Patent Application
20140329714
Throughout this application various publications are referred to by superscripts. Full citations for these references may be found at the end of the specification. The disclosures of these publications are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in the adult population and accounts for about 40% of newly diagnosed non-Hodgkin lymphoma cases.1 When treated with anthracyclin-based chemotherapy regimens such as a combination of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), the 5-year overall survival rate of DLBCL is approximately 50%.2 Addition of Rituximab to the standard CHOP regimens (R-CHOP) results in an improvement of overall survival rate by 10 to 15%.3 Nevertheless, a substantial number of patients still succumb to the disease and hence improvements in therapy remain a necessary and important task.
DLBCL is a biological and clinical heterogenous disease, which is rooted at least in part in the diversity of its normal cell counterparts.4 Based upon their gene expression similarities to either normal germinal center (GC) B cells or activated peripheral blood B cells, DLBCLs can be classified into two main subcategories: germinal center B-cell-like (GCB) DLBCL and activated B-cell-like (ABC) DLBCL.5,6 The GCB-DLCBL subtype represents transformed counterpart of normal GC centroblasts as both highly express the GC master regulator BCL6 and lack B cell activation features. In comparison, the ABC-DLBCL subtype likely corresponds to cells arrested at the late GC/pre-plasmablastic stage of maturation,6 is characterized by constitutively activated NF-κB and shows activation of Jak/STAT3 signaling.7-9 Signal transducer and activator of transcription 3 (STAT3) activation has been identified as an oncogenic event in multiple malignancies, and in ABC-DLBCL cell lines, inhibition of STAT3 signaling leads to tumor cell apoptosis.8-9
The biological difference between the GCB- and ABC-DLBCL subgroups also transpires to different responses to therapy, with GCB-DLBCL having significantly better overall survival rates when treated with the CHOP regimen.10 Although the survival outcome of ABC-DLBCL patients has been notably improved with the R-CHOP therapy, the survival difference between ABC- and GCB-DLBCL still persists.11-14 It is thus important to identify novel biomarkers that can risk-stratify the ABC-DLBCL patients in the R-CHOP era in order to guide development of targeted therapy.
Aberrantly activated STAT3 has been shown to be oncogenic in a number of malignancies. In normal cells, STAT3 activation in response to growth factor or cytokine receptor signaling is a transient and tightly controlled process due to rapid activation and self-inactivation cycles.15 In cancer, constitutive activation of the STAT3 signaling pathway promotes tumor cell growth, survival, angiogenesis, and metastasis.16 Through inflammatory mediators in the tumor microenvironment, tumor cells with activated STAT3 can evade immune surveillance by inhibiting anti-tumor immune responses.17 In lymphoid malignancies, a pathogenic role of STAT3 has been shown in multiple myeloma, Hodgkin's lymphoma, anaplastic large T-cell lymphoma, and recently, in ABC-DLBCL.8,9,18-21 The STAT3 gene is a direct target of BCL6-mediated transcription repression such that BCL6 positive normal GC B cells and GCB-DLBCLs are largely STAT3-low or negative.8 Furthermore, treating cultured ABC-DLCBL cells with specific siRNA against STAT3 or a Jak inhibitor induces cell cycle arrest and apoptosis.8,9 Analysis by Lam et al further suggested that, in ABC-DLBCL cells, constitutively activated NF-κB pathway may indirectly activate Jak/STAT3 pathway by upregulating the STAT3-activating cytokine IL-6 and/or IL-10.9
The present invention addresses the need, using STAT3 activation, for improved methods that can be used for prognosis and risk-stratified therapy of DLBCL patients.
The present invention provides methods of classifying a human patient with diffuse large B-cell lymphoma (DLBCL), the method comprising determining mRNA expression levels of human genes in a DLBCL biopsy specimen from the patient, wherein the genes comprise HSD17B4, RNF149, ZNF805, SLC2A13, RHEB, MT1X, NAT8L, C15orf29, ZNF420, PCNX and SLA, so as to classify the DLBCL patient based on expression levels.
The invention also provides methods for classifying a human patient with diffuse large B-cell lymphoma (DLBCL), the methods comprising determining mRNA expression levels of human genes in a DLBCL biopsy specimen from the patient, wherein the genes comprise Module A genes MEX3D, BATF, CAPN2, CCND2, CD2, CMTM3, DYNLT1, ELL2, GALNT1, GCA, GMFG, GYG1, GZMB, MAN1A1, MT1X, PERP, PLAGL1, PRF1, RAB27A, S100A6, SERPINB1, TTC39C, XK, ZBED2 and ZNRF1, and Module B genes BTLA, C13orf18, CFLAR, EV12A, HIST2H2AA3, IL16, IL2RA and PTGER4; and comparing the expression levels of Module A genes with the expression levels of Module B genes so as to classify the DLBCL patient based on the mRNA expression levels.
The invention also provides methods of determining the prognosis of a diffuse large B-cell lymphoma (DLBCL) patient undergoing treatment with rituximab in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), or treatment with rituximab in combination with cyclophosphamide, mitoxantrone, vincristine, and prednisone (R-CNOP), the method comprising determining the level of phospho-Tyr705-STAT3 (PY-STAT3) in a DLBCL biopsy specimen from the patient using immunohistochemistry, wherein PY-STAT3 positivity predicts a poor likelihood of survival in comparison to a patient with PY-STAT3 negativity.
The invention further provides methods of determining the prognosis of a diffuse large B-cell lymphoma (DLBCL) patient undergoing treatment with a combination of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), or with a combination of cyclophosphamide, mitoxantrone, vincristine, and prednisone (CNOP), the method comprising determining the level of phospho-Tyr705-STAT3 (PY-STAT3) and the level of BCL6 in a DLBCL biopsy specimen from the patient using immunohistochemistry, wherein PY-STAT3 positivity and BCL6 negativity predicts a poor likelihood of survival in comparison to a patient who is not PY-STAT3 positive and BCL6 negative.
