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WO2011137912A1 - Methods and systems for breast cancer prognosis - Google Patents

Methods and systems for breast cancer prognosis Download PDF

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Publication number
WO2011137912A1
WO2011137912A1 PCT/EP2009/050464 EP2009050464W WO2011137912A1 WO 2011137912 A1 WO2011137912 A1 WO 2011137912A1 EP 2009050464 W EP2009050464 W EP 2009050464W WO 2011137912 A1 WO2011137912 A1 WO 2011137912A1
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threshold level
predetermined
expression
expression level
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French (fr)
Inventor
Mathias Gehrmann
Christian VON TÖRNE
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Siemens Healthcare Diagnostics GmbH Germany
Siemens Healthcare Diagnostics Inc
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Siemens Healthcare Diagnostics GmbH Germany
Siemens Healthcare Diagnostics Inc
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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
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • 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
    • C12Q2600/118—Prognosis of disease development
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression

Definitions

  • the present invention relates to methods, kits and systems for the prognosis of cancer in cancer patients, preferably untreated breast cancer patients. More specifically, the pre ⁇ sent invention relates to the prognosis of cancer based on measurements of the expression levels of marker genes in tu- mor samples. Marker genes are disclosed which allow for an accurate classification of cancer patients into multiple risk classes, using a multi-step decision tree.
  • Breast cancer is one of the leading causes of cancer death in women in western countries. Breast cancer claims the lives of approximately 40,000 women and is diagnosed in approximately 200,000 women annually in the United States alone. Over the last few decades, adjuvant systemic therapy has led to mark ⁇ edly improved survival in early breast cancer. This clinical experience has led to consensus recommendations offering ad ⁇ juvant systemic therapy for the vast majority of breast can ⁇ cer patients. In breast cancer a multitude of treatment op- tions are available which can be applied in addition to the routinely performed surgical removal of the tumor and subse ⁇ quent radiation of the tumor bed.
  • a possible outcome of cancer in particular, breast cancer, in untreated patients.
  • This is also referred to the "progno- sis" of breast cancer in a patient (as opposed to e.g. the "prediction” of the possible outcome of a cancer therapy) .
  • Determination of the risk for an unfavorable outcome of a disease is valuable information when deciding on the best possible treatment strategy for a cancer patient.
  • Van ' t Veer et al identified a "prognostic signa ⁇ ture" consisting of 70 and 231 genes in a finding cohort of 78 sporadic breast cancers of node negative women younger than 53 years of age (Van't Veer et al . 2002. Gene expression profiling predicts clinical outcome of breast cancer. Nature 415 : 530-536; Van de Vijver et al . 2002. A gene-expression signature as a predictor of survival in breast cancer. N Eng J Med 347 : 1999-2009) .
  • WO 08/006517, WO 07/107254, WO 07/062777 disclose classification schemes for cancer tumors using a multi-step classifica ⁇ tion in a decision tree.
  • the specific decision tree of the present invention is not disclosed.
  • the pre ⁇ sent invention fulfills the need for advanced methods for the prognosis of breast cancer on the basis of readily accessible experimental data.
  • the present invention is based on the surprising finding that the outcome of cancer, preferably breast cancer in patients which do not receive chemotherapy, can be accurately pre ⁇ dicted from the expression levels of a small number of marker genes. Accordingly, the present invention relates to prog ⁇ nostic methods for the determination of the outcome of breast cancer in non-treated breast cancer patients, using informa ⁇ tion on the expression of a small number of highly informative marker genes. The method is based on multiple compari- sons of determined expression levels with predetermined threshold levels. Depending on the outcome of a first com ⁇ parison, further comparisons of expression levels with threshold values are performed, as shown in the decision tree in Figure 1.
  • the outcome of the prognostic method is a clas- sification of the tumor under investigation into one of several "risk classes".
  • Risk classes of the invention are pref ⁇ erably a "low risk of recurrence” class, an "intermediate risk of recurrence class” and a "high risk of recurrence class".
