EP1836313A2 - Verfahren zur beurteilung von patienten mit akuter myeloider leukämie - Google Patents

Verfahren zur beurteilung von patienten mit akuter myeloider leukämie

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Publication number
EP1836313A2
EP1836313A2 EP05854761A EP05854761A EP1836313A2 EP 1836313 A2 EP1836313 A2 EP 1836313A2 EP 05854761 A EP05854761 A EP 05854761A EP 05854761 A EP05854761 A EP 05854761A EP 1836313 A2 EP1836313 A2 EP 1836313A2
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gene
aml
expression
genes
patient
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French (fr)
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EP1836313A4 (de
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Mitch Raponi
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Janssen Diagnostics LLC
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Veridex LLC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/50—Detection characterised by immobilisation to a surface
    • C12Q2565/501—Detection characterised by immobilisation to a surface being an array of oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • This invention relates to diagnostics, prognostics, and treatments for acute myeloid leukemia (AML) based on the detection of molecular markers and/or gene expression analysis.
  • AML acute myeloid leukemia
  • Karyotyping is currently effective in providing prognostic value while it also serves to identify biologically distinct subtypes of AML.
  • mutations in genes such as FLT3, c-KIT, AMLl, GATAl, CEBPA and N-RAS are implicated in the pathogenesis of the disease. It is clear, that screening for FLT3 and CEBPA mutations can stratify groups that have different risks of relapse. Effective risk stratification can allow for the appropriate use of allogeneic stem cell transplantation or other adjuvant therapies.
  • VaIk et al. (2004) evaluated 285 patients (bone marrow or peripheral blood) on the Affymetrix U133A chip.
  • the patient samples encompassed a wide range of cytogenetic and molecular abnormalities. Only 16 clusters were identified indicating AML may not be as heterogeneous as previously thought. Several of the clusters corresponded well with the cytogenetically and molecularly defined subtypes of AML thus supporting their use in the WHO classification system. These clusters were also seen by Bullinger et al. (2004) and other previously published smaller studies. Schoch et al. (2002); Debernardi et al. (2003); and Kohlmann et al. (2003). These clusters, not surprisingly, correlated with prognosis since they were associated with well known prognostic karyotypes.
  • the farnesyl transferase (FTase) enzyme mediates the covalent attachment of a carbon farnesyl moiety to the C-terminal CAAX (C, cysteine; A, aliphatic residue; X, any amino acid) recognition motif. Reiss et al. (1990). This farnesylation is further processed by cleavage of the 3 terminal amino acids (AAX) and methylation of the C-terminal isoprenyl-cysteine. The inhibition of protein farnesylation abrogates the correct subcellular localization required for protein function. Originally, the oncogenic Ras protein was thought to be the target for the antiproliferative effects of FTIs in cancer biology. Reuter et al. (2000).
  • farnesylated proteins have been implicated as candidate targets that may mediate the antitumorigenic effects of FTIs including the small GTPase proteins Rho B, the centromere proteins CENP-E and CENP-F, the protein tyrosine phosphatase PTP-CAAX, and the nuclear membrane structural lamins A and B.
  • the inhibition of farnesylation of these proteins may lead to the antiproliferative effect of FTIs and also indirectly modulate several important signaling molecules including TGF ⁇ RII, MAPK/ERK, PI3K/AKT2, Fas (CD95), NF- ⁇ B, and VEGF.
  • the invention is a method of using one or more gene signatures for predicting prognosis in patients with acute myeloid leukemia (AML). These signatures can be used alone or in combination depending upon the type of drug treatment.
  • AML acute myeloid leukemia
  • the present invention provides a method of assessing acute myeloid leukemia (AML) status by obtaining a biological sample from an AML patient; and measuring Biomarkers associated with Marker genes corresponding to those selected from Table 3, Table 4, Table 5, Table 7, Table 8 or Table 9 where the expression levels of the Marker genes above or below pre-determined cut-off levels are indicative of AML status.
  • AML acute myeloid leukemia
  • the present invention provides a method of staging acute myeloid leukemia (AML) patients by obtaining a biological sample from an AML patient; and measuring Biomarkers associated with Marker genes corresponding to those selected from Table 3, Table 4, Table 5, Table 7, Table 8 or Table 9 where the expression levels of the Marker genes above or below pre-determined cut-off levels are indicative of AML survival.
  • AML acute myeloid leukemia
  • the present invention provides a method of determining acute myeloid leukemia (AML) patient treatment protocol by obtaining a biological sample from an AML patient; and measuring Biomarkers associated with Marker genes corresponding to those selected from Table 3, Table 4, Table 5, Table 7, Table 8 or Table 9 where the expression levels of the Marker genes above or below pre-determined cut-off levels are sufficiently indicative of response to therapy to enable a physician to determine the degree and type of therapy recommended to provide appropriate therapy.
  • AML acute myeloid leukemia
  • the present invention provides a method of treating a acute myeloid leukemia (AML) patient by obtaining a biological sample from an AML patient; and measuring Biomarkers associated with Marker genes corresponding to those selected from Table 3, Table 4, Table 5, Table 7, Table 8 or Table 9 where the expression levels of the Marker genes above or below pre-determined cut-off levels are indicate a response to therapy and; treating the patient with adjuvant therapy if they have a responder profile.
  • AML acute myeloid leukemia
  • the present invention provides a method of determining whether a acute myeloid leukemia (AML) patient is high or low risk of mortality by obtaining a biological sample from an AML patient; and measuring Biomarkers associated with Marker genes corresponding to those selected from Table 3 where the expression levels of the Marker genes above or below pre-determined cut-off levels are sufficiently indicative of risk of mortality to enable a physician to determine the degree and type of therapy recommended.
  • AML acute myeloid leukemia
  • the present invention provides a method of generating an acute myeloid leukemia (AML) prognostic patient report by determining the results of any one of the above-described methods; and preparing a report displaying the results and reports generated thereby.
  • the present invention provides a kit for conducting an assay to determine acute myeloid leukemia (AML) prognosis in a biological sample comprising: materials for detecting isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting of Marker genes corresponding to those selected from Table 3, Table 4, Table 5, Table 7, Table 8 or Table 9.
  • the present invention provides articles for assessing acute myeloid leukemia (AML) status comprising: materials for detecting isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting of Marker genes corresponding to those selected from Table 3, Table 4, Table 5, Table 7, Table 8 or Table 9.
  • the present invention provides a microarray or gene chip for performing the above-described methods.
  • the present invention provides a diagnostic/prognostic portfolio comprising isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting of Marker genes corresponding to those selected from Table 3, Table 4, Table 5, Table 7, Table 8 or
  • FIG. 1 Unsupervised clustering of relapsed and refractory AML patients.
  • the dendogram shows the unsupervised k-means clustering of 58 relapsed or refractory AML patients, where each column represents a patient and each row represents a gene.
  • the expression ratio for each gene was calculated by dividing the expression level of that gene in a patient by the mean of all other patients.
  • the color bar indicates the fold-change (log 2 ). Red is upregulated, blue is down-regulated. White indicates no change. The presence of 6 main clusters is shown.
  • FIG. 1 Real-time RT-PCR of 2 genes.
  • AHR and AKAP13 were measured by real-time RT-PCR.
  • the HPRT or PBGD control genes were used to normalize gene expression values. Error bars are standard deviations. The resulting values were plotted against the corresponding microarray data and linear regression analysis was performed.
  • Figure 3 depicts the predictive value of the AKAP 13 gene.
  • Panel A shows a 2x2 table generated from a LOOCV performed using AKAP 13 expression as a classifier on the responders (R) and non-responders (NR).
  • Panel B shows the AKAP 13 expression values for the same 58 patients. The P value indicates a significant difference in the gene expression between the mean values of each response group.
  • Panel C shows the Kaplan-Meier curves generated from patients classified by the AKAP 13 gene as being responders and non-responders.
  • Figure 4 provides identification of a minimal set of predictive markers. In Panel
  • a LOOCV was performed using a sensitivity of 100%. Independent classifiers were tested that contained from 1 to 19 genes. The resulting error rate is plotted.
  • Panel B shows a 2x2 table generated from a LOOCV performed using the 3-gene signature as a classifier on the responders (R) and non-responders (NR).
  • Panel C shows the scores generated from the 3-gene classifier. The P value indicates a significant difference in the gene expression between the response groups.
  • Panel D is the Kaplan-Meier curves generated from patients classified by the 3-gene signature as being responders and non-responders. Median survival times are also indicated. Figure 5. A Kaplan-Meier analysis was performed on patients classified by the
  • 3-gene signature as being predicted responders and non-responders.