The invention also provides a gene expression profile that is predictive of activation of signal transducer and activator of transcription 3 (STAT3), wherein the profile comprises expression of a plurality of, or all of, the following genes: MEX3D, BATF, CAPN2, CCND2, CD2, CMTM3, DYNLT1, ELL2, GALNT1, GCA, GMFG, GYG1, GZMB, MAN1A1, MT1X, PERP, PLAGL1, PRF1, RAB27A, S100A6, SERPINB1, TTC39C, XK, ZBED2, ZNRF1, BTLA, C13orf18, CFLAR, EV12A, HIST2H2AA3, IL16, IL2RA and PTGER4.
The invention provides a microarray for classifying a human patient with diffuse large B-cell lymphoma (DLBCL), where the microarray comprises nucleic acid probes for genes HSD17B4, RNF149, ZNF805, SLC2A13, RHEB, MT1X, NAT8L, C15orf29, ZNF420, PCNX and SLA.
The invention also provides a microarray for classifying a human patient with diffuse large B-cell lymphoma (DLBCL), where the microarray comprises nucleic acid probes for genes MEX3D, BATF, CAPN2, CCND2, CD2, CMTM3, DYNLT1, ELL2, GALNT1, GCA, GMFG, GYG1, GZMB, MAN1A1, MT1X, PERP, PLAGL1, PRF1, RAB27A, S100A6, SERPINB1, TTC39C, XK, ZBED2, ZNRF1, BTLA, C13orf18, CFLAR, EV12A, HIST2H2AA3, IL16, IL2RA and PTGER4.
The invention provides a gene expression-based method for classifying a human patient with diffuse large B-cell lymphoma (DLBCL), where the method comprises using nucleic acid probes for detecting expression of genes HSD17B4, RNF149, ZNF805, SLC2A13, RHEB, MT1X, NAT8L, C15orf29, ZNF420, PCNX and SLA.
The invention also provides a gene expression-based method for classifying a human patient with diffuse large B-cell lymphoma (DLBCL), where the method comprises using nucleic acid probes for detecting expression of genes MEX3D, BATF, CAPN2, CCND2, CD2, CMTM3, DYNLT1, ELL2, GALNT1, GCA, GMFG, GYG1, GZMB, MAN1A1, MT1X, PERP, PLAGL1, PRF1, RAB27A, S100A6, SERPINB1, TTC39C, XK, ZBED2, ZNRF1, BTLA, C13orf18, CFLAR, EV12A, HIST2H2AA3, IL16, IL2RA and PTGER4.
The invention provides a method of classifying a human patient with diffuse large B-cell lymphoma (DLBCL), the method comprising determining STAT3 mRNA expression level in a DLBCL biopsy specimen from the patient, and comparing the level of STAT3 mRNA expression from the patient with the level of expression of STAT3 mRNA from a cohort of DLBCL patients, wherein a patient with a level of STAT3 mRNA expression that is greater than 1 standard deviation above the mean level of STAT3 mRNA expression in the cohort has a less favorable survival outcome compared to patients having a level of STAT3 mRNA expression that is less than 1 standard deviation below the mean level of STAT3 mRNA expression in the cohort.
The present invention provides a method of classifying a human patient with diffuse large B-cell lymphoma (DLBCL), the method comprising determining mRNA expression levels of human genes in a DLBCL biopsy specimen from the patient, wherein the genes comprise HSD17B4, RNF149, ZNF805, SLC2A13, RHEB, MT1X, NAT8L, C15orf29, ZNF420, PCNX and SLA (i.e., the “11 gene signature”), so as to classify the DLBCL patient based on expression levels.
For example, the patient can be classified into a subgroup by comparing the expression of this 11-gene signature from the patient with the expression of the same signature from a cohort of DLBCL patients who have already been classified into expression subgroups. Preferred subgroups include quartiles. A patient classified into the bottom 50% subgroup has a more favorable outcome of survival compared to patients in the top gene expression quartile.
For another example, in the non-GCB/ABC subgroup, a patient classified into the bottom gene expression quartile has a more favorable survival outcome compared to patients in the other quartile subgroups.
The invention also provides a gene expression signature that is predictive of activation of signal transducer and activator of transcription 3 (STAT3), wherein the profile comprises expression of a plurality of, or all of, the following genes: HSD17B4, RNF149, ZNF805, SLC2A13, RHEB, MT1X, NAT8L, C15orf29, ZNF420, PCNX and SLA.
The expression levels of all genes described in the present invention can be normalized to the level of expression of a “housekeeping gene” that is required for the maintenance of basic cellular function. Examples of housekeeping genes include, but are not limited to, ACTB, GAPDH, and STAT1.
An average expression of the gene signature can be obtained, for example, by taking normalized microarray signals for each probe (e.g., as in Table 7), and calculating the mean value.
The invention also provides a method of classifying a human patient with diffuse large B-cell lymphoma (DLBCL), the method comprising
determining mRNA expression levels of human genes in a DLBCL biopsy specimen from the patient, wherein the genes comprise Module A genes MEX3D, BATF, CAPN2, CCND2, CD2, CMTM3, DYNLT1, ELL2, GALNT1, GCA, GMFG, GYG1, GZMB, MAN1A1, MT1X, PERP, PLAGL1, PRF1, RAB27A, S100A6, SERPINB1, TTC39C, XK, ZBED2 and ZNRF1, and Module B genes BTLA, C13orf18, CFLAR, EV12A, HIST2H2AA3, IL16, IL2RA and PTGER4; and
determining the expression levels of Module A genes and Module B genes so as to classify the DLBCL patient based on the expression levels.
Preferably, the genes for which expression is determined are predictive of activation of signal transducer and activator of transcription 3 (STAT3).
The DLBCL patient can be classified into a subgroup by comparing the expression of Module A and Module B genes from the patient with the expression of the same gene signature from a cohort of DLBCL patients who have already been classified into expression subgroups. The DLBCL patient can be classified into one of four clusters depending on the levels of expression of the Module A genes and of the Module B genes. For example, the patient can be classified in Cluster 1 if the majority of genes in Module A is downregulated and if the majority of genes in Module B is downregulated; the patient can be classified in Cluster 2 if the majority of genes in Module A is upregulated and if the majority of genes in Module B is not upregulated; the patient can be classified in Cluster 3 if the majority of genes in Module A is upregulated and if the majority of genes in Module B is upregulated; and/or the patient can be classified in Cluster 4 if the majority of genes in Module A is not upregulated and if the majority of genes in Module B is upregulated.