  • the outcome of the prognostic methods of the inven- tion provides useful information for the selection of the most suitable treatment regimen for the patient.
  • Methods of the invention are multi-step classification methods in which consecutive classification steps are performed, the classifiers used in each step (i.e. the marker genes and threshold levels applied) being dependent on the outcome of the previous classification step(s) . It has been found that this classification strategy is superior to other methods, using a single multi-variate classification step. Without being bound by theory, it is assumed that the superior per ⁇ formance of sequential classification schemes over single- step classification schemes is due to the fact that marker gene expression can be differently correlated with the dis- ease outcome in different subgroups of patients. For exam ⁇ ple, high expression of a marker gene A may be positively correlated with good disease outcome in patients having high expression of marker gene B, but may be negatively correlated with good disease outcome in patients having low expression of marker gene B. In multi-step classification methods, this can be taken into account by e.g. first classifying on the basis of the expression level of marker gene B, and then classifying on the basis of the correct correlation of marker gene A expression with the disease outcome.
  • a preferred method of the invention is a method of prognosis of cancer in a patient using the classification scheme shown in Figure 1.
  • the present invention relates to prognostic methods, methods of assessing the risk of recurrence, methods of establishing the likelihood of good/bad outcome of cancer and to systems for performing said methods.
  • Fig. 1 shows a classification scheme (a decision tree) used in methods of the invention.
  • prognosis within the meaning of the invention, shall be understood to be the determination of a likely outcome of a disease in a patient not receiving systemic chemotherapy.
  • prognosis is an estima ⁇ tion of the likelihood of metastasis fee survival of said pa ⁇ tient over a predetermined period of time, e.g. over a period of 5 years, or is an estimation of the risk of recurrence of cancer in said patient within a predetermined period of time.
  • Tumor sample shall be understood to be any sample taken from a tumor of the respective patient.
  • Tumor samples can be taken by biopsy, needle biopsy, by surgery or any by other method known to the person skilled in the art.
  • Formalin- fixed paraffin embedded (FFPE) tumor samples are preferred.
  • Fresh-frozen samples can also be used in methods of the in- vention.
  • Classification within the meaning of the invention, shall be understood as being the process of classifying objects into one of multiple classes, said classes being defined by certain properties of the objects therein.
  • tumors or patients are classified into one of sev ⁇ eral "risk classes", said risk classes being defined e.g. by the likelihood of recurrence of cancer in said patients after surgery .
  • Sequential classification steps within the meaning of the invention, shall be understood as being multiple classifica ⁇ tion steps in a sequence of classification steps, wherein the a subsequent classification step further classifies a class which was previously classified by a preceding classification step.
  • Sequential classification steps can be sequential classification steps in a "branched" classification scheme, such as the decision tree shown in Figure 1. Classification by sequential classification steps is effectively be imple- mented as a combination of the individual comparison steps, combined by the logical AND operator. The combination of the conditions of multiple classifiers using the AND operator shall be understood as being “sequential classification steps", within the meaning of the invention.
  • a “marker gene”, within the meaning of the invention, shall be understood as being any gene present in a patient, the ex ⁇ pression level of which gene provides information useful for the prognosis of cancer in said patient.
  • Preferred marker genes of the invention are those mentioned in the claims and in the examples section.
  • a "risk class”, within the meaning of the invention, shall be understood to relate to a class of patients which share a similar risk of favorable or unfavorable disease outcome.
  • the risk classes re- fer to the risk of recurrence of cancer in said patient within a predetermined period of time, e.g. within 5 years.
  • "Risk classes” of the current invention are preferably a "high risk” class, an "intermediate risk” class, and a "low risk” class.
  • “Risk of recurrence” shall be understood to be a measure for the likelihood, that a cancer patient develops at least one further tumor after the primary tumor is removed by surgery.
  • the risk of recurrence re- lates to the risk of developing distant metastases after re ⁇ moval of the primary tumor.
  • the risk of recurrence is pref ⁇ erably quantified by classifying said tumor and/or patient into one of several risk classes, such as a "high risk” class, an "intermediate risk” class, and "low risk” class.