  • the survival curve of patients who were clinically defined as non-responders but classified as responders using the 3-gene signature is shown. Median survival times are also indicated.
  • Figure 6 depicts over-expression of AKAPl 3 in an AML cell line. Cell counts were normalized to cultures with no drug (indicated at -12 log units) to give a percentage of control. Error bars indicate standard errors of the mean. Open data points indicate results from a second experiment exploring higher concentrations of drug.
  • Figure 7 provides a model of FTI action in relapsed or refractory AML. A.
  • IL3RA and AKAPl 3 genes are lowly expressed allowing for down-regulation of the ras, and RhoA, and lamin B pathways, respectively. Up-regulation of RhoH leads to increased inhibition of cellular transformation pathways. Together this allows for greater efficacy in FTI antitumorigenicity.
  • B. The opposite expression profile is seen in non-responders allowing for the expression of compensatory pathways.
  • FIG. 8 The Zarnestra predictive gene signature has superior utility to an independent prognostic gene signature.
  • panel A columns represent AML samples from relapsed or refractory patients and rows represent 167 probe sets that correspond to 103 of the 133 prognostic genes identified by Bullinger et al. ', ordered according to hierarchical clustering.
  • Panel B shows Kaplan-Meier survival estimates of the cluster-defined groups of patients.
  • panel C the 3-gene classifier has been used to identify responders of tipifarnib in the good and poor prognostic groups defined by the Bullinger signature. Kaplan-Meier survival curves are shown for patients identified as being responders to tipifarnib in the good (Zn+.clusterl) and poor (Zn+.cluster2) prognostic groups. The median survival times for each group are indicated.
  • Figure 9 is a flow chart depicting how the genes from Bullinger et al. (2004) were matched to 167 probe sets (103 unique genes) on the Affymetrix Ul 33 A chip.
  • Figure 10 shows the utility of the 167 probe set signature in relapsed or refractory AML patients.
  • columns represent AML samples from relapsed or refractory patients and rows represent 167 probe sets that correspond to 103 of the 133 prognostic genes identified by Bullinger et al. (2004), ordered according to hierarchical clustering.
  • Panel B shows Kaplan-Meier survival estimates of the cluster-defined groups of patients.
  • Figure 11 provides comparisons of prognostic and Zarnestra predictive gene signatures.
  • Panel A shows the Kaplan-Meier survival curves for the good and poor prognostic clusters as defined by the subset of 103 Bullinger et al. (2004) genes.
  • Panel B shows the Kaplan-Meier survival curves for the good and poor prognostic clusters as defined by the 3-gene signature that predicts response to Zarnestra.
  • Panel C shows the Kaplan-Meier survival curves for the good and poor prognostic clusters from Panel A further stratified by the 3-gene Zarnestra signature.
  • Panel D shows the Kaplan-Meier survival curves for patients who are predicted to have a poor prognosis and not respond to Zarnestra versus the remainder of patients.
  • Figure 12 Identification of a minimal set of predictive markers, a) A LOOCV was performed selecting for genes with a sensitivity of 100%, specificity of 40% and fold-change > 2. Independent classifiers were tested that contained from 1 to 8 genes ranked by the AUC . The resulting error rate is plotted, b) A 2x2 table generated from a LOOCV performed using AKAP 13 as a classifier on the responders (R) and non-responders (NR). c) The gene-expression values of AKAPl 3. The P value indicates a significant difference in the gene expression between the response groups, d) The Kaplan-Meier curves generated from patients classified by AKAPl 3 as being responders and non-responders. Median survival times are also indicated.
  • Figure 13 depicts an overview of gene expression analysis.
  • Figure 14 depicts AML samples maintain FTI-mediated global gene expression changes following termination of tipifarnib treatment.
  • Figure 15 depicts predictive expression profiles and testing of predictive classifiers in newly diagnosed AML.
  • Figure 16 depicts the 6-gene classifier stratifies newly diagnosed AML.
  • DETAILED DESCRIPTION OF THE INVENTION A subset of genes previously described to have prognostic value in newly diagnosed AML is shown here to have utility in relapsed and refractory AML patients treated with a molecularly targeted therapy (Zarnestra).
  • Zarnestra molecularly targeted therapy
  • current methods for understanding the prognosis of patients with AML is limited to histological subtype and karyotyping, both of which are not ideal markers for determining clinical outcome.
  • the current signatures expand upon these traditional technologies by providing better stratification of prognostic high risk and low risk patients.
  • US Patent Application Serial No. 10/883,436 demonstrates that a 3-gene classifier (including AHR, AKAPl 3 and MINA53) predicts relapsed, refractory
  • AML patient response to tipifarnib (Zarnestra ® , Rl 15777) with the lowest error rate. This was also seen when a leave-five-out cross validation was performed. When more genes were added the error rate increased indicating that additional genes introduced noise to the classifier.
  • the LOOCV demonstrated a sensitivity of 86% and specificity of 70% with an overall diagnostic accuracy of 74%. Kaplan-Meier analysis again showed a significant difference in survival between the predicted responder group and the non-responder group. Moreover, comparing the incorrectly classified non-responders to the correctly classified non-responders, the misclassified non-responders showed a better overall survival.
  • Zarnestra ® is an orally available non-peptidomimetic competitive farnesyl transferase inhibitor (FTI) that has been shown to inhibit the proliferation of a variety of human tumor cell lines both in vitro and in vivo. End et al. (2001); and Cox et al. (2002). A phase I clinical trial of tipifarnib demonstrated a 32% response rate in patients with refractory or relapsed acute myeloid leukemia. Karp et al. (2001). Activity has also been seen in early clinical trials for myelodysplastic syndrome (MDS) (Kurzrock et al. (2004)), multiple myeloma (MM) (Alsina et al.
  • MDS myelodysplastic syndrome
  • MM multiple myeloma
  • nucleic acid sequences having the potential to express proteins or peptides within the genome is not determinative of whether a protein or peptide is expressed in a given cell. Whether or not a given gene capable of expressing proteins or peptides or transcribing RNA does so and to what extent such expression or transcription occurs, if at all, is determined by a variety of complex factors. Nevertheless, assaying gene expression can provide useful information about the cellular response to a given stimulus such as the introduction of a drug or other therapeutic agent. Relative indications of the degree to which genes are active or inactive can be found in such gene expression profiles. In some instances, the presence of a molecular marker can, by itself or with the use of gene expression information, provide useful information about treatment efficacy too.
  • the gene expression profiles and molecular markers of this invention are used to identify and treat AML patients.
  • Cancers typically arise from mutations in a variety of genes.
  • the same type of cancer may arise as a result of, or coincident with, one or more mutations that differ from those of another patient having the same type of cancer.
  • the fact that there are often multiple molecular bases underlying the same cancers is consistent with the observation that some therapies that affect one patient do not necessarily equally affect another patient with the same type of cancer.
  • the presence of particular mutations such as translocations, deletions, or SNPs can have powerful implications.
  • such molecular markers are themselves useful indicators for diagnosis, prognosis, or treatment response determinations. This is particularly true where the molecular mutations can be associated with response to particular treatments.
  • a Biomarker is any indicia of the level of expression of an indicated Marker gene.
  • the indicia can be direct or indirect and measure over- or under-expression of the gene given the physiologic parameters and in comparison to an internal control, normal tissue or another carcinoma.
  • Biomarkers include, without limitation, nucleic acids (both over and under-expression and direct and indirect).
  • nucleic acids as Biomarkers can include any method known in the art including, without limitation, measuring DNA amplification, RNA, micro RNA, loss of heterozygosity (LOH), single nucleotide polymorphisms (SNPs, Brookes (1999)), microsatellite DNA, DNA hypo- or hyper-methylation.
  • Biomarkers can include any method known in the art including, without limitation, measuring amount, activity, modifications such as glycosylation, phosphorylation, ADP-ribosylation, ubiquitination, etc., imunohistochemistry (IHC).
  • Other Biomarkers include imaging, cell count and apoptosis markers.
  • a Marker gene may be associated with numerous cancer types but provided that the expression of the gene is sufficiently associated with one tumor or tissue type to be identified using the algorithm described herein to be specific for a lung cancer cell, the gene can be used in the claimed invention to determine cancer status and prognosis. Numerous genes associated with one or more cancers are known in the art. The present invention provides preferred Marker genes and even more preferred Marker gene combinations. These are described herein in detail.