The invention provides a method of classifying a human patient with diffuse large B-cell lymphoma (DLBCL), the method comprising
determining mRNA expression levels of human genes in a DLBCL biopsy specimen from the patient, wherein the genes comprise Module A genes MEX3D, BATF, CAPN2, CCND2, CD2, CMTM3, DYNLT1, ELL2, GALNT1, GCA, GMFG, GYG1, GZMB, MAN1A1, MT1X, PERP, PLAGL1, PRF1, RAB27A, S100A6, SERPINB1, TTC39C, XK, ZBED2 and ZNRF1, and Module B genes BTLA, C13orf18, CFLAR, EV12A, HIST2H2AA3, IL16, IL2RA and PTGER4; and
comparing the expression levels of Module A genes and Module B genes from the patient with the expression of the Module A genes and Module B genes from a cohort of DLBCL patients who have already been classified into expression subgroups,
wherein the patient is classified in Cluster 1 if the majority of genes in Module A is downregulated and if the majority of genes in Module B is down-regulated;
wherein the patient is classified in Cluster 2 if the majority of genes in Module A is upregulated and if the majority of genes in Module B is not upregulated;
wherein the patient is classified in Cluster 3 if the majority of genes in Module A is upregulated and if the majority of genes in Module B is upregulated; and
wherein the patient is classified in Cluster 4 if the majority of genes in Module A is not upregulated and if the majority of genes in Module B is upregulated,
so as to classify the DLBCL patient based on the expression levels.
The patient can be classified into one of four clusters, for example, by comparing the expression of Module A genes and Module B genes from the patient with the expression of Module A genes and Module B genes from a cohort of DLBCL patients who have already been classified into one of the four clusters. For example, this may be performed by comparing the gene expression pattern obtained from the patient to at least the expression profile associated with one of the four clusters, determining the degree of similarity between the gene expression pattern obtained from the patient and the expression profile associated with at least one of the four clusters, and based on the degree of similarity, classifying the patient into one of the four clusters. For example, a high degree of similarity between the gene expression pattern obtained from the patient and the expression profile for Cluster 1 will lead to classification of the patient into Cluster 1.
An example of one such cohort of subjects with DLBCL is a cohort of 233 DLBCL cases for which both gene expression profile and clinical data are available (National Center for Biotechnology Information (NCBI, Bethesda Md.) accession number GSE10846). See also Lenz et al. (2008).14 These subjects were treated with the R-CHOP regimen, which is rituximab (R) in combination with cyclophosphamide, hydroxydaunorubicin (doxorubicin), vincristine, and prednisone (CHOP).
The patient can also be classified into one of four clusters by determining ypred for Module A and ypred for Module B, where ypred=b0+b1x1+b2x2+ . . . +bnxn, wherein x1, x2 . . . xn is the expression value of each gene, and where the coefficients b0, b1 . . . +bn are set forth in Table 5;
wherein the patient is classified in Cluster 1 if ypred for Module A and ypred for Module B are both negative;
wherein the patient is classified in Cluster 2 if ypred for Module A is positive and if ypred for Module B is negative;
wherein the patient is classified in Cluster 3 if ypred for Module A and ypred for Module B are both positive; and
wherein the patient is classified in Cluster 4 if ypred for Module A is negative and if ypred for Module B is positive.
The invention provides a method of classifying a human patient with diffuse large B-cell lymphoma (DLBCL), the method comprising
determining mRNA expression levels of human genes in a DLBCL biopsy specimen from the patient, wherein the genes comprise Module A genes MEX3D, BATF, CAPN2, CCND2, CD2, CMTM3, DYNLT1, ELL2, GALNT1, GCA, GMFG, GYG1, GZMB, MAN1A1, MT1X, PERP, PLAGL1, PRF1, RAB27A, S100A6, SERPINB1, TTC39C, XK, ZBED2 and ZNRF1, and Module B genes BTLA, C 13orf18, CFLAR, EV12A, HIST2H2AA3, IL16, IL2RA and PTGER4; and
classifying the patient into one of four clusters by determining ypred for Module A and ypred for Module B, where ypred=b0+b1x1+b2×2+ . . . +bnxn, where x1, x2 . . . xn is the expression value of each gene, and where the coefficients b0, b1 . . . +bn are set forth in Table 5;
wherein the patient is classified in Cluster 1 if ypred for Module A and ypred for Module B are both negative;
wherein the patient is classified in Cluster 2 if ypred for Module A is positive and if ypred for Module B is negative;
wherein the patient is classified in Cluster 3 if ypred for Module A and ypred for Module B are both positive; and
wherein the patient is classified in Cluster 4 if ypred for Module A is negative and if ypred for Module B is positive,
so as to classify the DLBCL patient.
The invention provides a method of classifying a human patient with diffuse large B-cell lymphoma (DLBCL), the method comprising determining STAT3 mRNA expression level in a DLBCL biopsy specimen from the patient, and comparing the level of STAT3 mRNA expression from the patient with the level of expression of STAT3 mRNA from a cohort of DLBCL patients, wherein a patient with a level of STAT3 mRNA expression that is greater than 1 standard deviation above the mean level of STAT3 mRNA expression in the cohort has a less favorable survival outcome compared to patients having a level of STAT3 mRNA expression that is less than 1 standard deviation below the mean level of STAT3 mRNA expression in the cohort.
The Entrez Gene (National Center for Biotechnology Information) and HUGO Gene Nomenclature Committee (HGNC) gene identification numbers, and probe set, for Module A and Module B genes are as follows:
The levels of expression of these genes can be determined, for example, using standard gene expression microarray procedures. A microarray contains, for example, a plurality of nucleic acid probes coupled to the surface of a substrate in different known locations. Microarrays are well known in the art and can be obtained, for example from Affymetrix (Santa Clara, Calif.). Gene expression data can also be obtained using, for example, reverse transcription-polymerase chain reaction (RT-PCR).