  • the risk of recurrence preferably relates to the risk of re ⁇ currence within a predetermined period of time, preferably within a period of 5 years.
  • an "expression level”, within the present invention, shall be understood to be any measure for the strength of expression of a gene in a tissue sample. In preferred embodiments of the invention, the average expression of a gene in a tissue sample is measured. Expression levels can be determined by gene-chip based methods or real-time PCR methods, these meth- ods being well known to the person skilled in the art.
  • a "system for the prognosis of cancer”, within the meaning of the invention, is a system of equipment, hardware and/or software that is capable of performing a prognosis of cancer, e.g. in accordance to the present invention.
  • the system can be built from separate pieces of hardware (and software) , but can also be integral, i.e. combined in a single piece of hardware, said single piece of hardware having all necessary features combined within a single housing.
  • the present invention relates to methods for the prognosis of cancer in a patient, from a tumor sample of said patient, said method comprising:
  • Classification on the basis of sequential classification steps using the specific marker genes of the present inven ⁇ tion allows for an efficient and accurate prognosis of cancer in a patient. At least two sequential classification steps are preferably performed so that a useful prognosis can be obtained .
  • the invention further relates to the methods described above, wherein all expression levels in said group of expression levels are determined.
  • Determination of all expression levels allows for a classifi ⁇ cation of any sample, irrespective of the actual expression levels determined therein. Determination of less than all expression levels may not allow classification of the sample in any case.
  • the invention further relates to the methods described above, wherein said prognosis is the determination of the risk of recurrence of cancer in said patient within 5 years.
  • the invention further relates to the methods described above, wherein said prognosis is a classification of said patient into one of three distinct classes, said classes correspond- ing to a "high risk of recurrence” class, an "intermediate risk of recurrence” class and a "low risk or recurrence” class .
  • the invention further relates to the methods described above, wherein said cancer is breast cancer or ovarian cancer.
  • the invention further relates to the methods described above, wherein said determination of expression levels is in a formalin-fixed paraffin embedded sample or in a fresh-frozen sample .
  • Methods of the invention are optimized for analysis of forma ⁇ lin-fixed paraffin embedded samples and for fresh-frozen sam ⁇ ples, however, they can be applied to different samples as well .
  • the invention further relates to the methods described above, wherein said tumor is classified "low risk", if the ex ⁇ pression level of MLPH is equal to or above a predetermined first threshold level and the expression level of TOP2A is equal to or below a predetermined second threshold level and the expression level of UBE2C is equal to or below a prede- termined third threshold level, and
  • tumor is classified "low risk", if the expression level of MLPH is equal to or above said predeter ⁇ mined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is above a predetermined fourth threshold level, and
  • tumor is classified "intermediate risk", if the expression level of MLPH is equal to or above said prede- termined first threshold level and the expression level of
  • TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is equal to or below said predetermined fourth threshold level and the expression level of MMP1 is below a predetermined fifth threshold level, and
  • tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter- mined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is equal to or below said predetermined fourth threshold level and the expression level of MMP1 is equal to or above said predeter ⁇ mined fifth threshold level, and
  • tumor is classified "intermediate risk", if the expression level of MLPH is equal to or above said prede- termined first threshold level, and the expression level of TOP2A is above said predetermined second threshold level or the expression level of UBE2C is above is above said prede ⁇ termined third threshold level, and the expression level of UBE2C is equal to or above said predetermined third threshold level, and the median-normalized immunoglobulin expression is above a predetermined sixth threshold level, and
  • tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter ⁇ mined first threshold level, and the expression level of TOP2A is above said predetermined second threshold level or the expression level of UBE2C is above is above said prede ⁇ termined third threshold level, and the expression level of UBE2C is equal to or above said predetermined third threshold level, and the median-normalized immunoglobulin expression is equal to or below said predetermined sixth threshold level, and
  • tumor is classified "intermediate risk", if the expression level of MLPH is below said predetermined first threshold level and the median-normalized immunoglobu- lin expression is above said predetermined sixth threshold level, and
  • said tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter ⁇ mined first threshold level and the median-normalized immu- noglobulin expression is equal to or below said predetermined sixth threshold level.
