>1 under low stringency conditions wherein said nucleic acid molecule is expressed post-natally in poorly differentiated NPC but is substantially not expressed post-natally in either normal epithelial cells or in highly
differentiated NPC cells.
Still another aspect of the present invention, provides an agent such as and including a nucleotide sequence capable of modulating the state of methylation of the nucleotide sequence set forth in <400>1 or a promoter region operably linked to the nucleotide sequence set forth in <400>1 or a regulatory sequence required for expression of the nucleotide sequence set forth in <400>l .
Even yet another to this aspect of the present invention is directed to a nucleic acid molecule comprising at least about 10 nucleotides in length wherein said nucleic acid molecule is capable of interacting with a corresponding portion of a mRNA molecule transcribed from a genetic sequence defined by the nucleotide sequence set forth in <400>1 and wherein said interaction substantially reduces the level of single stranded mRNA transcribed from <400>1.
Another aspect of the present invention provides a nucleic acid molecule corresponding to all or a part of the nucleotide sequence as set forth in <400>1 wherein said nucleic acid molecule is capable of inducing co-suppression of the nucleotide sequence set forth in <400>1.
Still another aspect of the present invention contemplates a method of detecting poorly differentiated NPC in a mammal such as a human, said method comprising obtaining a cancer tissue biopsy and screening the cells of said cancer tissue for the presence of mRNA transcript from <400>1 wherein the presence of such mRNA is indicative of a poorly differentiated NPC.
Yet another aspect of the present invention provides a composition for the treatment or prophylaxis of undifferentiated NPC in a mammal such as a human, said composition comprising an effective amount of an agent which:-
(i) promotes modulation of methylation of D-l l or its promoter or its 3' or 5' flanking regions to thereby modulate D-l 1 expression;
(ii) acts as an antisense molecule to all or part of the D-l 1 mRNA transcript to thereby
down-regulate D-l 1 expression;
and one or more pharmaceutically acceptable carriers and/or diluents.
The nucleotide sequence set forth in <400>1 is referred to herein as "D-l l". The D-l 1 gene is deemed herein to be the same as the H-19 gene. Reference herein to the nucleotide sequence defined by <400>1 or D-l 1 includes reference to derivatives, homologues and analogues of this sequence.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a photographic representation of Northern blot analysis of CNE-1, CNE-2, HK-1 and HT-3 cells using cDNA clone D-l l .
Figure 2 is a diagrammatic representation of a genetic map showing the relationship between clone D-l 1 and its sub-clones and the H-19 gene.
Figure 3 is a representation of the nucleotide sequence of the H-19 gene with the nucleotide sequence of the D-l l clone within the H-19 sequence underlined.
Figure 4 is a photographic representation of Northern blot analysis of various cells using D-l 1 nucleotide sequence. D-l l is highly expressed in CNE-2 cells.
Figure 5 is a photographic representation of Northern blot analysis of (A) human adult tissue and (b) fetal tissue using the D-l l clone.
Figure 6 is a photographic representation of in situ hybridization showing D-l l expression in two poorly differentiated NPC biopsies (NPC-1 and NPC-2).
Figure 7 is a photographic representation of β-actin in situ hybridization showing its presence in normal tissue and two NPC biopsies, NPC-1 and NPC-2. This represents a positive control to the in situ hybridization shown in Figure 6.
Figure 8 is a photographic representation showing a Northern blot of cancer cell lines with and without treatment of 5-azacytosine to induce hypomethylation.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is predicated in part on the identification of a genetic sequence involved in carcinogenesis such as but not limited to NPC and cervical carcinogenesis. The identification of this genetic sequence was conveniently accomplished using differential cDNA screening. The inventors sought to identify genes which are differentially expressed in poorly differentiated NPC cells relative to either normal epithelial cells or highly differentiated NPC cells. It should be noted that 90% of clinically detected NPC is of the poorly differentiated type. The inventors made the surprising discovery that a differentially expressed clone, designated herein D-l 1, is identical to the gene H-19. The H-19 gene is highly expressed in embryonic tissue of endodermal and mesodermal origin but is dramatically down regulated after birth. Clone D-l l exhibits the same expression pattern as H-19, however, is expressed in poorly differentiated NPC but not in either normal epithelial tissue or highly differentiated NPC tissue. Although the present invention is particularly directed to the detection of H-19 in relation to H-19, the present invention extends to the gene in other cancers such as cervical cancer.
Accordingly, one aspect of the present invention is directed to an isolated nucleic acid molecule comprising a sequence of nucleotides which is expressed post-natally in poorly differentiated rumor cells but is substantially not expressed post-natally in either normal tissue corresponding to said tumor cells or in highly differentiated forms of said tumor cells.