Classification of the DLBCL patient can aid in predicting the treatment that may be most beneficial for the patient.
In one embodiment, a patient classified in Cluster four is predicted to be the least likely to benefit from therapy with rituximab in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), compared to a patient in Cluster one, two or three.
In one embodiment, a DLBCL patient undergoing therapy with a combination of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) classified in Cluster two is predicted to have a more favorable likelihood of survival compared to a patient classified in Cluster three.
In one embodiment, a patient classified in Cluster one or three is predicted to benefit more from therapy with rituximab in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), compared to a patient in Cluster two or four.
The invention also provides a method of determining the prognosis of a diffuse large B-cell lymphoma (DLBCL) patient undergoing treatment with rituximab in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), or treatment with rituximab in combination with cyclophosphamide, mitoxantrone, vincristine, and prednisone (R-CNOP), the method comprising determining the level of phospho-Tyr705-STAT3 (PY-STAT3) in a DLBCL biopsy specimen from the patient using immunohistochemistry, wherein PY-STAT3 positivity predicts a poor likelihood of survival in comparison to a patient with PY-STAT3 negativity.
The invention also provides a method of determining the prognosis of a diffuse large B-cell lymphoma (DLBCL) patient undergoing treatment with rituximab in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), or treatment with rituximab in combination with cyclophosphamide, mitoxantrone, vincristine, and prednisone (R-CNOP), the method comprising determining the level of phospho-Tyr705-STAT3 (PY-STAT3) in a DLBCL biopsy specimen from the patient using immunohistochemistry; and
determining PY-STAT3 positivity or negativity by scoring the intensity of PY-STAT3 staining using a 4-tiered scale (0, 3, 6, 9), scoring the percentage of PY-STAT3 stained DLBCL tumor cells using a 10-tiered scale (0-9), and multiplying the two scores together to obtain a case score for the patient, where a case score with a value of 15 or greater is considered positive and a case score with a value below 15 is considered negative;
wherein PY-STAT3 positivity predicts a poor likelihood of survival in comparison to a patient with PY-STAT3 negativity.
An antibody for PY-STAT3 can be obtained, for example, from Cell Signaling Technology (Catalog #9131). Double immunostaining for PY-STAT3 and CD20 can be performed to obtain tumor cell-specific PY-STAT3 expression. CD20 antibody can be obtained, for example, from Dako, Carpinteria, Calif. or from LabVision (Clone L26).
The patient can be a non-germinal center B-cell-like (non-GCB) DLBCL patient. DLBCL patients can be classified as germinal center B-cell-like- (GCB-) and non-GCB-DLBCL patients using, for example, expression of CD10, BCL6 and MUM1 as described by Hans et al. (2004).22
The invention further provides a method of determining the prognosis of a diffuse large B-cell lymphoma (DLBCL) patient undergoing treatment with a combination of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), or with a combination of cyclophosphamide, mitoxantrone, vincristine, and prednisone (CNOP), the method comprising determining the level of phospho-Tyr705-STAT3 (PY-STAT3) and the level of BCL6 in a DLBCL biopsy specimen from the patient using immunohistochemistry, wherein PY-STAT3 positivity and BCL6 negativity predicts a poor likelihood of survival in comparison to a patient who is not PY-STAT3 positive and BCL6 negative.
Preferably, PY-STAT3 positivity or negativity is determined by scoring the intensity of PY-STAT3 staining using a 4-tiered scale (0, 3, 6, 9), scoring the percentage of PY-STAT3 stained DLBCL tumor cells using a 10-tiered scale (0-9), and multiplying the two scores together to obtain a case score for the patient, where a case score with a value of 15 or greater is considered positive and a case score with a value below 15 is considered negative. Preferably, the patient is considered BCL6 positive if 30% or more of the DLBCL tumor cells stain positive for BCL6, and BCL6 negative if less than 30% of the DLBCL tumor cells stain positive for BCL6.
BCL6 antibody can be obtained, for example, from Santa Cruz Biotechnology, Santa Cruz, Calif. (Catalog number sc-858).
The invention provides a method of determining the prognosis of a diffuse large B-cell lymphoma (DLBCL) patient undergoing treatment with a combination of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), or with a combination of cyclophosphamide, mitoxantrone, vincristine, and prednisone (CNOP), the method comprising determining the level of phospho-Tyr705-STAT3 (PY-STAT3) and the level of BCL6 in a DLBCL biopsy specimen from the patient using immunohistochemistry; and
determining PY-STAT3 positivity or negativity by scoring the intensity of PY-STAT3 staining using a 4-tiered scale (0, 3, 6, 9), scoring the percentage of PY-STAT3 stained DLBCL tumor cells using a 10-tiered scale (0-9), and multiplying the two scores together to obtain a case score for the patient, where a case score with a value of 15 or greater is considered positive and a case score with a value below 15 is considered negative, and wherein the patient is considered BCL6 positive if 30% or more of the DLBCL tumor cells stain positive for BCL6, and BCL6 negative if less than 30% of the DLBCL tumor cells stain positive for BCL6;
wherein PY-STAT3 positivity and BCL6 negativity predicts a poor likelihood of survival in comparison to a patient who is not PY-STAT3 positive and BCL6 negative.
For the methods disclosed herein, the steps of determining mRNA expression levels, determining the level of phospho-Tyr705-STAT3 (PY-STAT3), and determining the level of BCL6 in a DLBCL biopsy specimen from a patient require an experimental determination that involves the use of a machine and/or involves a physical and/or chemical transformation.
The invention also provides a gene expression profile or signature that is predictive of activation of signal transducer and activator of transcription 3 (STAT3), wherein the profile comprises expression of a plurality of, or all of, the following genes: MEX3D, BATF, CAPN2, CCND2, CD2, CMTM3, DYNLT1, ELL2, GALNT1, GCA, GMFG, GYG1, GZMB, MAN1A1, MT1X, PERP, PLAGL1, PRF1, RAB27A, S100A6, SERPINB1, TTC39C, XK, ZBED2, ZNRF1, BTLA, C13orf18, CFLAR, EV12A, HIST2H2AA3, IL16, IL2RA and PTGER4.