  • a Marker gene corresponds to the sequence designated by a SEQ ID NO when it contains that sequence.
  • a gene segment or fragment corresponds to the sequence of such gene when it contains a portion of the referenced sequence or its complement sufficient to distinguish it as being the sequence of the gene.
  • a gene expression product corresponds to such sequence when its RNA, mRNA, or cDNA hybridizes to the composition having such sequence (e.g. a probe) or, in the case of a peptide or protein, it is encoded by such mRNA.
  • a segment or fragment of a gene expression product corresponds to the sequence of such gene or gene expression product when it contains a portion of the referenced gene expression product or its complement sufficient to distinguish it as being the sequence of the gene or gene expression product.
  • Marker genes include one or more Marker genes.
  • Marker or “Marker gene” is used throughout this specification to refer to genes and gene expression products that correspond with any gene the over- or under-expression of which is associated with a tumor or tissue type.
  • the preferred Marker genes are described in more detail in Table 8.
  • the present invention provides a method of assessing acute myeloid leukemia (AML) status by obtaining a biological sample from an AML patient; and measuring Biomarkers associated with Marker genes corresponding to those selected from Table 3, Table 4, Table 5, Table 7, Table 8 or Table 9 where the expression levels of the Marker genes above or below pre-determined cut-off levels are indicative of AML status.
  • AML acute myeloid leukemia
  • the present invention provides a method of staging acute myeloid leukemia (AML) patients by obtaining a biological sample from an AML patient; and measuring Biomarkers associated with Marker genes corresponding to those selected from Table 3, Table 4, Table 5, Table 7, Table 8 or Table 9 where the expression levels of the Marker genes above or below pre-determined cut-off levels are indicative of AML survival.
  • AML acute myeloid leukemia
  • the present invention provides a method of determining acute myeloid leukemia (AML) patient treatment protocol by obtaining a biological sample from an AML patient; and measuring Biomarkers associated with Marker genes corresponding to those selected from Table 3, Table 4, Table 5, Table 7, Table 8 or Table 9 where the expression levels of the Marker genes above or below pre-determined cut-off levels are sufficiently indicative of response to therapy to enable a physician to determine the degree and type of therapy recommended to provide appropriate therapy.
  • AML acute myeloid leukemia
  • the present invention provides a method of treating a acute myeloid leukemia (AML) patient by obtaining a biological sample from an AML patient; and measuring Biomarkers associated with Marker genes corresponding to those selected from Table 3, Table 4, Table 5, Table 7, Table 8 or Table 9 where the expression levels of the Marker genes above or below pre-determined cut-off levels are indicate a response to therapy and; treating the patient with adjuvant therapy if they have a responder profile.
  • AML acute myeloid leukemia
  • the present invention provides a method of determining whether a acute myeloid leukemia (AML) patient is high or low risk of mortality by obtaining a biological sample from an AML patient; and measuring Biomarkers associated with Marker genes corresponding to those selected from Table 3 where the expression levels of the Marker genes above or below pre-determined cut-off levels are sufficiently indicative of risk of mortality to enable a physician to determine the degree and type of therapy recommended.
  • AML acute myeloid leukemia
  • the method provided herein may further include, contain or utilize measuring the expression level of at least one gene constitutively expressed in the sample.
  • the method provided herein results in a specificity of at least about 40%.
  • the method provided herein results in a sensitivity of at least at least about 80%.
  • the method provided herein results in a p-value of less than 0.05.
  • the method provided herein may be performed by measuring gene expression on a microarray or gene chip.
  • the microarray can be a cDNA array or an oligonucleotide array and may further contain one or more internal control reagents.
  • the method provided herein may be performed by determining gene expression by nucleic acid amplification conducted by polymerase chain reaction (PCR) of RNA extracted from the sample.
  • PCR polymerase chain reaction
  • the PCR can be reverse transcription polymerase chain reaction (RT-PCR) and can further contain one or more internal control reagent.
  • the method provided herein may be performed by measuring or detecting a protein encoded by the gene.
  • Te protein can be detected by an antibody specific to the protein.
  • the method provided herein may be performed by measuring a characteristic of the gene. Characteristics include, without limitation, DNA amplification, methylation, mutation and allelic variation.
  • the present invention provides a method of generating an acute myeloid leukemia (AML) prognostic patient report by determining the results of any one of the above-described methods; and preparing a report displaying the results and reports generated thereby.
  • the report may contain an assessment of patient outcome and/or probability of risk relative to the patient population and/or likelihood or response to chemotherapy.
  • the present invention provides a kit for conducting an assay to determine acute myeloid leukemia (AML) prognosis in a biological sample comprising: materials for detecting isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting of Marker genes corresponding to those selected from Table 3, Table 4, Table 5, Table 7, Table 8 or Table 9.
  • the kit can further contain reagents for conducting a microarray analysis and/or a medium through which said nucleic acid sequences, their complements, or portions thereof are assayed.
  • the present invention provides articles for assessing acute myeloid leukemia (AML) status containing materials for detecting isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting of Marker genes corresponding to those selected from Table 3, Table 4, Table 5, Table 7, Table 8 or Table 9.
  • the articles can further contain reagents for conducting a microarray analysis and/or a medium through which said nucleic acid sequences, their complements, or portions thereof are assayed.
  • the present invention provides a microarray or gene chip for performing the above-described methods.
  • the microarray may contain isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting of Marker genes corresponding to those selected from Table 3, Table 4, Table 5, Table 7, Table 8 or Table 9.
  • the microarray provides a measurement or characterization at least 1.5-fold over- or under-expression.
  • the microarray provides a measurement with a statistically significant p-value over- or under-expression. More preferably, the p-value is less than 0.05.
  • the microarray can be any known in the art including, without limitation, cDNA array or an oligonucleotide array and can further contain internal control reagents.
  • the present invention provides a diagnostic/prognostic portfolio comprising isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting of Marker genes corresponding to those selected from Table 3, Table 4, Table 5, Table 7, Table 8 or Table 9.
  • the measurement or characterization is at least 1.5-fold over- or under-expression.
  • the measurement provides a statistically significant p-value over- or under-expression. More preferably, the p-value is less than 0.05.
  • Preferred methods for establishing gene expression profiles include determining the amount of RNA that is produced by a gene that can code for a protein or peptide.
  • RT-PCR reverse transcriptase PCR
  • competitive RT-PCR competitive RT-PCR
  • real time RT-PCR real time RT-PCR
  • differential display RT-PCR Northern Blot analysis and other related tests. While it is possible to conduct these techniques using individual PCR reactions, it is best to amplify complementary DNA (cDNA) or complementary RNA (cRNA) produced from mRNA and analyze it via microarray.
  • cDNA complementary DNA
  • cRNA complementary RNA
  • Patents such as: 5,445,934; 5,532,128; 5,556,752; 5,242,974; 5,384,261 ; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327; 5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554,501 ; 5,561,071; 5,571,639; 5,593,839; 5,599,695; 5,624,711; 5,658,734; and 5,700,637.
  • Microarray technology allows for the measurement of the steady-state mRNA level of thousands of genes simultaneously thereby presenting a powerful tool for identifying effects such as the onset, arrest, or modulation of uncontrolled cell proliferation.
  • Two microarray technologies are currently in wide use. The first are cDNA arrays and the second are oligonucleotide arrays. Although differences exist in the construction of these chips, essentially all downstream data analysis and output are the same.
  • the product of these analyses are typically measurements of the intensity of the signal received from a labeled probe used to detect a cDNA sequence from the sample that hybridizes to a nucleic acid sequence at a known location on the microarray.
  • the intensity of the signal is proportional to the quantity of cDNA, and thus mRNA, expressed in the sample cells.
  • mRNA mRNA
  • Analysis of the expression levels is conducted by comparing such signal intensities. This is best done by generating a ratio matrix of the expression intensities of genes in a test sample versus those in a control sample. For instance, the gene expression intensities from a diseased tissue can be compared with the expression intensities generated from benign or normal tissue of the same type. A ratio of these expression intensities indicates the fold-change in gene expression between the test and control samples.
  • Gene expression profiles can also be displayed in a number of ways. The most common method is to arrange raw fluorescence intensities or ratio matrix into a graphical dendogram where columns indicate test samples and rows indicate genes. The data are arranged so genes that have similar expression profiles are proximal to each other. The expression ratio for each gene is visualized as a color. For example, a ratio less than one (down-regulation) may appear in the blue portion of the spectrum while a ratio greater than one (indicating up-regulation) may appear as a color in the red portion of the spectrum.