For the purposes of exemplifying the present invention, the tumor cells correspond to NPC. This is done, however, with the understanding that the present invention extends to any cancer cells and in particular cancers of pharyngeal tissue such as the nasopharynx region as well as related cancers and/or cancers in surrounding tissue which possess genetic sequences differentially expressed in poorly differentiated cancer tissue relative to normal tissue or tumor cells which are highly differentiated. Furthermore, the expression of the nucleotide sequence shown to be prevented by methylation is, for example, differentiated NPC cell lines and in cervical cancer cell lines.
Accordingly, another aspect of the present invention is directed to an isolated nucleic acid
molecule comprising a sequence of nucleotides which is substantially expressed in tumor cells when the nucleotide sequences are subjected to a hypomethylation.
A further aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides which is expressed post-natally in poorly differentiated NPC cells but is substantially not expressed post-natally in normal epithelial cells or in highly differentiated NPC cells.
Yet a further aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides which is expressed in cervical cancer cells when the nucleotide sequences are subject to hypomethylation.
The tumor cells and in particular the NPC cells are generally of human origin. However, the present invention extends to cells and in particular NPC-like cells from other mammals such as primates, livestock animals (e.g. sheep, pigs, cows, horses, donkeys), laboratory test animals (e.g. mice, rats, rabbits, guinea pigs, hamsters) companion animals (e.g. dogs, cats) and captive wild animals.
The term "expressed" includes and encompasses the transcription of the D-l l genetic material to mRNA, referred to herein as D-l l transcript. Expression does not necessarily require the translation of the mRNA into an amino acid sequence. The preferred differentially expressed D-l l genetic sequence of the present invention acts at the RNA level and is not translated into a polypeptide. Reference herein to a genetic sequence differentially expressed in one tissue relative to another tissue means that it is at least transcribed into mRNA in the first mentioned tissue but is substantially not transcribed in the second mentioned tissue.
The preferred nucleic acid molecule of the present invention is referred to herein as D-l 1. The genetic sequence D-l l is also referred to as a "gene" and is sometimes referred to as "H-19". A "gene" may be a genomic sequence with exons and introns or may correspond to a cDNA coding sequence. The nucleotide sequence of D-l 1 is shown in Figure 3 and in <400>1. The present invention extends to derivatives, homologues and analogues of D-l l as well as
nucleotide sequences having at least about 50% similarity to at least 30 contiguous nucleotides of D-l l, and preferably at least about 50% similarity to entire sequence of D-l l, as well as nucleotide sequences capable of hybridizing to <400>1 under low stringency conditions. Reference herein to D-l 1, H-19 or <400>1 includes any and all derivatives, homologues and analogues of this genetic sequence. A "derivative" includes mutants, parts and fragments of the nucleotide sequence as well as single and multiple nucleotide substitutions, additions and/or deletions to <400>1.
The term "similarity" as used herein includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, "similarity" includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, "similarity" includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide and sequence comparisons are made at the level of identity rather than similarity.
Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence similarity", "sequence identity", "percentage of sequence similarity", "percentage of sequence identity", "substantially similar" and "substantial identity". A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e. only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or
deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et α/.(1). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al.i2).
The terms "sequence similarity" and "sequence identity" as used herein refers to the extent that sequences are identical or functionally or structurally similar on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity", for example, is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) or the identical amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence identity" will be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity.
Reference herein to a low stringency includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions. Generally, low stringency is at from about 25-30°C to about 42°C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions. Alternative stringency conditions may be applied where necessary, such as medium
stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31%o v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions. In general, washing is carried out Tm = 69.3 + 0.41 (G+C)% (Marmur and Doty(3)). However, the n of a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatch base pairs (Bonner and Laskey(4)). Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6 x SSC buffer, 0.1% w/v SDS at 25-42°C; a moderate stringency is 2 x SSC buffer, 0.1% w/v SDS at a temperature in the range 20°C to 65°C; high stringency is 0.1 x SSC buffer, 0.1% w/v SDS at a temperature of at least 65°C.
The D-l 1 nucleic acid molecule of the present invention may be single or double stranded DNA, RNA, mRNA or a RNA/DNA hybrid.
Another aspect of the present invention is directed to an isolated nucleic acid molecule comprising a nucleotide sequence substantially as set forth in <400>1 or a nucleotide sequence having at least 50% similarity to at least about 30 contiguous nucleotides of <400>1 or a nucleotide sequence capable of hybridizing to <400>1 under low stringency conditions wherein said nucleic acid molecule is expressed post-natally in poorly differentiated NPC but is substantially not expressed post-natally in either normal epithelial cells or in highly differentiated NPC cells.
Preferably, the level of sequence similarity to <400>1 is at least about 60%>, more preferably at least about 70%, still more preferably at least about 80%, even still more preferably at least about 90% or greater such as about 95-97% or 96-99% or 98-100%.
The D-l 1 nucleic acid molecule of the present invention may be present as naked DNA or in RNA form or present in a genetic construct comprising a vector such as an expression vector.