  • tumor is classified "low risk", if the expression level of MLPH is equal to or above a predetermined first threshold level and the expression level of TOP2A is equal to or below a predetermined second threshold level and the expression level of UBE2C is equal to or below a prede ⁇ termined third threshold level, or
  • tumor is classified "low risk", if the ex- pression level of MLPH is equal to or above said predeter ⁇ mined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is above a predetermined fourth threshold level, or
  • tumor is classified "intermediate risk", if the expression level of MLPH is equal to or above said prede ⁇ termined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is equal to or below said predetermined fourth threshold level and the expression level of MMP1 is below a predetermined fifth threshold level, or
  • said tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter ⁇ mined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is equal to or below said predetermined fourth threshold level and the expression level of MMP1 is equal to or above said predeter ⁇ mined fifth threshold level, or wherein said tumor is classified "intermediate risk", if the expression level of MLPH is equal to or above said prede ⁇ termined first threshold level, and the expression level of TOP2A is above said predetermined second threshold level or the expression level of UBE2C is above is above said prede ⁇ termined third threshold level, and the expression level of UBE2C is equal to or above said predetermined third threshold level, and the median-normalized immunoglobulin expression is above a predetermined sixth threshold level, or
  • tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter ⁇ mined first threshold level, and the expression level of TOP2A is above said predetermined second threshold level or the expression level of UBE2C is above is above said prede- termined third threshold level, and the expression level of
  • UBE2C is equal to or above said predetermined third threshold level, and the median-normalized immunoglobulin expression is equal to or below said predetermined sixth threshold level, or
  • tumor is classified "intermediate risk", if the expression level of MLPH is below said predetermined first threshold level and the median-normalized immunoglobu ⁇ lin expression is above said predetermined sixth threshold level, or
  • said tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter ⁇ mined first threshold level and the median-normalized immu ⁇ noglobulin expression is equal to or below said predetermined sixth threshold level.
  • an exact classification may in some cases not be possi ⁇ ble, if the determined expression levels lie outside the se ⁇ lected branches of the tree.
  • the invention further relates to the methods described above, wherein said predetermined first threshold level is about 2000,
  • predetermined second threshold level is about 150
  • predetermined third threshold level is about 1000
  • said predetermined fourth threshold level is about 600
  • said predetermined fifth threshold level is about 200
  • said predetermined sixth threshold level is about 1.95.
  • the invention further relates to the methods described above, wherein said predetermined threshold levels are exactly the ones mentioned above.
  • threshold values are optimized threshold values for the purpose of the current method, and for the specific classification scheme shown in Figure 1, it is en- visaged that different threshold values can also be used.
  • the expression level of a co-regulated gene is equivalent to information provided by the original marker gene.
  • the invention further relates to the methods described above, wherein an expression level of a marker gene is substituted with the expression level of a substitute marker gene, said substitute marker gene being co-regulated with said marker gene .
  • the invention further relates to systems for the prognosis of cancer in a patient from samples taken from said patient, said system comprising
  • (c) means for performing multiple sequential classifi- cation steps on the basis of the outcome of said comparison steps under (b) , and for classifying said tumor into one of at least two distinct risk classes.
  • Systems of the invention can comprise various features. They may comprise a gene-chip system for the determination of the expression levels of preferred marker genes, and a personal computer, adapted to perform a classification of a tumor sam- pie according to the present invention. Systems of the in ⁇ vention, however, may also not comprise means for determining the expression level of marker genes of the invention, but consist only of the means for comparing given expression levels of marker genes of the invention with marker-gene spe- cific threshold levels.
  • the invention thus further relates to systems for the progno ⁇ sis of cancer in a patient said system comprising
  • (c) means for performing multiple sequential classifi ⁇ cation steps on the basis of the outcome of said comparison steps under (b) , and for classifying said tumor into one of at least two distinct risk classes.