  • Commercially available computer software programs are available to display such data including "GENESPRING” from Silicon Genetics, Inc. and “DISCOVERY” and "INFER” software from Partek, Inc.
  • protein levels can be measured by binding to an antibody or antibody fragment specific for the protein and measuring the amount of antibody-bound protein.
  • Antibodies can be labeled by radioactive, fluorescent or other detectable reagents to facilitate detection. Methods of detection include, without limitation, enzyme-linked immunosorbent assay (ELISA) and immunoblot techniques.
  • ELISA enzyme-linked immunosorbent assay
  • Modulated Markers used in the methods of the invention are described in the Examples.
  • the genes that are differentially expressed are either up regulated or down regulated in patients with various lung cancer prognostics. Up regulation and down regulation are relative terms meaning that a detectable difference (beyond the contribution of noise in the system used to measure it) is found in the amount of expression of the genes relative to some baseline. In this case, the baseline is determined based on the algorithm.
  • the genes of interest in the diseased cells are then either up- or down-regulated relative to the baseline level using the same measurement method.
  • Assays for the gene expression status of a cell also can determine normal/abnormal tissue distribution for diagnostic purposes using techniques such as immunohistochemical analysis (IHC).
  • IHC immunohistochemical analysis
  • Any method known in the art can be used, for example in the case of the LBC oncogene, the antibodies to LBC protein may be used in conjunction with both fresh-frozen and formalin-fixed, paraffin-embedded tissue blocks prepared for study by IHC. Each tissue block may consist of 50 mg of residual "pulverized" tumor.
  • frozen-sections may be prepared by rehydrating 50 ng of frozen pulverized tumor at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and pelleting again by centrifugation; snap-freezing in -70°C isopentane; cutting the plastic capsule and removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and cutting 25-50 serial sections containing intact tumor cells.
  • PBS phosphate buffered saline
  • OCT viscous embedding medium
  • Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hr fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and embedding the block in paraffin; and cutting up to 50 serial permanent sections.
  • the sections are overlaid with a blocking solution containing: 3% bovine serum albumin (BSA) in PBS or other blocking reagents.
  • BSA bovine serum albumin
  • the blocking reagents include non-specific serum or dry milk. Blocking is allowed to proceed for 1 hr at room temperature.
  • Anti-LBC protein antibody is diluted with PBS buffer containing 3% BSA, 0.1% TritonXTM-100 and t-octylphenoxypolyethoxyethanol, at a ratio of 1 : 100.
  • the sample sections are generally overlaid with the antibody solution for 16 hr at 4°C. The duration and temperature conditions may be varied according to the antibody selected and the material tested. The optimal conditions are determined empirically.
  • the antibody treated sections are then washed three times in PBS for 15 min. each to remove unbound antibody and then overlaid with PBS containing 3% BSA and a secondary antibody at a dilution of 1 :2000.
  • the secondary antibodies may be coupled to a chromogenic enzyme such as: horseradish peroxidase, alkaline phosphatase, fluorescein isothiocyanate, or other suitable enzymes.
  • the secondary antibody may be conjugated to biotin and used in conjunction with chromophore-labeled avidin.
  • in situ hybridization comprises the following major steps: (1) fixation of tissue or biological structure to be analyzed; (2) prehybridization treatment of the biological structure to increase accessibility of target DNA, and to reduce nonspecific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological structure or tissue; (4) post-hybridization washes to remove nucleic acid fragments not bound in the hybridization and (5) detection of the hybridized nucleic acid fragments.
  • the reagent used in each of these steps and the conditions for use vary depending on the particular application.
  • a hybridization solution comprising at least one detectable nucleic acid probe capable of hybridizing to a gene (at its chromosomal locus) is contacted with the cell under hybridization conditions. Any hybridization is then detected and compared to a predetermined hybridization pattern from normal or control cells.
  • the probes are alpha-centromeric probes. Such probes can be made commercially available from a number of sources (e.g., from Visys Inc., Downers Grove, IL).
  • the hybridization solution contains a multiplicity of probes, specific for an area on the chromosome that corresponds to the translocation of the sequences that make up the chimera (e.g., 15q24-25).
  • Hybridization protocols suitable for use with the methods of the invention are described, e.g., in Albertson (1984); Pinkel (1988); EP No. 430,402; and Methods in Molecular Biology, Vol. 33: In Situ Hybridization Protocols, Choo, ed., Humana Press, Totowa, NJ (1994), etc.
  • the hybridization protocol of Pinkel et al. (1998) or of Kallioniemi (1992) is used. Methods of optimizing hybridization conditions are well known (see, e.g., Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 24: Hybridization With Nucleic Acid Probes, Elsevier, NY).
  • background signal is reduced by the use of a detergent (e.g., C-TAB) or a blocking reagent (e.g., sperm DNA, cot-1 DNA, etc.) during the hybridization to reduce non-specific binding.
  • a detergent e.g., C-TAB
  • a blocking reagent e.g., sperm DNA, cot-1 DNA, etc.
  • the hybridization is performed in the presence of about 0.1 to about 0.5 mg/ml DNA (e.g., cot-1 DNA).
  • the probes may be prepared by any method known in the art, including synthetically or grown in a biological host. Synthetic methods include but are not limited to oligonucleotide synthesis, riboprobes, and PCR.
  • the probe may be labeled with a detectable marker by any method known in the art.
  • Methods for labeling probes include random priming, end labeling, PCR and nick translation. Enzymatic labeling is conducted in the presence of nucleic acid polymerase, three unlabeled nucleotides, and a fourth nucleotide which is either directly labeled, contains a linker arm for attaching a label, or is attached to a hapten or other molecule to which a labeled binding molecule may bind.
  • Suitable direct labels include radioactive labels such as 32 P, 3 H, and 35 S and non-radioactive labels such as fluorescent markers, such as fluorescein, Texas Red, AMCA blue, lucifer yellow, rhodamine, and the like; cyanin dyes which are detectable with visible light; enzymes and the like. Labels may also be incorporated chemically into DNA probes by bisulfite-mediated transamination or directly during oligonucleotide synthesis.
  • Fluorescent markers can readily be attached to nucleotides with activated linker arms incorporated into the probe.
  • Probes may be indirectly labeled by the methods disclosed above, by incorporating a nucleotide covalently linked to a hapten or other molecule such as biotin or digoxygenin, and performing a sandwich hybridization with a labeled antibody directed to that hapten or other molecule, or in the case of biotin, with avidin conjugated to a detectable label.
  • Antibodies and avidin may be conjugated with a fluorescent marker, or with an enzymatic marker such as alkaline phosphatase or horseradish peroxidase to render them detectable.
  • Conjugated avidin and antibodies are commercially available from companies such as Vector
  • Probes can be detected through a colorimetric reaction by providing a substrate for the enzyme. In the presence of various substrates, different colors are produced by the reaction, and these colors can be visualized to separately detect multiple probes. Any substrate known in the art may be used. Preferred substrates for alkaline phosphatase include 5-bromo-4-chloro-3-indolylphosphate (BCIP) and nitro blue tetrazolium (NBT). The preferred substrate for horseradish peroxidase is diaminobenzoate (DAB). Fluorescently labeled probes suitable for use in the in situ hybridization methods of the invention are preferably in the range of 150-500 nucleotides long. Probes may be DNA or RNA, preferably DNA.
  • Hybridization of the detectable probes to the cells is conducted with a probe concentration of 0.1-500 ng/ ⁇ L, preferably 5-250 ng/ ⁇ L.
  • the hybridization mixture will preferably contain a denaturing agent such as formamide.
  • hybridization is carried out at 25°C-45°C, more preferably at 32°C-40°C, and most preferably at 37°C-38°C.
  • the time required for hybridization is about 0.25-96 hours, more preferably 1 -72 hours, and most preferably for 4-24 hours.
  • Hybridization time will vary based on probe concentration and hybridization solution content which may contain accelerators such as hnRNP binding protein, trialkyl ammonium salts, lactams, and the like.
  • a denaturing agent such as formamide
  • concentrations of sodium chloride or in any solution that removes unbound and mismatched probe will vary depending on the stringency of hybridization desired. For example, high stringency washes may be carried out at 42°C-68°C, while intermediate stringency may be in the range of 37°C-55°C, and low stringency may be in the range of 30°C-37°C.
  • Salt concentration for a high stringency wash may be 0.5-1 times SSC (0.15M NaCl, 0.015M Na citrate), while medium stringency may be 1-4 times, and low stringency may be 2-6 times SSC.