The nucleic acid molecule of the present invention may also be provided in a carrier agent such as in a virus or integrated into an animal, plant, bacterial or insect chromosome or in an artificial chromosome such as a YAC, BAC, PAC or HAC.
Accordingly, the present invention clearly encompasses genetic constructs comprising the subject D-l 1 nucleic acid molecule or its complementary form in an expressible format suitable for the preparation of mRNA or antisense mRNA. The production of antisense mRNA is useful in a genetic vaccine.
For expression constructs, the D-l l nucleic acid molecule or its complementary form is operably connected to a promoter sequence which thereby regulates expression of the nucleic acid molecule in a suitable prokaryotic or eukaryotic cell. As discussed below, if the subject D-l l nucleic acid molecule is methylated, it will be necessary to demethylate the nucleotides in order for transcription to occur. This may be done chemically or, more conveniently, the nucleic acid molecule can be cloned into a microorganism incapable of methylation. The resulting clone, when isolated, will not be methylated.
The present invention extends to the H-19 promoter (i.e. a homologous promoter) or another promoter (i.e. a heterologous promoter).
The genetic construct may optionally further comprises a terminator sequence. The term "terminator" refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. A "terminator" is a nucleotide sequence, generally located within the 3 '-non-translated region of a gene or mRNA, comprising a polyadenylation signal to facilitate the post-transcriptional addition of a polyadenylate sequence to the 3'-end of a primary mRNA transcript. Terminator sequences or heterologous promoter sequences may be isolated from the genetic sequences of bacteria, fungi, viruses, animals and/or plants. Terminators and promoters active in animal cells are known and described in the literature.
In a preferred embodiment, the genetic construct is a cloning or expression vector, such as a plasmid, cosmid or phage, comprising the D-l 1 nucleic acid molecule of the present invention
or its complementary form. The present invention also extends to host cells transformed or transfected with such a construct or with naked D-l l DNA or RNA.
The genetic constructs of the present invention are particularly useful for producing recombinant D-l l nucleic acid molecules or derivatives thereof or complementary forms thereof for use in a therapeutic composition or as diagnostic agents as herein described.
The present invention further provides the antisense molecules directed to the nucleotide sequence set forth in <400>1 or its corresponding mRNA transcript.
According to this aspect of the present invention, there is provided a nucleic acid molecule comprising at least about 10 nucleotides in length wherein said nucleic acid molecules is capable of interacting with a corresponding portion of a mRNA molecule transcribed from a genetic sequence defined by the nucleotide sequence set forth in <400>1 and wherein said interaction substantially reduces the level of single stranded mRNA transcribed from <400> 1.
The nucleic acid molecule comprising at least about 10 nucleotides is considered to be an antisense molecule. The length of the antisense molecule may vary from about 10 nucleotides to substantially corresponding to the entire mRNA transcript of the D-l l genetic sequence. Particularly useful antisense molecules are at least about 13 nucleotides, at least about 18 nucleotides and at least about 20-40 nucleotides in length.
The term "interacting" as applied to the antisense nucleic acid molecule includes and encompasses forming a duplex with D-l l mRNA or otherwise associating with D-l l mRNA in a manner sufficient to reduce the levels of single stranded D-l 1 mRNA. This term may have an effect on differentiation of the NPC or related cancer or on progression and development of NPC or related cancers.
Substantially reducing levels of the D-l 1 mRNA sequence includes and encompasses reducing the levels of single stranded D-l 1 transcript. Generally, at least a 30% reduction is observed, more preferably at least about a 40% reduction and even more preferably at least about a 50%
or greater reduction in D-l 1 mRNA transcript.
The present invention further extends to sense molecules corresponding to all or a portion of D- 11 as defined in <400>1. Sense molecules are useful in co-suppression to reduce expression of a target sequence. Generally, a suitable nucleic acid molecules useful in co-suppression of D- 11 expression corresponds to the full length D-l l gene or is an internal genetic fragment or corresponds to a 3'-terminal or 5'-terminal portion of the D-l 1 genetic sequence.
According to this aspect of the present invention, there is provided a nucleic acid molecule corresponding to all or a part of the nucleotide sequence as set forth in <400>1 wherein said nucleic acid molecule is capable of inducing co-suppression of the D-l l gene.
The present invention still further extends to ribozyme constructs capable of acting on D- 11 mRNA transcript.
Although not intending to limit the present invention to any one theory or mode of action, it is proposed that in the normal course of events, the D-l l genetic sequence is subjected to methylation at birth resulting in substantial down-regulation of this gene. It is further proposed, in accordance with the present invention, that in undifferentiated NPC or related cancers, the D-l l sequence is transcriptionally re-activated resulting in expression of this sequence. The gene still remains substantially silent in highly differentiated forms of the cancer and in normal epithelial cells. Another example where this occurs is in cervical cancer.
Methylation may affect the entire mRNA-coding sequence of D-l l or may only affect the promoter and/or 5' or 3' flanking regions of the D-l 1 nucleotide sequence.