  • Expression levels can be obtained by manual input e.g. on a keyboard, or by reading expression levels from a storage medium, such as a diskette, hard drive, a memory, etc.. Expression levels can also be transferred from the means for determining the ex- pression levels via a cable connection or a wireless connec ⁇ tion, or via the internet. Accordingly, means for obtaining multiple expression levels can be a computer keyboard, a touch-screen, a cable connection, a wireless connection or any other type of suitable interface.
  • the invention further relates to a system as described above, wherein said means for determining multiple expression levels is a gene-chip system, a real time PCR system.
  • the invention further relates to a system as described above, wherein said means for performing multiple sequential classi ⁇ fication steps is a separate personal computer, or a computer integral with the remaining components of the system. Use of a separate personal computer simplifies the manufacturing process, whereas the use of a built-in computer, integral with the remaining hardware parts of the system provides a more compact, space-saving system for use in e.g. a labora ⁇ tory .
  • the invention further relates to a system as described above, wherein the computer receives expression level measurements directly from said means for determining the expression level .
  • the invention further relates to a system as described above, wherein said means for performing multiple sequential classi ⁇ fication steps is adapted to classify said tumor,
  • tumor is classified "low risk", if the expression level of MLPH is equal to or above a predetermined first threshold level and the expression level of TOP2A is equal to or below a predetermined second threshold level and the expression level of UBE2C is equal to or below a prede ⁇ termined third threshold level, and
  • tumor is classified "low risk", if the expression level of MLPH is equal to or above said predeter- mined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is above a predetermined fourth threshold level, and
  • said tumor is classified "intermediate risk", if the expression level of MLPH is equal to or above said prede ⁇ termined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is equal to or below said predetermined fourth threshold level and the expression level of MMP1 is below a predetermined fifth threshold level, and wherein said tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter ⁇ mined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is equal to or below said predetermined fourth threshold level and the expression level of MMP1 is equal to or above said predeter ⁇ mined fifth threshold level, and
  • tumor is classified "intermediate risk", if the expression level of MLPH is equal to or above said prede ⁇ termined first threshold level, and the expression level of TOP2A is above said predetermined second threshold level or the expression level of UBE2C is above is above said prede- termined third threshold level, and the expression level of
  • UBE2C is equal to or above said predetermined third threshold level, and the median-normalized immunoglobulin expression is above a predetermined sixth threshold level, and
  • said tumor is classified "high risk", if the ex- pression level of MLPH is equal to or above said predeter ⁇ mined first threshold level, and the expression level of TOP2A is above said predetermined second threshold level or the expression level of UBE2C is above is above said prede ⁇ termined third threshold level, and the expression level of UBE2C is equal to or above said predetermined third threshold level, and the median-normalized immunoglobulin expression is equal to or below said predetermined sixth threshold level, and
  • tumor is classified "intermediate risk", if the expression level of MLPH is below said predetermined first threshold level and the median-normalized immunoglobu ⁇ lin expression is above said predetermined sixth threshold level, and
  • the invention further relates to a system as described above, wherein said means for performing multiple sequential classi ⁇ fication steps is adapted to classify said tumor,
  • tumor is classified "low risk", if the ex- pression level of MLPH is equal to or above a predetermined first threshold level and the expression level of TOP2A is equal to or below a predetermined second threshold level and the expression level of UBE2C is equal to or below a prede ⁇ termined third threshold level, or
  • tumor is classified "low risk", if the ex ⁇ pression level of MLPH is equal to or above said predeter ⁇ mined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is above a predetermined fourth threshold level, or
  • tumor is classified "intermediate risk", if the expression level of MLPH is equal to or above said prede ⁇ termined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is equal to or below said predetermined fourth threshold level and the expression level of MMP1 is below a predetermined fifth threshold level, or
  • tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter ⁇ mined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is equal to or below said predetermined fourth threshold level and the expression level of MMP1 is equal to or above said predeter ⁇ mined fifth threshold level, or
  • tumor is classified "intermediate risk", if the expression level of MLPH is equal to or above said prede ⁇ termined first threshold level, and the expression level of TOP2A is above said predetermined second threshold level or the expression level of UBE2C is above is above said prede ⁇ termined third threshold level, and the expression level of UBE2C is equal to or above said predetermined third threshold level, and the median-normalized immunoglobulin expression is above a predetermined sixth threshold level, or
  • tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter ⁇ mined first threshold level, and the expression level of TOP2A is above said predetermined second threshold level or the expression level of UBE2C is above is above said prede ⁇ termined third threshold level, and the expression level of UBE2C is equal to or above said predetermined third threshold level, and the median-normalized immunoglobulin expression is equal to or below said predetermined sixth threshold level, or
  • tumor is classified "intermediate risk", if the expression level of MLPH is below said predetermined first threshold level and the median-normalized immunoglobu ⁇ lin expression is above said predetermined sixth threshold level, or
  • said tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter ⁇ mined first threshold level and the median-normalized immu ⁇ noglobulin expression is equal to or below said predetermined sixth threshold level.