  • the detection incubation steps should preferably be carried out in a moist chamber at 23°C-42°C, more preferably at 25°C-38°C and most preferably at 37-38°C.
  • Labeled reagents should preferably be diluted in a solution containing a blocking reagent, such as BSA, non-fat dry milk, or the like. Dilutions may range from 1 :10-1 :10,000, more preferably 1 :50-1 :5,000, and most preferably at 1 :100-
  • Slides may then be mounted and analyzed by microscopy in the case of a visible detectable marker, or by exposure to autoradiographic film in the case of a radioactive marker.
  • slides are preferably mounted in a solution that contains an antifade reagent, and analyzed using a fluorescence microscope. Multiple nuclei may be examined for increased accuracy of detection.
  • assays for the expression product of the LBC oncogene can also be used to determine whether the LBC oncogene mutation has occurred.
  • assays are immunoassays.
  • Immunoassays in their most simple and direct sense, are binding assays.
  • Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art.
  • ELISAs enzyme linked immunosorbent assays
  • RIA radioimmunoassays
  • protein-specific antibodies are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition containing the desired antigen, such as a clinical sample, is added to the wells. After binding and washing to remove non-specif ⁇ cally bound immune complexes, the bound antigen may be detected. Detection is generally achieved by the addition of another antibody, specific for the desired antigen, that is linked to a detectable label.
  • ELISA is a simple "sandwich ELISA.” Detection may also be achieved by the addition of a second antibody specific for the desired antigen, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
  • ELISA techniques are well known. Pn one such variation, the samples containing the desired antigen are immobilized onto the well surface and then contacted with the antibodies of the invention. After binding and appropriate washing, the bound immune complexes are detected. Where the initial antigen specific antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first antigen specific antibody, with the second antibody being linked to a detectable label.
  • gene expression portfolios are most preferred.
  • a portfolio of genes is a set of genes grouped so that expression information obtained about them provides the basis for making a clinically relevant judgment such as a diagnosis, prognosis, or treatment choice.
  • gene expression portfolios can be fashioned to help make therapeutic decisions regarding AML patients.
  • Diseased in this context, refers to an alteration of the state of a body that interrupts or disturbs, or has the potential to disturb, proper performance of bodily functions as occurs with the uncontrolled proliferation of cells.
  • someone is diagnosed with a disease when some aspect of that person's genotype or phenotype is consistent with the presence of the disease.
  • the act of conducting a diagnosis or prognosis may include the determination of disease/status issues such as determining the likelihood of relapse, type of therapy and therapy monitoring.
  • therapy monitoring clinical judgments are made regarding the effect of a given course of therapy by comparing the expression of genes over time to determine whether the gene expression profiles have changed or are changing to patterns more consistent with normal tissue.
  • Genes can be grouped so that information obtained about the set of genes in the group provides a sound basis for making a clinically relevant judgment such as a diagnosis, prognosis, or treatment choice. These sets of genes make up the portfolios of the invention. As with most diagnostic markers, it is often desirable to use the fewest number of markers sufficient to make a correct medical judgment. This prevents a delay in treatment pending further analysis as well unproductive use of time and resources.
  • One method of establishing gene expression portfolios is through the use of optimization algorithms such as the mean variance algorithm widely used in establishing stock portfolios. This method is described in detail in US patent publication number 20030194734.
  • the method calls for the establishment of a set of inputs (stocks in financial applications, expression as measured by intensity here) that will optimize the return (e.g., signal that is generated) one receives for using it while minimizing the variability of the return.
  • Many commercial software programs are available to conduct such operations.
  • "Wagner Associates Mean- Variance Optimization Application” referred to as “Wagner Software” throughout this specification, is preferred.
  • This software uses functions from the “Wagner Associates Mean- Variance Optimization Library” to determine an efficient frontier and optimal portfolios in the Markowitz sense is one option.
  • Use of this type of software requires that microarray data be transformed so that it can be treated as an input in the way stock return and risk measurements are used when the software is used for its intended financial analysis purposes.
  • the process of selecting a portfolio can also include the application of heuristic rules.
  • such rules are formulated based on biology and an understanding of the technology used to produce clinical results. More preferably, they are applied to output from the optimization method.
  • the mean variance method of portfolio selection can be applied to microarray data for a number of genes differentially expressed in subjects with cancer. Output from the method would be an optimized set of genes that could include some genes that are expressed in peripheral blood as well as in diseased tissue. If samples used in the testing method are obtained from peripheral blood and certain genes differentially expressed in instances of cancer could also be differentially expressed in peripheral blood, then a heuristic rule can be applied in which a portfolio is selected from the efficient frontier excluding those that are differentially expressed in peripheral blood.
  • the rule can be applied prior to the formation of the efficient frontier by, for example, applying the rule during data pre-selection.
  • Other heuristic rules can be applied that are not necessarily related to the biology in question. For example, one can apply a rule that only a prescribed percentage of the portfolio can be represented by a particular gene or group of genes.
  • Commercially available software such as the Wagner Software readily accommodates these types of heuristics. This can be useful, for example, when factors other than accuracy and precision (e.g., anticipated licensing fees) have an impact on the desirability of including one or more genes.
  • the gene expression profiles of this invention can also be used in conjunction with other non-genetic diagnostic methods useful in cancer diagnosis, prognosis, or treatment monitoring.
  • diagnostic power of the gene expression based methods described above with data from conventional markers such as serum protein markers (e.g., Cancer Antigen 27.29 ("CA 27.29”))-
  • CA 27.29 Cancer Antigen 27.29
  • blood is periodically taken from a treated patient and then subjected to an enzyme immunoassay for one of the serum markers described above. When the concentration of the marker suggests the return of tumors or failure of therapy, a sample source amenable to gene expression analysis is taken.
  • FNA fine needle aspirate
  • Kits made according to the invention include formatted assays for determining the gene expression profiles. These can include all or some of the materials needed to conduct the assays such as reagents and instructions and a medium through which Biomarkers are assayed.
  • Preferred methods for establishing gene expression profiles include determining the amount of RNA that is produced by a gene that can code for a protein or peptide or transcribe RNA. This is best accomplished by reverse transcription PCR (RT- PCR), competitive RT-PCR, real time RT-PCR, differential display RT-PCR, Northern Blot analysis and other related tests. While it is possible to conduct these techniques using individual PCR reactions, it is often desirable to amplify copy DNA (cDNA) or copy RNA (cRNA) produced from mRNA and analyze it via microarray.
  • cDNA copy DNA
  • cRNA copy RNA
  • Microarray technology measures steady-state mRNA levels of thousands of genes simultaneously thereby presenting a powerful tool for identifying AML patient gene expression profiles.
  • Two microarray technologies are currently in wide use. The first are cDNA arrays and the second are oligonucleotide arrays. Although differences exist in the construction of these chips, essentially all downstream data analysis and output are the same.
  • the products of these analyses are typically measurements of the intensity of the signal received from a labeled probe used to detect a cDNA sequence from the sample that hybridizes to a nucleic acid sequence at a known location on the microarray. Typically, the signal intensity is proportional to the cDNA quantity, and thus mRNA, expressed in the sample cells.
  • a large number of such techniques are available and useful. Preferred methods can be found in US Patents 6,271,002; 6,218,122; 6,218,1 14; and 6,004,755.
  • Analysis of the expression levels is conducted by comparing such intensities. This is best done by generating a ratio matrix of the expression intensities of genes in a test sample versus those in a control sample. For instance, the gene expression intensities from a tissue that has been treated with a drug can be compared with the expression intensities generated from the same tissue that has not been treated with the drug. A ratio of these expression intensities indicates the fold-change in gene expression between the test and control samples.
  • Gene expression profiles can be displayed in a number of ways. A common method is to arrange a ratio matrix into a graphical dendogram where columns indicate test samples and rows indicate genes. The data are arranged so genes that have similar expression profiles are proximal to each other. The expression ratio for each gene is visualized as a color.
  • a ratio less than one may appear in the blue portion of the spectrum while a ratio greater than one (indicating up-regulation) may appear as a color in the red portion of the spectrum.
  • Commercially available computer software programs are available to display such data including "GENESPR1NT” from Silicon Genetics, Inc. and “DISCOVERY” and “INFER” software from Partek, Inc.
  • the differentially expressed genes are either up regulated or down regulated in diseased cells, as deduced by an assessment of gene expression as described above.
  • Up regulation and down regulation are relative terms meaning that a detectable difference (beyond the contribution of noise in the system used to measure it) is found in the amount of expression of the genes relative to some baseline.