At the initiation of development of undifferentiated NPC, the D-l 1 sequence and/or its promoter or 5' or 3' flanking regions are subjected to demethylation. This may be induced by a foreign agent such as an environmental pollutant, carcinogen or by a virus or result from transcriptional activation by a genetic alteration in the chromosome. The present invention extends to naturally occurring and synthetic agents capable of inducing methylation or demethylation of D-l 1 or its
flanking sequences as well as genetic regions of the epithelial cell chromosome which are capable of inducing methylation or demethylation of D-l 1 and/or its flanking sequences.
According to this aspect of the present invention, there is provided an agent including a nucleotide sequence, capable of modulating the state of methylation of the nucleotide sequence set forth in <400>1 or a promoter region operably linked to the nucleotide sequence set forth in <400>1 or a regulatory sequence required for expression of the nucleotide sequence set forth in <400>l.
The differentially expressed D- 11 nucleic acid molecule or its transcript of the present invention is useful in the detection of NPC or related cancers and in distinguishing poorly differentiated NPC from highly differentiated NPC. The nucleic acid molecule also represents the coding sequence for an oncofetal mRNA transcript which provides a means of targeting fetal cells or post-natal cancer cells.
Accordingly, another aspect of the present invention contemplates a method of detecting poorly differentiated NPC in a mammal such as a human, said method comprising obtaining a cancer tissue biopsy and screening the cells of said cancer tissue for the present of D-l l mRNA transcript wherein the presence of such mRNA is indicative of a poorly differentiated NPC.
This aspect of the present invention may be performed in any number of ways such as contacting the cancer cells or their extracts with a labelled nucleic acid probe capable of interacting with D-l l mRNA transcript and then screening for the label. Alternatively, or in addition to, the nucleic acid from the cancer cells may be subjected to polymerase chain reactions to amplify potential target nucleotide sequences or to insert a label into the amplified target sequences.
As used herein, the term "probe" refers to a nucleic acid molecule which is generally but not necessarily derived from the nucleotide sequence set forth in <400>1 and which is capable of being used in the detection of this sequence or sequences related thereto at the 50% similarity level or at the low stringency hybridization level. Probes may comprise DNA (single-stranded
or double-stranded) or RNA (e.g., riboprobes) or analogues thereof.
Means for detecting D-l 1 expression may be any nucleic acid-based detection means such as, for example, nucleic acid hybridization techniques or paper chromatography hybridization assay (PACHA), or an amplification reaction such as PCR, or nucleic acid sequence-based amplification (NASBA) system. The invention further encompasses the use of different assay formats of the nucleic acid-based detection means, including restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), single-strand chain polymorphism (SSCP), amplification and mismatch detection (AMD), interspersed repetitive sequence polymerase chain reaction (IRS-PCR), inverse polymerase chain reaction (iPCR), in situ polymerase chain reaction and reverse transcription polymerase chain reaction (RT-PCR), amongst others.
Where the detection means is a nucleic acid hybridization technique, the probe can be labelled with a reporter molecule capable of producing an identifiable signal (e.g. a radioisotope such as 32P or 35S, or a biotinylated molecule). According to this embodiment, those skilled in the art will be aware that the detection of the reporter molecule provides for identification of the probe and that, following the hybridization reaction, the detection of the corresponding nucleotide sequences in the biological sample is facilitated. Additional probes can be used to confirm the assay results obtained using a single probe.
A variation of the nucleic acid hybridization technique contemplated by the present invention is the paper chromatography hybridization assay (PACHA) described by Reinhartz et al.{5), and equivalents thereof, wherein a target nucleic acid molecule is labelled with a reporter molecule such as biotin, applied to one end of a nitrocellulose or nylon membrane filter strip and subjected to chromatography under the action of capillary or other forces (e.g. an electric field) for a time and under conditions sufficient to promote migration of said target nucleic acid along the length of said membrane to a zone at which a DNA probe is immobilized thereto such as, for example, in the middle region. According to this detection format, labelled target nucleic acid comprising the D-l l mRNA nucleotide sequences complementary to the probe will hybridize thereto and become immobilized in that region of the membrane to which the probe
is bound. Non-complementary sequences to the probe will diffuse past the site at which the probe is bound. The target nucleic acid may comprise a crude or partially-pure extract of DNA or RNA or, alternatively, an amplified or purified DNA. Additional variations of this detection means which utilize the nucleotide sequences described herein are clearly encompassed by the present invention.
Wherein the detection means is a RFLP, nucleic acid derived from the biological sample, in particular DNA, is digested with one or more restriction endonuclease enzymes and the digested DNA is subjected to electrophoresis, transferred to a solid support such as, for example, a nylon or nitrocellulose membrane, and hybridized to a probe optionally labelled with a reporter molecule as hereinbefore defined. According to this embodiment, a specific pattern of DNA fragments is displayed on the support, wherein the pattern is specific for D-l l.