STAT3 activation can be positively correlated with expression of one or more of, or with all of, MEX3D, BATF, CAPN2, CCND2, CD2, CMTM3, DYNLT1, ELL2, GALNT1, GCA, GMFG, GYG1, GZMB, MAN1A1, MT1X, PERP, PLAGL1, PRF1, RAB27A, S100A6, SERPINB1, TTC39C, XK, ZBED2 and ZNRF1.
STAT3 activation can be positively correlated with expression of one or more of, or with all of, MEX3D, BATF, CAPN2, CCND2, CD2, CMTM3, DYNLT1, ELL2, GALNT1, GCA, GMFG, GYG1, GZMB, MAN1A1, MT1X, PERP, PLAGL1, PRF1, RAB27A, S100A6, SERPINB1, TTC39C, XK, ZBED2, ZNRF1, BTLA, C13orf18, CFLAR, EV12A, HIST2H2AA3, IL16, IL2RA and PTGER4.
The gene expression profile can be obtained by determining mRNA expression in a human diffuse large B-cell lymphoma (DLBCL) biopsy specimen. The biopsy specimen can be, for example, from a subject diagnosed as having DLBCL before the subject undergoes treatment for DLBCL, e.g., prior to undergoing treatment with CHOP or R-CHOP.
The invention provides a gene expression profile or signature for classifying a human patient with diffuse large B-cell lymphoma (DLBCL), where the signature comprises nucleic acid probes for genes HSD17B4, RNF149, ZNF805, SLC2A13, RHEB, MT1X, NAT8L, C15orf29, ZNF420, PCNX and SLA.
The invention also provides a gene expression profile or signature for classifying a human patient with diffuse large B-cell lymphoma (DLBCL), where the signature comprises nucleic acid probes for genes MEX3D, BATF, CAPN2, CCND2, CD2, CMTM3, DYNLT1, ELL2, GALNT1, GCA, GMFG, GYG1, GZMB, MAN1A1, MT1X, PERP, PLAGL1, PRF1, RAB27A, S100A6, SERPINB1, TTC39C, XK, ZBED2, ZNRF1, BTLA, C13orf18, CFLAR, EV12A, HIST2H2AA3, IL16, IL2RA and PTGER4.
The invention provides a microarray for classifying a human patient with diffuse large B-cell lymphoma (DLBCL), where the microarray comprises nucleic acid probes for genes HSD17B4, RNF149, ZNF805, SLC2A13, RHEB, MT1X, NAT8L, C15orf29, ZNF420, PCNX and SLA.
The invention also provides a microarray for classifying a human patient with diffuse large B-cell lymphoma (DLBCL), where the microarray comprises nucleic acid probes for genes MEX3D, BATF, CAPN2, CCND2, CD2, CMTM3, DYNLT1, ELL2, GALNT1, GCA, GMFG, GYG1, GZMB, MAN1A1, MT1X, PERP, PLAGL1, PRF1, RAB27A, S100A6, SERPINB1, TTC39C, XK, ZBED2, ZNRF1, BTLA, C13orf18, CFLAR, EV12A, HIST2H2AA3, IL16, IL2RA and PTGER4.
The microarray can comprise probes attached, for example, via surface engineering to a solid surface by a covalent bond to a chemical matrix (via, in non-limiting examples, epoxy-silane, amino-silane, lysine, polyacrylamide). Suitable solid surface can be, in non-limiting examples, glass or a silicon chip, a solid bead forms of, for example, polystyrene. Microarrays can include solid-phase microarrays and bead microarrays. In an embodiment, the microarray is a solid-phase microarray. In an embodiment, the microarray is a plurality of beads microarray. In an embodiment, the microarray is a spotted microarray. In an embodiment, the microarray is an oligonucleotide microarray. The oligonucleotide probes of the microarray may be of any convenient length necessary for unique discrimination of targets. In non-limiting examples, the oligonucleotide probes are 20 to 30 nucleotides in length, 31 to 40 nucleotides in length, 41 to 50 nucleotides in length, 51 to 60 nucleotides in length, 61 to 70 nucleotides in length, or 71 to 80 nucleotides in length. In an embodiment, the target sample, or nucleic acids derived from the target sample, such as mRNA or cDNA, are contacted with a detectable marker, such as one or more fluorophores, under conditions permitting the fluorophore to attach to the target sample or nucleic acids derived from the target sample. In non-limiting examples the fluorophores are cyanine 3 or cyanine 5. In an embodiment, the target hybridized to the probe can be detected, for example, by conductance, MS, or electrophoresis. The microarray can be manufactured by any method known in the art including, for example, by photolithography, pipette, drop-touch, piezoelectric (ink-jet), and electric techniques.
STAT3 activation for prognosis of patients with DLBCL can be combined with the use of additional biomarkers, e.g., BCL6 expression for the CHOP treatment and the non-GCB immunophenotype for the R-CHOP regimen.
This invention will be better understood from the Examples, which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.
A retrospective analysis of DLBCL patients treated with R-CHOP was performed focusing on understanding the prognostic significance of STAT3 activation. By quantitating the levels of phospho-Tyr705-STAT3 (PY-STAT3) in tumor cells, it was demonstrated that PY-STAT3 positivity predicted poor survival in DLBCL patients, especially in the non-GCB subgroup. In addition, a 33-gene PY-STAT3 gene expression profiling (GEP) signature can stratify R-CHOP treated DLBCL patients into four subgroups with different immunophenotypes and survival outcomes.