  • the baseline is the measured gene expression of a normal cell.
  • the genes of interest in the diseased cells are then either up regulated or down regulated relative to the baseline level using the same measurement method.
  • levels of up and down regulation are distinguished based on fold changes of the intensity measurements of hybridized microarray probes. A 1.5 fold difference is preferred for making such distinctions.
  • the treated cell is found to yield at least 1.5 times more, or 1.5 times less intensity than the untreated cells.
  • a 1.7 fold difference is more preferred and a 2 or more fold difference in gene expression measurement is most preferred.
  • One method of the invention involves comparing gene expression profiles for various genes to determine whether a person is likely to respond to the use of a therapeutic agent. Having established the gene expression profiles that distinguish responder from non-responder, the gene expression profiles of each are fixed in a medium such as a computer readable medium as described below.
  • a patient sample is obtained that contains diseased cells (such as hematopoietic blast cells in the case of AML) is then obtained.
  • the samples are of bone marrow and are extracted from the patient's sternum or iliac crest according to routine methods.
  • the bone marrow aspirate is processed to enrich for leukemic blast cells using routine methods.
  • RNA is then obtained and amplified from the diseased patient cells and a gene expression profile is obtained, preferably (in the case of a large gene portfolio) via micro-array, for genes in the appropriate portfolios.
  • the expression profiles of the samples are then compared to those previously analyzed for prognostic outcome.
  • a simple nucleic acid amplification and detection scheme is the most preferred method of measuring gene modulation.
  • PCR, NASBA, rolling circle, LCR, and other amplification schemes known to skilled artisans can be used with PCR being most preferred.
  • the portfolios include a large number of genes or it is desirable to measure the expression of numerous other genes then it is preferred to assess the expression patterns based on intensity measurements of microarrays as described above.
  • gene expression profile analysis can be conducted to monitor treatment response.
  • gene expression analysis as described above is conducted on a patient treated with any suitable treatment at various periods throughout the course of treatment. If the gene expression patterns are consistent with a positive outcome the patient's therapy is continued. If it is not, the patient's therapy is altered as with additional therapeutics, changes to the dosage, or elimination of the current treatment.
  • Such analysis permits intervention and therapy adjustment prior to detectable clinical indicia or in the face of otherwise ambiguous clinical indicia.
  • Methylation of genomic regions can affect gene expression levels. For example, hypermethylation of gene promoter regions can constitutively down-regulate gene expression whereas hypomethylation can lead to an increase in steady-state mRNA levels. As such, detection of methylated regions associated with genes predictive of drug response, prognosis or status can be used as an alternative method for diagnosing gene expression levels. Such methods are known to those skilled in the art. Alternatively, single nucleotide polymorphisms (SNPs) that are present in promoter regions can also affect transcriptional activity of a gene.
  • SNPs single nucleotide polymorphisms
  • Articles of this invention are representations of the gene expression profiles useful for treating, diagnosing, prognosticating, staging, and otherwise assessing diseases. Preferably they are reduced to a medium that can be automatically read such as computer readable media (magnetic, optical, and the like).
  • the articles can also include instructions for assessing the gene expression profiles in such media.
  • the articles may comprise a CD ROM having computer instructions for comparing gene expression profiles of the portfolios of genes described above.
  • the articles may also have gene expression profiles digitally recorded therein so that they may be compared with gene expression data from patient samples. Alternatively, the profiles can be recorded in different representational format. Clustering algorithms such as those incorporated in "DISCOVERY” and "INFER” software from Partek, Inc. mentioned above can best assist in the visualization of such data.
  • kits for conducting the assays described above Each such kit would preferably include instructions in human or machine readable form as well as the reagents typical for the type of assay described. These can include, for example, nucleic acid arrays (e.g. cDNA or oligonucleotide arrays), as described above, configured to discern the gene expression profiles of the invention.
  • nucleic acid arrays e.g. cDNA or oligonucleotide arrays
  • reagents used to conduct nucleic acid amplification and detection including, for example, reverse transcriptase, reverse transcriptase primer, a corresponding PCR primer set, a thermostable DNA polymerase, such as Taq polymerase, and a suitable detection reagent(s), such as, without limitation, a scorpion probe, a probe for a fluorescent probe assay, a molecular beacon probe, a single dye primer or a fluorescent dye specific to double-stranded DNA, such as ethidium bromide.
  • reagents used to conduct nucleic acid amplification and detection including, for example, reverse transcriptase, reverse transcriptase primer, a corresponding PCR primer set, a thermostable DNA polymerase, such as Taq polymerase, and a suitable detection reagent(s), such as, without limitation, a scorpion probe, a probe for a fluorescent probe assay, a molecular beacon probe, a single dye primer or a fluorescent dye specific to double-stranded
  • Kits for detecting surface antigens contain staining materials or are antibody based including components such as buffer, anti-antigenic antibody, detection enzyme and substrate such as Horse Radish Peroxidase or biotin-avidin based reagents.
  • Kit components for detecting blast cells generally include reagents for conducting flow cytometry, blast cell adhesion assays, and other common blast cell assays.
  • Conventional anti-cancer agents include, without limitation, tyrosine kinase inhibitors, MEK kinase inhibitors, P13K kinase inhibitors, MAP kinase inhibitors, apoptosis modulators and combinations thereof.
  • Exemplary drugs that are most preferred among these are the "GLEEVEC” tyrosine kinase inhibitor of Novartis, U-0126 MAP kinase inhibitor, PD-098059 MAP kinase inhibitor, SB-203580 MAP kinase inhibitor, and antisense, ribozyme, and DNAzyme, BcI-XL, and anti- apoptotics.
  • Examples of other useful drugs include, without limitation, the calanolides of US Patent 6,306,897; the substituted bicyclics of US Patent 6,284,764; the indolines of US Patent 6,133,305; and the antisense oligonucleotides of US Patent 6,271,210; platinum coordination compounds for example cisplatin or carboplatin, taxane compounds for example paclitaxel or docetaxel, camptothecin compounds for example irinotecan or topotecan, anti-tumor vinca alkaloids for example vinblastine, vincristine or vinorelbine, anti-tumor nucleoside derivatives for example 5-fluorouracil, gemcitabine or capecitabine, nitrogen mustard or nitrosourea alkylating agents for example cyclophosphamide, chlorambucil, carmustine or lomustine, anti-tumor anthracycline derivatives for example daunorubicin, doxorubicin or idar
  • Anti-cancer agents can also include therapeutics directed to gene therapy or antisense therapy or RNA interference. These include, without limitation, oligonucleotides with sequences complementary to an mRNA sequence can be introduced into cells to block the translation of the mRNA, thus blocking the function of the gene encoding the mRNA. The use of oligonucleotides to block gene expression is described, for example, in, Strachan and Read, Human Molecular Genetics, 1996. These antisense molecules may be DNA, stable derivatives of DNA such as phosphorothioates or methylphosphonates, RNA, stable derivatives of RNA such as 2'-O-alkylRNA, or other antisense oligonucleotide mimetics. Antisense molecules may be introduced into cells by microinjection, liposome encapsulation or by expression from vectors harboring the antisense sequence.
  • the gene of interest can be ligated into viral vectors that mediate transfer of the therapeutic DNA by infection of recipient host cells.
  • suitable viral vectors include retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, polio virus and the like.
  • therapeutic DNA can be transferred into cells for gene therapy by non-viral techniques including receptor-mediated targeted DNA transfer using ligand-DNA conjugates or adenovirus-ligand-DNA conjugates, lipofection membrane fusion or direct microinjection. These procedures and variations thereof are suitable for ex vivo as well as in vivo gene therapy. Protocols for molecular methodology of gene therapy suitable for use with the gene is described in Gene Therapy Protocols, edited by Paul D. Robbins, Human press, Totowa NJ, 1996.
  • the current study was part of an open label, multicenter, non-comparative phase 2 clinical study in which patients with relapsed or refractory AML (Harousseau et al. (2003)) were treated with tipifarnib at a starting oral dose of 600 mg bid for the first 21 consecutive days of each 28-day cycle.
  • Patients were enrolled into 2 cohorts, those with relapsed AML and those with refractory AML.
  • a total of 252 patients (135 relapsed and 1 17 refractory) were treated.
  • Eighty patients chose to provide bone marrow samples for RNA microarray analysis, for which a separate informed consent was required. The overall response rate was relatively low in this study.