Wherein the detection means is an amplification reaction such as, for example, a polymerase chain reaction or a nucleic acid sequence-based amplification (NASBA) system or a variant thereof, one or more nucleic acid primer molecules of at least 15 contiguous nucleotides in length derivable from <400>1 or its complementary nucleotide sequence, or a homologue, analogue or derivative thereof, is hybridized to nucleic acid derived from a biological sample, and nucleic acid copies of the hemolysin-encoding genetic sequences in said sample, or a part or fragment thereof, are enzymically-amplified.
Those skilled in the art will be aware that there must be a sufficiently high percentage of nucleotide sequence identity between the primers and the sequences in the biological sample template molecule to which they hybridize (i.e., the "template molecule"). As stated previously, the stringency conditions can be selected to promote hybridization.
Preferably, each primer is at least about 95% identical to a region of <400>1 or its complementary nucleotide sequence in the template molecule to which it hybridizes.
Those skilled in the art will also be aware that, in one format, PCR provides for the hybridization of non-complementary primers to different strands of the template molecule, such
that the hybridized primers are positioned to facilitate the 5'→ 3' synthesis of nucleic acid in the intervening region, under the control of a thermostable DNA polymerase enzyme. As a consequence, PCR provides an advantage over other detection means in so far as the nucleotide sequence in the region between the hybridized primers may be unknown and unrelated to any known nucleotide sequence.
In an alternative embodiment, wherein the detection means is AFLP, the primers are selected such that, when nucleic acid derived from the biological sample, in particular DNA, is amplified, different length amplification products are produced. The amplification products can be subjected to electrophoresis, transferred to a solid support such as, for example, a nylon or nitrocellulose membrane, and hybridized to a probe optionally labelled with a reporter molecule as hereinbefore described. According to this embodiment, a specific pattern of amplified DNA fragments is displayed on the support, said pattern being specific for D-l l.
The technique of AMD facilitates, not only the detection of D- 11. DNA in a biological sample, but also the determination of nucleotide sequence variants which differ from the primers and probes used in the assay format. Wherein the detection means is AMD, the probe is end-labelled with a suitable reporter molecule and mixed with an excess of the amplified template molecule. The mixtures are subsequently denatured and allowed to renature to form nucleic acid "probe:template hybrid molecules" or "hybrids", such that any nucleotide sequence variation between the probe and the temple molecule to which it is hybridized will disrupt base-pairing in the hybrids. These regions of mismatch are sensitive to specific chemical modification using hydroxylamine (mismatched cytosine residues) or osmium tetroxide (mismatched thymidine residues), allowing subsequent cleavage of the modified site using piperidine. The cleaved nucleic acid may be analysed using denaturing polyacrylamide gel electrophoresis, followed by standard nucleic acid hybridization as described in Reinhartz et α/.(5) to detect D-l 1 nucleotide sequences. Those skilled in the art will be aware of the means of end-labelling a genetic probe according to the performance of the invention described in this embodiment.
According to this embodiment, the use of a single end-labelled probe allows unequivocal localization of the sequence variation. The distance between the point(s) of sequence variation
and the end-label is represented by the size of the cleavage product.
In an alternative embodiment of AMD, the probe is labelled at both ends with a reporter molecule, to facilitate the simultaneous analysis of both DNA strands.
Wherein the detection means is RT-PCR, the nucleic acid sample comprises an RNA molecule which is a transcription product of D-l 1 DNA or a homologue, analogue or derivative thereof. According to this embodiment, the RNA sample is reverse-transcribed to produce the complementary single-stranded DNA which is subsequently amplified using standard procedures.
The present invention clearly extends to the use of any and all detection means referred to Reinhartz et al.(5) for the purposes of detecting D-l l expression which is an indicator of undifferentiative NPC.
Another aspect of the present invention provides a composition for the treatment or prophylaxis of undifferentiated NPC in a mammal such as a human, said composition comprising an effective amount of an agent which:-
(i) promotes modulation of methylation of D-l l or its promoter or its 3' or 5' flanking regions to thereby modulate D-l 1 expression;
(ii) acts as an antisense molecule to all or part of the D-l 1 mRNA transcript to thereby down-regulate D-l 1 expression; and/or
(iii) promotes methylation of genetic sequences in NPC cells and one or more pharmaceutically acceptable carriers and/or diluents.
In relation to point (iii) above, NPC cells may be specifically targeted for methylation or methylation may be non-specific for genetic sequences in any cell.
The term "modulation" in relation the methylation means either inducing methylation or inducing de-methylation.
The formulation of compositions is generally known in the art and reference can conveniently be made to Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pennsylvania, USA.
A particularly useful form of the composition is a recombinant composition produced, for example, in a vaccine vector, such as but not limited to a cell transfected with a vaccinia virus vector or a bacterial cell capable of expressing an antisense molecule to the D-l 1 transcript.