Patient Information and Gene Expression Profiles:
The study population included 309 patients with de novo DLBCL who were diagnosed and treated with rituximab plus standard CHOP or CHOP-like therapy (R-CHOP). Among these patients, 99 were treated at the Nebraska Lymphoma Study Group, while the rest of the cases were treated at the other LLMPP affiliated institutions. This study was approved by the institutional review boards of University of Nebraska Medical Center and of other respective institutions, and all patients gave written informed consent. Gene expression profiling (GEP) information for 222 of these patients (contain 12 Nebraska cases) was previously published and publicly available.14 The mRNA expression of STAT3 was evaluated using the averaged intensity of three probe-sets (208991_at, 208992_s_at, and 225289at) from the GEP datasets. Expression of the 3 probe-sets significantly correlated with each other (Pearson correlation, P<0.001).
Tissue Microarray and Immunohistochemistry (IHC):
The methods of tissue processing and tissue microarray (TMA) construction have been described previously.13 A classification of GCB- and non-GCB-DLBCL was utilized based on an algorithm described by Hans et al.22 Double immunostaining for PY-STAT3 and CD20 was performed to measure tumor cell-specific PY-STAT3 expression (
STAT3 siRNA Experiment:
SiRNA-mediated knock-down experiments were performed using 4 human PY-STAT3 positive DLBCL cell lines: Ly3, Ly10, HBL1, and Pfeiffer. The first three lines express constitutively activated STAT38,9 while Pfeiffer has moderate levels STAT3 activation (data not shown). All cell lines were transiently transfected with either STAT3 siRNA or a control oligo-nucleotides in triplicate as described previously.8 Substantial knock-down of the STAT3 protein was achieved at 48 hrs. At this time, endogenous STAT3 was significantly down-regulated with little or no signs of apoptosis (
Generation of the 33 Gene PY-STAT3 Signature:
GEP data from the STAT3 siRNA experiment were extracted and normalized using the BRB-Array Tools (National Cancer Institute, NIH). The SAM23 algorithm was used to identify genes that were differentially expressed between STAT3 and control siRNA treated samples. To develop a STAT3-based gene expression signature that has prognostic value, genes that were significantly altered by STAT3 siRNA and differentially expressed between PY-STAT3 positive and negative cases (P<0.05) were used. Semi-supervised prediction (SSP) method was used to regress the differentially expressed STAT3 targets by patient overall survival based on the Cox proportional hazard model with a significance of 0.05.24 The leave-one-out approach was used for cross-validation.25
Survival Analysis:
Clinical and pathological characteristics of patients in different categories were compared by chi-square test. Kaplan-Meier method was used to estimate the overall survival (OS) and event-free survival (EFS) distributions, and the differences were compared using the log-rank test. Cox proportional-hazards regression model was used to evaluate predictors of the survival distributions while adjusting for international prognostic index (IPI) and COO subgroups. All reported P-values are two-sided and those <0.05 were considered statistically significant.
Clinical Characteristics of Patients:
There were a total of 309 DLBCL cases in this study. The median age of the entire cohort was 62.2 years (range, 16.7 to 92.0 years), and the male to female ratio was 1.4 (180/129). Of the 185 cases examined for PY-STAT3 by IHC, 69 (37.3%) cases were positive and 116 (62.7%) cases were negative. The clinical features were not significantly different between the PY-STAT3-positive and -negative cases (Table 1), except a weak association of PY-STAT3 with the IPI high-risk (3-5) group (32.8% vs 20.0%, P=0.094).
STAT3 Activation is Significantly Associated with ABC-DLBCL:
The cohort of 87 Nebraska cases only with IHC defined COO subgroups, PY-STAT3 positivity was significantly associated with the non-GCB subgroup compared to the GCB subgroup (63.9%, 23/36 vs 41.1%, 21/51, P=0.037). For the rest cases with GEP defined COO subgroup status, PY-STAT3 was marginally enriched in ABC-DLBCLs relative to the GCB-DLBCL subgroup (33.3%, 14/42 vs 17.8%, 8/45, P=0.096). Among all patients within the Nebraska and LLMPP cohorts, the ABC-DLBCL (or non-GCB) subgroup contained significantly more PY-STAT3 positive cases compared to the GCB-subgroup (47.4%, 37/78 vs 30.2%, 29/96, P=0.030). Since Mum1/IRF4 is a hallmark of ABC-DLBCL, as expected, PY-STAT3 positivity also showed significant association with MUM1/IRF4 (P=0.041, Table 1). Consistent with previous reports on two different cohorts8,9, high level STAT3 mRNA expression preferentially occurred in the ABC subgroup (Table 2).