  • response to tipifarnib was defined as patients who had an objective response (complete remission [CR], complete remission with incomplete platelet recovery [CRp] or partial remission [PR]), a hematological response (decrease of >50% of leukemic blast cells in bone marrow) as determined by either central review or by the clinical site, or stable disease (no hematological response but no progression of the disease) as determined by both central review and the clinical site.
  • Complete remission with incomplete platelet recovery was defined similarly, except for a platelet count less than 100,000/ ⁇ L sufficient to ensure transfusion independence.
  • Partial remission was defined as at least a 50% decrease in bone marrow blasts with partial neutrophil (>500/ ⁇ L) and platelet count (>50,000/ ⁇ L) recovery. Response had to be confirmed at least 4 weeks after first documentation.
  • RNA yield for several samples was too low to obtain enough labeled cRNA for chip hybridization using one round of amplification.
  • 11 ⁇ g ofcRNA were fragmented randomly by incubation at 94°C for 35 minutes in 40 mM Tris-acetate, pH 8.1 , 100 mM potassium acetate, and 30 mM magnesium acetate. Fragmented cRNA was hybridized to U133A arrays at 45°C for 16 hours in a rotisserie oven set at 60 rpm.
  • arrays were washed (with 6x SSPE and 0.5x SSPE containing Triton X-100 [0.005%]), and stained with streptavidin- phycoerythrin (SAPE; Molecular Probes, Eugene, OR). Quantification of bound labeled probe was conducted using the Agilent G2500A GeneArray scanner (Agilent Technologies, Palo Alto, CA).
  • Chip performance was quantitated by calculating a signal to noise ratio (raw average signal/noise). Chips were removed from further analysis if their signal-to- noise ratio was less than 5. Genes were only included in further analysis if they were called "present” in at least 10% of the chips. Eleven thousand seven hundred twenty three Affymetrix probe sets remained following this cut-off. The quality of the gene expression data were further controlled by identifying outliers based on principal components analysis and by analyzing the normal distributions of the gene intensities (Partek Pro V5.1). Statistical Analysis
  • TaqMan ® real-time RT-PCR was employed to verify the microarray results of the AHR and AKAP 13 genes.
  • cDNA was produced using T7 oligo(dT) primer and Superscript II reverse transcriptase according to the manufacturer's instructions (Invitrogen).
  • Primers and MGB-probes for AKAP 13 gene and control gene PBGD were designed using Primer Express (Applied Biosystems), while those for AHR gene and control gene HPRT were available as Assays-on-Demand from ABI.
  • Primer/probe sequences for AKAP 13 were as follows: AKAPl 3 forward, 5'ggtcagatgtttgccaaggaa3' (SEQ ID NO: 1); AKAP 13 reverse, 5'tcttcagaaacacactcccatc-3' (SEQ ID NO: 2); AKAP 13 probe, 6FAM-tgaaacggaagaagcttgtA-3' (SEQ ID NO: 3).
  • RT-PCR amplification mixtures contained 100 ng template cDNA, 2x TaqMan ® universal PCR master mix (12.5 ⁇ L; Applied Biosystems), 500 nM forward and reverse primers, and 250 nM probe. Reactions were run on an ABI PRISM 7900HT Sequence Detector (Applied Biosystems).
  • the cycling conditions were: 2 min of AmpErase UNG activation at 5O 0 C, 10 min of polymerase activation at 95 0 C and 50 cycles at 95 0 C for 15 sec and annealing temperature (59 0 C or 6O 0 C) for 60 sec.
  • a standard curve and a no-template control along with template cDNA were included in triplicates for the gene of interest and control gene.
  • the relative quantity of each gene was calculated based on the standard curve, and was normalized with the quantity of the control gene.
  • the median coefficient of variation (based on calculated quantities) of triplicate samples was 8%.
  • the correlation between repeated runs using independently diluted templates from the stock was above 0.95. Samples were only compared with microarray data if duplicate TaqMan ® experiments showed reproducible results.
  • the AKAPl 3 vectors, oncoLBC and protoLBC, and vector control (pSRalpha- neo) were obtained from Dr. Deniz Toksoz. Zheng et al. (1995).
  • the HL60 cell line was obtained from the American Tissue Culture Collection and grown in RPMI 1640 with 10% FBS. Cells were transiently transfected with each vector using the Effectene reagent (Qiagen) according to the manufacturer's instructions and kept under G418 (600 ⁇ g/mL) for 7 days. Tipifarnib was then added in various concentrations (0, 1.5, 3.1, 6.3, 13, 25, 50, 100, 200, 1000, and 10,000 nM) to duplicate cultures (1.5x10 5 cells/mL). Cells were counted at Day 6. Cell counts were normalized to cultures with no drug to give a percent of viable control cells. Results
  • FTIs were originally designed to specifically inhibit FTase activity, thereby blocking the oncogenic ras pathways. Therefore, we initially analyzed DNA from the bone marrow of 80 patients with relapsed or refractory AML for activating ras mutations and investigated the possible correlation between ras mutation and the response to tipifarnib. While 26% of the AML samples harbored N-ras mutations, mutation status did not correlate with objective response or overall survival. Harousseau et al. (2003). We therefore performed gene expression profiling to identify novel signatures that could be used to predict response to the FTI tipifarnib. Bone marrow samples were obtained for gene expression analysis from 80 patients prior to treatment with tipifarnib. Table 1 shows the patient information. Table 1. Patient Information
  • the gene expression data were integrated with the clinical information and retrospective analyses were performed to identify genes that could separate responders from non-responders with a high level of sensitivity.
  • the data went through several filtering steps before identification of differentially expressed genes.
  • genes that were not expressed in at least 10% of the samples were removed. This reduced the number of genes from approximately 22,000 to 1 1,723 genes.
  • genes that showed little variation in expression across the dataset were also excluded and quantile normalization was applied to the remaining 5,728 genes.
  • an unsupervised k-means clustering analysis was performed to identify any differences between patients based on their global gene expression profiles. Six main clusters of patients were identified using this technique.
  • TaqMan real time RT-PCR was performed on cDNA that was used for generating the labeled target cRNA for microarray hybridization. Two genes were selected to verify the gene expression data. The AHR and AKAP 13 genes were chosen because the use of these genes resulted in the highest level of specificity for responders. The correlation coefficient was 0.74 for AHR and 0.94 for AKAP 13 indicating that the microarray gene expression data could be validated by PCR ( Figure 2). Identification of the AKAP 13 Gene as a Marker of Resistance AKAP 13 was over-expressed in patients who were resistant to tipifarnib.
  • the overall diagnostic accuracy was 69% and positive likelihood ratio of responding was 2.4. Therefore, stratification of this patient population based on AKAP 13 gene expression increased the response rate from 24% (14/58) in the entire group to 43% (13/30) among those patients with low expression of the gene.
  • LOOCV was used to identify a candidate set of gene markers that could predict response to tipifarnib with an improved accuracy compared to AKAPl 3 alone.
  • the AKAP 13 gene was the most robust marker of resistance to tipifarnib. We therefore investigated its involvement in FTI biology by over-expressing the oncoLBC and protoLBC variants of this gene in the HL60 cell line. Transient transfectants were then tested for sensitivity to tipifarnib. Over-expression of both AKAP 13 variants in this AML cell line model led to an approximate 20-fold increase in resistance to tipifarnib compared to control cells ( Figure 3). Both the LBC oncogene and proto-oncogene increased the resistance to tipifarnib to the same extent, as seen by a parallel rightward shift of the kill curves by more than one log- unit compared to control. Discussion
  • AKAP 13 lymphoid blast crisis oncogene
  • RhoB and lamin B are farnesylated and are candidate targets of FTIs.
  • AKAP 13 is also a proto-oncogene, because loss of its 3-prime end causes cellular transformation. Sterpetti et al. (1999). While it was originally identified from a patient with chronic myeloid leukemia, its expression has not been documented in AML.
  • Rho proteins are potentially important antitumorigenic targets for FTIs. Sahai et al. (2002); and Lancet et al. (2003). RhoB, RhoA, and RhoC have been found to be over-expressed in multiple cancer types. Sahai et al. (2002).
  • RhoH is frequently re-arranged in tumors of myeloid origin, and this may lead to its over-expression.
  • Pasqualucci et al. 2001. While most of these Rho proteins are geranygeranylated, they interact closely with each other and the farnesylated ras, RhoE, and RhoB small GTPases. Sahai et al. (2002); and Li et al. (2002).
  • RhoH, RhoB, and RhoE can act in an antagonistic fashion to the transforming abilities of RhoA and RhoG. Li et al. (2002).