In the production of a recombinant DNA vaccine described herein, it is necessary to express DNA in a suitable vector system. For the present purpose, an antisense D-l l transcript can be produced by: -
(i) placing an isolated nucleic acid molecule in an expressible format, said nucleic acid molecule comprising the complementary sequence of the coding region of the nucleotide sequence set forth in <400>1 or a homologue or derivative of <400>1 selected from the group consisting of:-
(a) nucleotide sequences that have at least about 50% sequence identity to <400> 1 ; and
(b) nucleotide sequences that hybridize under at least low stringency hybridization, preferably under at least moderate stringency conditions, and even more preferably under high stringency conditions, to <400>1;
(ii) introducing the isolated nucleic acid molecule of (i) in an expressible format into a suitable vector; and
(iii) incubating or growing the vector for a time and under conditions sufficient for
expression of antisense D-l l mRNA transcript encoded by said nucleic acid molecule to occur.
As used herein, a "nucleic acid molecule in an expressible format" is a mRNA-encoding region of a nucleic acid molecule placed in operable connection with a promoter or other regulatory sequence capable of regulating expression in the vector system.
Reference herein to a "promoter" is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue- specific manner. In the present context, the term "promoter" is also used to describe a recombinant, synthetic or fusion molecule, or derivative which confers, activates or enhances the expression of a nucleic acid molecule to which it is operably connected, and which encodes the antisense mRNA transcript. Preferred promoters can contain additional copies of one or more specific regulatory elements to further enhance expression and/or to alter the spatial expression and/or temporal expression of the said nucleic acid molecule.
Placing a nucleic acid molecule under the regulatory control of i.e., "in operable connection with" a promoter sequence means positioning the said molecule such that expression is controlled by the promoter sequence. Promoters are generally, but not necessarily, positioned
5' (upstream) to the genes that they control. In the construction of heterologous promoter/ structural gene combinations it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, i.e., the gene from which the promoter is derived. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene. As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e., the genes from which it is derived. Again, as is known in the art, some variation in this distance can also
occur.
Means for introducing the isolated nucleic acid molecule or a genetic construct comprising same into a cell for expression of the mRNA transcript of the composition are well-known to those skilled in the art. The technique used for a given organism depends on the known successful techniques. Means for introducing recombinant DNA into animal cells includes microinjection, transfection mediated by DEAE-dextran, transfection mediated by liposomes such as by using lipofectamine (Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation and microparticle bombardment such as by using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA) amongst others.
For DNA vaccines, a preferred amount for the treatment of undifferentiating NPC or related cancers is from about 0.1 μg/ml to about 5 mg/ml in a volume of about 1 to about 5 ml. The DNA can be present in "naked" form or it can be administered together with an agent facilitating cellular uptake (e.g. in liposomes or cationic lipids). The important feature is to administer sufficient D-l l antisense mRNA transcript to induce a protective therapeutic response. The above amounts can be administered as stated or calculated per kilogram of body weight. The dosage regime can be adjusted to provide the optimum therapeutic response. For example, several divided doses can be administered or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation. Booster administration may also be required.
The composition of the present invention can be administered in a convenient manner such as by oral, intravenous (where water soluble), intramuscular, subcutaneous, intranasal, intradermal or suppository routes or by implantation (e.g. using slow release technology). Depending on the route of administration, the immunogenic component may be required to be coated in a material to protect it from the action of enzymes, acids and other natural conditions which may inactivate it, such as those in the digestive tract.
The composition may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, or in oils. Under
ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms. Alternatively, the vaccine composition can be stored in lyophilized form to be rehydrated with an appropriate vehicle or carrier prior to use.
The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents such as, for example,, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents such as, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption such as, for example,, aluminum monostearate and gelatin.
The present invention further relates to the use of D-l 1 (or H-19) or derivatives, homologues or analogues thereof or complementary or mRNA forms thereof in the manufacture of a medicament for the treatment of undifferentiated NPC or related cancer.
Another aspect of the present invention is directed to the use of D-l 1 (or H-19) or derivatives, homologues or analogues thereof or complementary or transcript forms thereof in the manufacture of diagnostic reagents for the detection of undifferentiated NPC or related cancer.
The present invention further provides diagnostic kits comprising D-l l (or H- 19) or derivatives, homologues or analogues thereof or complementary or transcript forms thereof for use in detecting undifferentiated NPC or related cancer.
The present invention is further described by the following non-limiting Examples.
EXAMPLE 1
Cell lines and tissue culture
Human NPC cell lines CNE-1, CNE-2 and HK-1 have been previously described. CNE-1 and CNE-2 cells are obtained from the Cancer Institute, Sun Yat-sen University of Medical Sciences, Guangzhou, People's Republic of China. The NPC/HK1 cell line was obtained from The Prince of Wales Hospital, Shatin, Hong Kong. CNE-2 is derived from undifferentiated nasopharyngeal carcinoma, while CNE-1 and HK-1 are cell lines derived from well- differentiated squamous carcinoma. All other tumor cell lines were obtained from the American Type Culture Collection (ATCC) unless otherwise stated. Cells were grown in RPMI medium (Gibco, Grand Island, NY) supplemented with 10% v/v FCS (Hyclone, Logan, UT), 0.1 mM non-essential amino acids, 4 mM L-glutamine, 1 mM sodium pyruvate.