STAT3 Activation Predicts Poor Survival in DLBCL and ABC-DLBCL:
As expected, the IPI and the GCB/non-GCB classifiers (defined by either TMA or GEP) showed significant association with OS and EFS in the entire cohort (
Patients with PY-STAT3+/BCL6—Phenotype Had Inferior Survival with the CHOP Regimen:
Since BCL6 is an important criterion in the Hans classifier scheme and BCL6 and PY-STAT3 appear to be independently regulated, these two markers were combined in survival analysis (only the Nebraska CHOP and R-CHOP cases were used for this test). In the CHOP group (n=89), 9 patients had PY-STAT3+/BCL6-phenotype and showed a poor OS and EFS (POS=0.033; PEFS=0.087,
High Level STAT3 mRNA is an Adverse Risk Factor in DLBCL:
Since the level of STAT3 mRNA significantly correlated with the PY-STAT3 IHC score (Pearson correlation, P<0.001), the prognostic value of this biomarker was also examined DLBCL cases were divided into low (<group mean−standard deviation, S.D, n=37), high (>mean+S.D., n=29), and intermediated (the rest cases, n=156) groups based on the average intensity of the three STAT3 probe-sets (Table 2). Clinical characteristics of patients in these 3 groups were not significantly different. Pathologically, high levels of STAT3 mRNA were correlated with the ABC subtype, Mum1/IRF4 expression, and PY-STAT3 positivity. Similar to the observations on PY-STAT3, cases with high levels of STAT3 mRNA had significantly worse OS and EFS (POS=0.004; PEFS=0.003,
A GEP-Based PY-STAT3 Signature is a Predictor of Survival in DLBCL:
In order to evaluate the generality and reproducibility of the prognostic finding on PY-STAT3, a GEP-based PY-STAT3 signature was constructed based on the test cases described above. This signature was subsequently applied to a large public available GEP dataset that comes with treatment response information. GEP of DLBCL lines was obtained 48 hr after STAT3 siRNA treatment. At this time, endogenous STAT3 was significantly down-regulated with little or no signs of apoptosis based on PARP cleavage (
Using an unsupervised hierarchy clustering method, this 33-gene PY-STAT3 signature was applied to the GEP dataset that comprises 233 clinically well-characterized DLBCL cases treated with R-CHOP.14 The PY-STAT3 signature stratified the cohort into 4 clusters, each corresponding to one of four possible combinations of Module A and Module B (
To investigate the underlying biological basis for different survival response, a comparison was made of the relatively enriched genes in the ABC-DLBCL cases (62 in Cluster 3 and 25 in Cluster 4) using several previously curated gene-expression signatures. The Pan-T-cell signature14 was expressed at significantly higher levels in Cluster 3 than in Cluster 4 (T-test of median relative mRNA expression, P=0.007,
The Four PY-STAT3 Clusters Demonstrate Distinct Rituximab Sensitivity:
The 33-gene PY-STAT3 signature was also applied to a dataset of 181-case cohort treated with the CHOP therapy.14 Analyses showed that this 33-gene PY-STAT3 signature can similarly stratify this cohort into 4 subgroups with enrichment of Pan-T, proliferation, and plasmablastic signatures identical to those observed in the 233-case R-CHOP cohort (
PLSR Model for the 4-Cluster DLBCL Data Classification:
An algorithm was developed to classify DLBCL cases into the 4 PY-STAT3 clusters using the 33-gene signature. This classifier is based on the partial least square regression (PLSR) model:
Y=Xb,
or
y
pred
=b
0
+b
1
x
1
+b
2
x
2
+ . . . +b
n
x
n
where ypred is the predicted value and x1, x2, . . . xn is the expression value of each gene.
The PLSR model was applied for Module A and Module B genes, respectively. For Module A, the predicted covariate (Y) for Module A positive cases (Cluster 2 and 3) was set as 1, while predicted covariate (Y) for Module A negative cases (Cluster 1 and 4) was set as −1. The predicting covariates (X) were the expression values of the 25 genes in Module A. The same setting was applied for the Module B genes.
X and Y data were centered by their mean values before analysis, then PLSR was performed. The first PLS component was extracted from X and Y. For the Module A genes, the first PLS component stands for 25.5% of variance in X, and 55.0% of variance of Y. For the Module B genes, the first PLS component stands for 47.4% of variance in X, and 52.6 of variance of Y. The coefficient vector b for Module A and Module B genes is shown in Table 5.
For each DLBCL case, if ypred >0, it is the Module A/B positive case, otherwise, it is the Module A/B negative case. Predictive accuracy for Module A and B classifier is 90.6% and 85.4%, respectively. Then the predicted Cluster of each case is obtained based on the prediction result of Module A and B positivity. As shown in Table 6, total predictive accuracy is 76.8% (179/233) for the 233 DLBCL cases. The predictive accuracy for Cluster 1-4 is 71.4% (55/77), 51.6% (16/31), 86.5% (83/96), and 86.2% (25/29), respectively.
The studies described herein demonstrate that STAT3 activation has prognostic significance in patients with DLBCL, and its predictive power is much more significant when used in combination with other biomarkers, i.e. non-GCB immunophenotype for the R-CHOP regimen. This is believed to be the largest study to date demonstrating the prognostic significance of STAT3 activation in DLBCL patients treated with R-CHOP. In addition to providing strong and direct evidence that STAT3 activation is an independent prognostic biomarker in patients with DLBCL, the studies indicate that targeting STAT3 pathway may provide a novel therapeutic approach for patients with DLBCL.
The 33-gene PY-STAT3 GEP signature stratified R-CHOP treated DLBCL cases into 4 subgroups which have different immunophenotypes and, more importantly, exhibit marked differences in overall survival. The findings contradict the study by Lam et al. which reported no predictive value of a 23-gene STAT3 signature for DLBCL patients treated with CHOP regimen.9 For a direct comparison, the same cohort of patients was also analyzed using the 33-gene PY-STAT3 signature. The present GEP signature similarly stratified this cohort of DLBCL patients into 4 subgroups with different immunophenotypes and clinical outcomes (
An interesting property of the 33-gene PY-STAT3 signature is the fact that it contains two sub-Modules which were independently regulated across the entire phenotypic spectrum of DLBCL (
Analysis of relative enrichment for the signatures also provided tantalizing clues regarding the tumor B cell-microenvironment interactions. Three types of microenvironment influences were evaluated in relation to the 4 PY-STAT3 clusters: a pan-T cell signature, and the two stromal signatures (
The biological insights uncovered in this study have direct implications for ongoing and future DLBCL clinical trials. The data showed that the cases least responsive to R-CHOP belonged to Cluster 4, the cluster showing plasmablastic features. Since Rituximab targets the CD20 molecule and normal plasma cells are typically CD20 negative, it is tempting to speculate that reduced CD20 expression may be responsible for the inferior outcome of Cluster 4 cases managed with R-CHOP. However, the analysis of CD20 mRNA expression does not support this theory (not shown). It cannot be ruled out that CD20 protein expression at the cell surface may be reduced in Cluster 4. From an experimental therapeutics perspective, the plasmablastic feature of Cluster 4 DLBCL suggests a new opportunity. Published mechanistic studies have shown that plasma cell differentiation is intrinsically linked to proteasomal overload and hence explains the exquisite sensitivity of multiple myeloma cells to proteasome inhibitor—containing therapies.31,32 In a recent phase I trial involving 49 DLBCL patients, it was found that the combination of DA-EPOCH plus bortezomib was efficacious only in ABC-DLBCL but not in GCB-DLBCL.33 Based on these clinical observations and the results from this study, it is predicted that the ABC-DLBCL patients with plasmablastic tumors may benefit the most from the ongoing phase II trial of R-CHOP plus bortezomib.34 Compared to Clusters 1 and 2, Cluster 3 patients also showed an adverse response to both CHOP and R-CHOP. Tumors in Cluster 3 features strong PY-STAT3 activation and a microenvironment highly enriched in T cells. Persistent STAT3 activation in ABC-DLBCL cells is oncogenic.8,9 Thus, for patients with a Cluster 3 phenotype, an attractive direction for future clinical trials is to test the efficacy of Jak/STAT3 inhibitors, such as those currently in clinical trials for myeloid proliferative diseases.35 Recently, Lam et al have also shown that inhibition of both NF-κB and STAT3 pathways may be synergetic in enhancing tumor cell apoptosis in DLBCL cell lines with STAT3 activation9.