  • RhoA guanine nucleotide exchange factor lymphoid blast crisis oncogene
  • AKAP13 may increase mitotic activity by activating lamin B via protein kinase A. Foisner et al. (1991).
  • the IL3 receptor activates ras pathways. Testa et al. (2004). Therefore, as indicated in Figure 7, the increased activity of IL3RA and AKAP 13, and the decrease in RhoH expression could lead to an increased cellular profile of transformation.
  • FTIs may be more effective in blocking these pathways.
  • AKAP 13 both oncoLBC and protoLBC variants
  • IC 50 of the HL60 AML cell line increased by approximately 20-fold. This indicates that over-expression of AKAP 13 is a relevant marker of resistance and that it may also be a useful alternative drug target for patients who are resistant to tipifarnib.
  • the 3-gene signature can predict prognosis irrespective of the type of drug treatment. To determine this, we first evaluated a gene-expression signature recently identified in newly diagnosed AML patients who were treated with conventional chemotherapy. Bullinger et al. (2004). This signature was defined using a cDNA array and therefore we first matched these genes with the probes present on the Affymetrix gene chip. Of the 133 predictive genes identified by Bullinger et al., 167 probe sets (corresponding to 103 unique genes) were matched to the Affymetrix U133A chip. The 3 genes identified in our present analysis are not present in the
  • the current study was an open label, multicenter, non-comparative Phase 2 study investigating the efficacy and safety of farnesyl transferase inhibition with tipifarnib administered as a single agent, at a starting oral dose of 600 mg b.i.d. for the first 21 days of each 28 day cycle in AML.
  • Subjects were enrolled into two cohorts, those with relapsed AML and those with refractory AML. A total of 252 patients (135 relapsed and 117 refractory) were treated.
  • CR, CRp, or PR objective response
  • hematological response decrease of >50% of leukemic blast cells
  • Sample Collection and Microarray Processing All samples were obtained from patients who had consented to the described processing and analyses. Bone marrow samples were collected from patients before treatment with tipifarnib, diluted with PBS and centrifuged with Ficoll-diatrizoate (1.077g/ml).
  • RNA quality was checked using the Agilent Bioanalyzer. Synthesis of cDNA and cRNA were performed according to Affymetrix (Santa Clara, CA) protocols. Two rounds of linear amplification were performed because the RNA yield for several samples was too low to obtain enough labeled cRNA for chip hybridization using one round of amplification.
  • Quantification of bound labeled probe was conducted using the Agilent G2500A GeneArray scanner (Agilent Technologies, Palo Alto, CA). The total fluorescence intensity for each array was scaled to the uniform value of 600. Chip performance was quantitated by calculating a signal to noise ratio (raw average signal/noise). Chips were removed from further analysis if their signal-to- noise ratio was less than 5. Genes were only included in further analysis if they were called "present" in at least 10% of the chips. Approximately 12,000
  • Affymetrix probe sets remained following this cut-off. Gene expression data quality was further controlled by identifying outliers based on principal components analysis and by analyzing the normal distributions of the gene intensities (Partek Pro V5.1). Statistical Analysis
  • Unsupervised hierarchical clustering and clustering was performed in Omniviz. Kaplan-Meier analysis was performed using S-Plus.
  • a prognostic signature identified in de novo AML has utility in relapsed and refractory patients
  • the 167 probe sets are listed in the Sequence Listing Table and designated the SEQ
  • the prognostic signature is independent of a 3-gene signature that predicts response to tipifarnib.
  • AKAP13 The expression value for AKAP13 in each patient is shown in Fig. 12c. Therefore, for the group of patients with low expression of AKAP 13, the response rate to tipifarnib was 31% (8/26) compared to 18% (10/54) in the current patient population. Using the AKAPl 3 gene, Kaplan-Meier analysis showed a significant difference in survival between the predicted responder group and the non-responder group (Fig. 12d).
  • AUC area under the curve from receiver operator characteristic analysis. This is an indication of the overall diagnostic accuracy.
  • Tipifarnib (ZARNESTRA®, Rl 15777), has demonstrated clinical response in patients with hematological disease. While the inhibition of protein farnesylation is the primary mechanism of action (MOA), the level of farnesyl inhibition is not a reliable pharmacodynamic marker of response, nor is it clear what genetic markers can be employed to predict response.
  • Bone marrow samples were analyzed using the Affymetrix U133A GeneChip® array. Global gene expression signatures revealed that tipifarnib treatment resulted in gene expression changes that were maintained for up to 120 days following treatment termination. Pretreatment vs post-treatment samples identified approximately 500 genes that had significant changes (False Discovery Rate [FDR] ⁇ 0.005) in gene expression following farnesyl transferase inhibition, including several genes associated with farnesylation (eg K-ras, FNTA). Many of the modulated genes were identified as those significantly involved in protein biosynthesis, DNA replication, intracellular signaling, and cell cycle pathways, thus, reflective of inhibition of cellular proliferation.
  • FDR Fealse Discovery Rate
  • Affymetrix U133A chip which can probe for approximately 22,000 genes (Fig. 13). Chip data were pre-filtered to remove poor quality data and genes that were not expressed in at least 10% of the patient samples. In addition genes that did not vary across the dataset were removed (CV ⁇ 40%). Approximately, 8000 genes remained for further analysis. A total of 79 chips passed quality control measures and also had associated clinical response data.
  • LBC oncogene-derived peptide is synthesized, coupled to keyhole limpet hemocyanin, and used to immunize rabbits for production of polyclonal antibodies.
  • the sera are tested for reactivity against the corresponding peptide with ELlSA, and the positive batches are affinity-purified.
  • the purified antibody specifically detects the peptide that has the epitope in tissue sections. This is verified by complete abolishment of the signal if the corresponding peptide is added simultaneously with the antibody.
  • monoclonal antibodies able to detect the protein in its natural fold are produced.
  • a purified antigen produced in mammalian cells to ensure natural fold and posttranslational modifications, is generated.
  • the antigen LBC onco protein-IgG constant part fusion protein
  • the antigen is expressed in mouse myeloma cells, and the protein is purified using the Fc part as bait.
  • This purified antigen is recognized in Western blot by the C-terminal polyclonal antibody.
  • the antigen is used to generate mouse monoclonal antibodies against LBC peptides by selecting out of the positive clones those that produce antibodies that react against LBC peptide instead of the IgG constant part. Kits for the clinical identification of LBC oncogene can be readily fashioned employing these and similar antibodies. Such kits would include antibodies directed to LBC peptide identification (and hence,
  • LBC oncogene LBC oncogene
  • appropriate indicator reagents e.g., enzymes, labels, and the like
  • other reagents useful in the clinical application of such a kit such as dilution buffers, stabilizers, and other materials typically used in such assays.
  • An affinity-purified polyclonal antibody against the C-terminal peptide of LBC oncogene is used for the IHC detection and localization of LBC oncogene.
  • Four ⁇ m sections from formalin-fixed and paraffin embedded normal and tumor tissue is mounted on 3-aminopropyl-triethoxy-silane (APES, Sigma, St. Louis, MO) coated slides.
  • the sections are deparaffinized and rehydrated in graded concentrations of ethanol and treated with methanolic peroxide (0.5% hydrogen peroxide in absolute methanol) for 30 minutes at room temperature to block the endogenous peroxidase activity.
  • Antigen retrieval is done in a microwave oven twice for 5 minutes (650W).
  • An Elite ABC Kit (Vectastain, Vector Laboratories, Burlingame, CA) is used for immunoperoxidase staining.
  • the LBC peptide antibody is used at an optimal dilution of 1 :2000.
  • Both the biotinylated second antibody and the peroxidase- labeled avidin-biotin complex are incubated on the sections for 30 minutes.
  • the dilutions are made in PBS (pH 7.2), and all incubations are carried out in a moist chamber at room temperature. Between the different staining steps the slides are rinsed three times with PBS.
  • the peroxidase staining is visualized with a 3-amino- 9-ethylcarbazole (Sigma) solution (0.2 mg/ml in 0.05 M acetate buffer containing 0.03% hydrogen peroxide, pH 5.0) at room temperature for 15 minutes. Finally, the sections are lightly counterstained with Mayer's haematoxylin and mounted with aqueous mounting media (Aquamount, BDH). In control experiments the primary antibodies are replaced with the IgG fraction of normal rabbit serum or the primary antibody was preabsorbed with the LBC peptide. These stainings indicate the presence of the LBC oncogene in a subset of cells.
  • RhoH is GTPase deficient and modulates activities of other Rho GTPases by an inhibitory function

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