  • the invention further relates to systems as described above, wherein said predetermined first threshold level is about 2000,
  • said predetermined second threshold level is about 150
  • said predetermined third threshold level is about 1000
  • said predetermined fourth threshold level is about 600
  • said predetermined fifth threshold level is about 200
  • said predetermined sixth threshold level is about 1.95.
  • the following marker genes are used in methods of the inven ⁇ tion. They are identified by their respective entries in the UniProtKB/Swiss-Prot database (see e.g.
  • Synaptotagmin-like protein 2a Sip homolog lacking C2 domains a
  • Prognosis Example 1 In a tumor sample of a patient the expression levels of MLPH, TOP2A, UBE2C, CXCL13, MMP1, and a median-normalized expres ⁇ sion value of IG was determined.
  • the determined expression levels were 2523 for MLPH, 188 for TOP2A, 576 for UBE2C, 840 for CXCL13, and 1.8 for IG.
  • Another tumor sample was analyzed.
  • the expression levels of MLPH, TOP2A, UBE2C, CXCL13, MMP1, and a median-normalized ex ⁇ pression value of IG were determined.

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Abstract

The present invention relates to methods, kits and systems for the prognosis of cancer in untreated cancer patients. More preferably, the present invention relates to the prognosis of breast cancer based on measurements of the expression levels of marker genes in tumor samples of breast cancer patients using a multi-step classification scheme, following a decision tree. A set of marker genes is disclosed which allow for an accurate prognosis of breast cancer in breast cancer patients.

Description

METHODS AND SYSTEMS FOR BREAST CANCER PROGNOSIS Technical Field The present invention relates to methods, kits and systems for the prognosis of cancer in cancer patients, preferably untreated breast cancer patients. More specifically, the pre¬ sent invention relates to the prognosis of cancer based on measurements of the expression levels of marker genes in tu- mor samples. Marker genes are disclosed which allow for an accurate classification of cancer patients into multiple risk classes, using a multi-step decision tree.
Background of the Invention
Breast cancer is one of the leading causes of cancer death in women in western countries. Breast cancer claims the lives of approximately 40,000 women and is diagnosed in approximately 200,000 women annually in the United States alone. Over the last few decades, adjuvant systemic therapy has led to mark¬ edly improved survival in early breast cancer. This clinical experience has led to consensus recommendations offering ad¬ juvant systemic therapy for the vast majority of breast can¬ cer patients. In breast cancer a multitude of treatment op- tions are available which can be applied in addition to the routinely performed surgical removal of the tumor and subse¬ quent radiation of the tumor bed.
Yet most, if not all of the different drug treatments have numerous potential adverse effects which can severely impair patients' quality of life. This makes it mandatory to select the treatment strategy on the basis of a careful risk assess¬ ment for the individual patient to avoid over- as well as un¬ der treatment.