EXAMPLE 2 Tissue specimens
Human NPC tumor tissues were obtained from patients at the Department of ENT of the Singapore General Hospital. These patients had not received any treatment (radiotherapy and/or chemotherapy) before the biopsies were taken. Histo-pathological diagnosis was confirmed in paraffin sections. Tumor biopsies taken from patients were immediately snap-frozen and stored in liquid nitrogen until being studied.
EXAMPLE 3 Construction ofcDNA library and differential screening
Total RNA was isolated from the poorly differentiated human NPC cell line CNE-2 and the human cervical cancer cell line HT-3. Poly(A)~ mRNA selected using the Fast- Track mRNA isolation kit from Invitrogen (Invitrogen Corp., San Diego, CA). A unidirectional cDNA library was prepared from the CNE-2 poly(A)+ RNA using the pcDNAII vector and cDNA cloning reagents (Librarian II cDNA construction kit, Invitrogen Corp.). Approximately 1 x 105 colonies were replicated plated onto four filters. Two filters were probed with 32P-labelled single-
stranded cDNA prepared from poly(A)+ RNA isolated from the HT-3 cells while the remaining two filters were probed with radiolabelled single-stranded cDNA prepared from poly(A)+ RNA of CNE-2 cells. The filters were hybridized in 100 ml of hybridization solution (6 X SSC, 5 X Denhardt's solution, 0.5% w/v SDS and 100 μg/ml of sonciated salmon sperm DNA) for 42 hrs at 65°C. After hybridization, the filters were washed twice at 65°C with 2 X SSC, 0.5% w/v SDS for 30 min, followed by washing twice at 65°C in 0.3 X SSC, 0.5% SDS for 60 min. Colonies that hybridized specifically with the CNE-2 probe, but not the HT-3 probe were picked for further studies. The cDNA insert in each colony was sequenced by the Ml 3 dideoxy chain termination method using dideoxy-sequencing kit from United States Biochemical Corporation (USB) using both T7 and T3 primers. Sequences thus obtained were used to search the databases using the BLAST network service.
EXAMPLE 4
RNA extraction and Northern blotting
Total RNA was isolated by the guanidinium/caesium chloride method. Poly(A)+ RNA was selected by using the Fast-Track mRNA isolation kit from Invitrogen Corp., San Diego, CA). For Northern blotting analysis, polyA+ RNA (5 μg) was loaded in each lane of a 1% w/v agarose gel containing 0.7% v/v formaldehyde and 5 mM iodoacetamide and subjected to electrophoresis. RNA was transferred to nylon membrane by capillary transfer and employed for hybridization. DNA was used to probe Northern blots was labelled using the random prime method. The filters employed for the adult human and human fetal multiple tissue Northern blot were purchased from Clontech Laboratories (Clontech Laboratories Inc., Palo Alto, CA).
EXAMPLE 5
In situ hybridization
Frozen biopsies were cryostat sectioned to 10 μm and hybridized with non-radioactive probes which were labelled by random primed incorporation of digoxigenin-labelled dUTP (Boehringer Mannheim, Mannheim, Germany). The hybridized digoxigenin-labelled probes were detected with a peroxidase-conjugated antibody recognizing digoxienin and subsequent enzyme-
catalyzed colour reaction with 5-bromo-4-chloto-3-indolyl phosphate and nitro blue tetrazolium salt (Boehringer Mannheim). Photographs shown were taken at 40 X magnification.
EXAMPLE 6 Isolation ofcDNAfrom substruction cDNA library
To isolate cDNAs preferentially expressed in NPC, the inventors differentially screened a cDNA library derived from the poorly differentiated human NPC cell line CNE-2 using 32P-labelled total cDNAs synthesized from total mRNAs of the CNE-2 cells and the human cervical epithelial cancer cell line HT-3 cells as hybridization DNA probes. Over one hundred colonies which preferentially hybridized to the CNE-2 derived cDNAs and not to cDNAs derived from HT-3 cells were selected. These colonies were secondary screened by 32P-labelled total cDNAs from CNE-2 and HT-3 cells. Over 80 clones which again hybridized strongly to cDNAs derived from CNE-2 cells but poorly to cDNAs of HT-3 cells were isolated for further analysis and sequencing. One of such cDNA clone selected was D-l l. Northern blot study demonstrated that D-l 1 hybridized strongly to total mRNA isolated from CNE-2 cells to give a single 2.3 kb mRNA species (Figure 1). No positive signal was obtained when D-l l was hybridized to total mRNA isolated from the well-differentiated human NPC cell lines CNE-1 and NPC/HK-1, and the HT-3 cells.