Patients Information and Gene Expression Profiling Analysis:
The same as described in Example A.
Identification of Candidate Genes for Prediction of STAT3 Activation Status in DLBCL:
As illustrated in
Construction of the 11-Gene STAT3 Activation Signature for DLBCL Prognostication:
The above STAT3 target set was trained for prognostic prediction using the semi-supervisory (SSP) algorithm, with leave-one-out cross validation avoiding over-fitting.24 Eleven probe-sets were selected from the 347-probe-set pool of STAT3 target genes by fitting the clinical outcome (OS) with the Cox proportional hazards model (P<0.05)24, and comparing the consistency of their expression between the patients and cell line GEP data (Table 7). Four of these genes were validated by qRT-PCR in two ABC-DLBCL cell lines treated with STAT3 siRNA (
Characteristics of the 11-Gene Signature:
As expected, known STAT3 target genes, such as CD48, CD96, IRF1, IL10, BCL3, and IL2RB were highly expressed in the PY-STAT3 positive tumors40-42, whereas the PY-STAT3 negative tumors express high levels of RAC1, MAPK1 and AKT2 (
The 11-Gene PY-STAT3 Signature Predicted Survival in DLBCL Patients Treated with R-CHOP and CHOP.
A previously published cohort of 222 DLBCL cases14 was divided into 4 quartiles using the average expression of this 11-gene predictor. To confirm that this quartile approach is biologically valid, the distribution of PY-STAT3 expression was examined in the quartile subgroups for a cohort of 98 cases for which both PY-STAT3 IHC score and GEP data were available. As shown in Table 8, the PY-STAT3 positive cases were significantly correlated with the expression of the STAT3 signature for the whole cohort (Chi-square test, P=0.001) as well as the ABC subgroup (Chi-square test, P=0.005). Most significantly, the PY-STAT3 signature separated the entire cohort of 222 patients into prognostically distinct quartile subgroups with 5-year OS rates of 84%, 81%, 57%, and 48%, and 5-year EFS rates of 81%, 77%, 51%, and 40% (POS<0.001; PEFS<0.001,
Additional insights into mechanism of resistance to the R-CHOP therapy may also be gleaned from the PY-STAT3 signature itself. Of the 11 genes in the signature, 6 have been studied functionally to various extents. HSD17B4 is a dehydrogenase involved in the peroxisomal fatty acid beta-oxidation. Its overexpression was recently reported to be a poor prognosticator in prostate cancer patients.44 SLC2A13 encodes a H+-myo-inositol transporter that has been suggested to be a marker for cancer stem cells in an oral squamous cell carcinoma.45 Aberrant expression of MT1X, which encodes metallothionein isoform 1, has been observed in several kinds of carcinomas, and its overexpression was correlated with enhanced drug resistance and shorter survival46,47 SLA encodes a Src-like adaptor protein (SLAP) that negatively regulates antigen-stimulated immune response.48 It has not been implicated in lymphomas previously. RHEB is a key regulator in the PI3K/Akt/mTOR pathway that directly activates mTOR1 activity.49 Cell type-specific oncogenic activity has been shown for RHEB especially in the context of PTEN haploinsufficiency.5° This is particular interesting in light of the previous report that PTEN loss occurs in 11% of GCB-DLBCL.51 Finally, ZNF420 encodes the KRAB-type zinc finger protein, Apak, which has been implicated in DNA damage and oncogene-induced stress response.52
With the PY-STAT3-based gene signature model, strong associations were found with OS and EFS in a published cohort of 222 patients treated with R-CHOP. While the overall conclusion parallels the findings with PY-STAT3 IHC, this gene expression based model is amenable to future technologies such as diagnostic gene chips at the point-of-care. Prior to this report, two GEP-based DLBCL prognostic models have been reported by the LLMPP consortium, namely the bivariate GCB/ABC model5,6 and the trivariate model derived from the GCB, stromal-1 and stromal-2 GEP signatures14. Interestingly, although the current 11-gene signature is a much simpler univariate predictor, its survival predictive power is quite comparable to the trivariate model specifically constructed to incorporate tumor stromal contribution. One possible explanation for the advantage of the current model is the fact that STAT3 activation within the tumor cells is not only influenced by cell intrinsic genetic alterations, it also incorporates cytokine and growth factor cues in the tumor microenvironment. In other words, STAT3 activation is a holistic readout of the entire tumor tissue.
It is pertinent to point out here that Lam et al have previously classified a group of CHOP-treated ABC-DLBCL patients into STAT3-high and STAT3-low subgroups using a Lymphochip-derived GEP signature but did not observe prognostic differences between these two subgroups.9 In this regard, the 11-gene PY-STAT3 signature developed in the current study has at least three benefits compared to the signature used by Lam et al: 1) the current signature was cross-validated for correlation with PY-STAT3 expression in primary tumors; 2) direct STAT3 target genes are selected with the requirement of high affinity STAT3 binding site(s) in the promoter region; and most importantly, 3) the ability to predict survival among R-CHOP treated patients was used as a filtering criteria.
This application claims the benefit of U.S. Provisional Patent Application No. 61/564,423, filed Nov. 29, 2011, the content of which is herein incorporated by reference in its entirety.
This invention was made with government support under grant numbers CA85573 and CA114778 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US12/66782 | 11/28/2012 | WO | 00 | 5/23/2014 |
| Number | Date | Country | |
|---|---|---|---|
| 61564423 | Nov 2011 | US |