Of particular importance, in this regard, is an assessment of a possible outcome of cancer, in particular, breast cancer, in untreated patients. This is also referred to the "progno- sis" of breast cancer in a patient (as opposed to e.g. the "prediction" of the possible outcome of a cancer therapy) . Determination of the risk for an unfavorable outcome of a disease (e.g. risk of tumor recurrence within a predetermined period of time) is valuable information when deciding on the best possible treatment strategy for a cancer patient.
Expression levels of marker genes were used for cancer prog¬ nosis by several investigators using supervised analysis methods. Van ' t Veer et al . identified a "prognostic signa¬ ture" consisting of 70 and 231 genes in a finding cohort of 78 sporadic breast cancers of node negative women younger than 53 years of age (Van't Veer et al . 2002. Gene expression profiling predicts clinical outcome of breast cancer. Nature 415 : 530-536; Van de Vijver et al . 2002. A gene-expression signature as a predictor of survival in breast cancer. N Eng J Med 347 : 1999-2009) . They used a case versus control sta¬ tistics, with development of metastasis within five years de¬ fined as case and disease free survival of more than five years as control, and found that the expression values of at least 70 genes could be used to calculate an average "good prognosis" profile. Unknown tumor samples were classified by correlation of the gene expression of these 70 genes to the good prognosis signature. This method, however, does not ap- ply a multi-step classification procedure, in which a subject is classified by at least two distinct classifiers (branches of a decision tree) . Multi-step methods, as explained below, allow for a more accurate prognosis. WO 08/006517, WO 07/107254, WO 07/062777 disclose classification schemes for cancer tumors using a multi-step classifica¬ tion in a decision tree. However, the specific decision tree of the present invention is not disclosed. In view of the continuing need for materials and methods use¬ ful in making clinical decisions on cancer therapy, the pre¬ sent invention fulfills the need for advanced methods for the prognosis of breast cancer on the basis of readily accessible experimental data.
Summary of the Invention
The present invention is based on the surprising finding that the outcome of cancer, preferably breast cancer in patients which do not receive chemotherapy, can be accurately pre¬ dicted from the expression levels of a small number of marker genes. Accordingly, the present invention relates to prog¬ nostic methods for the determination of the outcome of breast cancer in non-treated breast cancer patients, using informa¬ tion on the expression of a small number of highly informative marker genes. The method is based on multiple compari- sons of determined expression levels with predetermined threshold levels. Depending on the outcome of a first com¬ parison, further comparisons of expression levels with threshold values are performed, as shown in the decision tree in Figure 1. The outcome of the prognostic method is a clas- sification of the tumor under investigation into one of several "risk classes". Risk classes of the invention are pref¬ erably a "low risk of recurrence" class, an "intermediate risk of recurrence class" and a "high risk of recurrence class". The outcome of the prognostic methods of the inven- tion provides useful information for the selection of the most suitable treatment regimen for the patient.
Methods of the invention are multi-step classification methods in which consecutive classification steps are performed, the classifiers used in each step (i.e. the marker genes and threshold levels applied) being dependent on the outcome of the previous classification step(s) . It has been found that this classification strategy is superior to other methods, using a single multi-variate classification step. Without being bound by theory, it is assumed that the superior per¬ formance of sequential classification schemes over single- step classification schemes is due to the fact that marker gene expression can be differently correlated with the dis- ease outcome in different subgroups of patients. For exam¬ ple, high expression of a marker gene A may be positively correlated with good disease outcome in patients having high expression of marker gene B, but may be negatively correlated with good disease outcome in patients having low expression of marker gene B. In multi-step classification methods, this can be taken into account by e.g. first classifying on the basis of the expression level of marker gene B, and then classifying on the basis of the correct correlation of marker gene A expression with the disease outcome.
A preferred method of the invention is a method of prognosis of cancer in a patient using the classification scheme shown in Figure 1.
The present invention relates to prognostic methods, methods of assessing the risk of recurrence, methods of establishing the likelihood of good/bad outcome of cancer and to systems for performing said methods.