EXAMPLE 7
Nucleotide sequence ø/D-11 cDNA clone is identical to the H-19 gene
The cDNA insert of clone D-l 1 was subcloned into pUC18 and M13 vectors. Shorter cDNA fragments of original clone D-l 1 were generated and employed for DNA sequencing. A genetic map showing the relationship of clone D-l l and its sub-clones (derived for genomic sequencing) to the reported H-19 clone is outlined in Figure 2. The nucleotide sequence of D-l 1 was determined and was found to be of 1432 bp in length. The sequence of D-l l was then employed for computer search against Genbank and EMBL DNA databases. It was found that clone D-l 1 was identical in sequence to the human H-19 gene that has been implicated to play
a role in paternal imprinting. The alignment of the nucleotide sequence of clone D-l l to the human H-19 gene is shown in Figure 3. The longest open reading frame of the D-l 1 clone predicts an mRNA sequence identical to the reported sequence of the human H-19 gene. To further confirm that clone D-l l is the human H-19 gene, the inventors demonstrated strong hybridization of the D-l l clone to the H-19 gene.
EXAMPLE 8
D-ll is strongly expressed in poorly differentiated human
NPC tumor cells and human fetal tissues
To examine whether the expression of D-l 1 is unique to human NPC cells, the inventors carried out Northern blot analysis employing total mRNAs purified from 19 different human tumor cell lines of diverse origins. Although the level of expression varied between different cell lines, it was found that D-l l hybridized only to mRNAs isolated from human cells lines of either nasopharyngeal, pharynx or larynx origin (Figure 4). D- 11 was highly expressed in the poorly differentiated human NPC cell line CNE-2 cells, weakly expressed in Hep-2 cells (epidermoid carcinoma of larynx), Detroit 562, and Fadu cells (Figure 4). The latter two are cell lines derived from pharyngeal carcinoma. None of the other human cancer cell lines tested hybridized to the D-l l probe (Figure 4).
Northern blot analysis of polyA+ mRNA isolated from human adult (Figure 5 A) and fetal (Figure 5B) tissues were also performed. It was found that D-l l is only expressed in human placenta (Figure 5 A) and fetal liver (Figure 5B) and was not detected in all of the other normal human tissues examined.
EXAMPLE 9
D-ll is specifically expressed in non-differentiated primary human NPC biopsies
To study whether the expression of D-l 1 in primary human NPC tissues correlates with the expression detected in poorly differentiated human NPC cell lines, in situ hybridization studies
of human normal nasopharyngeal and NPC biopsies were performed. In situ hybridization studies revealed that D-l l is expressed strongly in the two poorly differentiated human NPC biopsies (Figure 6). In comparison, D-l l was not detected in normal human biopsy taken similarly from the nasopharyngeal region (Figure 6). The non-radioactively, digoxigenin- labelled probe identified positively D-l 1 mRNA expressing cells by their grey-brown colour. As a positive control, the β-actin probe hybridized to all three sections studied (Figure 7). Two additional human poorly differentiated primary NPC biopsies were studied and they gave the same results.
EXAMPLE 10
Methylation of D-ll
The present study demonstrates high levels of expression of the D-l l gene only in poorly and not in well differentiated human NPC cells. The differential expression of the D-l 1 gene in different NPC cells which exhibit different degrees of differentiation is relatable to the pathogenesis associated with the disease. Similar results were obtained with biopsies from NPC patients (Figure 6). It has been proposed that CpG methylation patterns over certain genes can modify their expression. In this context, it is noted that on examining the gene sequence of the D-l l gene, it contains many CpG sites within the 5'-flanking/promoter regions which can potentially be modified by methylation. The differences in methylation patterns may be responsible for, or at least related to, the observed de-regulation of D-l 1 gene expression.
EXAMPLE 11 H-19 and cancer cell lines
The chemical 5-azacytosine has been well known for its ability to induce hypo-methylation in vivo. To explore the possibility that methylation may play a role in regulating the activity of the H-19 gene, the inventors used 5-azacytosine to treat several tumor cell lines that did not express the H-19 mRNA to determine if the H-19 mRNA can be induced by hypo-methylation. Two well differentiated NPC cell lines (CNE-1 and HK-1), a cervical cancer cell line (Hela), a human breast cancer cell line (NIH OVCAR-3), and a human colorectal cancer cell line (SW480) were
cultured for one week with 12.5 μM 5-azacytosine. After culturing, total RNA was extracted and the level of H-19 mRNA was determined by Northern blot and hybridization (see Figure 8).
It was observed that H-19 mRNA can be induced specifically in the two well differentiated NPC cell lines HK-1 and CNE-1, and the human cervical cancer cell line Hela. It is therefore likely that the methylation of certain CpG sites within the H-19 gene is responsible for the down- regulation of the H-19 mRNA in these cancer cell lines. This down-regulation by methylation is reversible by the addition of 5-azacytosine in these cancer cell lines.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
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