Short description of the Figures
Fig. 1 shows a classification scheme (a decision tree) used in methods of the invention.
Detailed description of the Invention
"Prognosis", within the meaning of the invention, shall be understood to be the determination of a likely outcome of a disease in a patient not receiving systemic chemotherapy. In preferred methods of the invention, prognosis is an estima¬ tion of the likelihood of metastasis fee survival of said pa¬ tient over a predetermined period of time, e.g. over a period of 5 years, or is an estimation of the risk of recurrence of cancer in said patient within a predetermined period of time.
A "tumor sample" shall be understood to be any sample taken from a tumor of the respective patient. Tumor samples can be taken by biopsy, needle biopsy, by surgery or any by other method known to the person skilled in the art. Formalin- fixed paraffin embedded (FFPE) tumor samples are preferred. Fresh-frozen samples can also be used in methods of the in- vention.
"Classification", within the meaning of the invention, shall be understood as being the process of classifying objects into one of multiple classes, said classes being defined by certain properties of the objects therein. In methods of the invention, tumors or patients are classified into one of sev¬ eral "risk classes", said risk classes being defined e.g. by the likelihood of recurrence of cancer in said patients after surgery .
"Sequential classification steps", within the meaning of the invention, shall be understood as being multiple classifica¬ tion steps in a sequence of classification steps, wherein the a subsequent classification step further classifies a class which was previously classified by a preceding classification step. Sequential classification steps can be sequential classification steps in a "branched" classification scheme, such as the decision tree shown in Figure 1. Classification by sequential classification steps is effectively be imple- mented as a combination of the individual comparison steps, combined by the logical AND operator. The combination of the conditions of multiple classifiers using the AND operator shall be understood as being "sequential classification steps", within the meaning of the invention.
A "marker gene", within the meaning of the invention, shall be understood as being any gene present in a patient, the ex¬ pression level of which gene provides information useful for the prognosis of cancer in said patient. Preferred marker genes of the invention are those mentioned in the claims and in the examples section. A "risk class", within the meaning of the invention, shall be understood to relate to a class of patients which share a similar risk of favorable or unfavorable disease outcome. In a preferred embodiment of the invention, the risk classes re- fer to the risk of recurrence of cancer in said patient within a predetermined period of time, e.g. within 5 years. "Risk classes" of the current invention are preferably a "high risk" class, an "intermediate risk" class, and a "low risk" class.
"Risk of recurrence" shall be understood to be a measure for the likelihood, that a cancer patient develops at least one further tumor after the primary tumor is removed by surgery. In one aspect of the invention, the risk of recurrence re- lates to the risk of developing distant metastases after re¬ moval of the primary tumor. The risk of recurrence is pref¬ erably quantified by classifying said tumor and/or patient into one of several risk classes, such as a "high risk" class, an "intermediate risk" class, and "low risk" class. The risk of recurrence preferably relates to the risk of re¬ currence within a predetermined period of time, preferably within a period of 5 years.
An "expression level", within the present invention, shall be understood to be any measure for the strength of expression of a gene in a tissue sample. In preferred embodiments of the invention, the average expression of a gene in a tissue sample is measured. Expression levels can be determined by gene-chip based methods or real-time PCR methods, these meth- ods being well known to the person skilled in the art.
A "system for the prognosis of cancer", within the meaning of the invention, is a system of equipment, hardware and/or software that is capable of performing a prognosis of cancer, e.g. in accordance to the present invention. The system can be built from separate pieces of hardware (and software) , but can also be integral, i.e. combined in a single piece of hardware, said single piece of hardware having all necessary features combined within a single housing.
The present invention relates to methods for the prognosis of cancer in a patient, from a tumor sample of said patient, said method comprising:
(a) determining in said tumor sample multiple expres¬ sion levels selected from the group consisting of the expres¬ sion level of MLPH, the expression level of TOP2A, the ex- pression level of UBE2C, the expression level of CXCL13, the expression level of MMP1, and the median-normalized immu¬ noglobulin expression level;
(b) comparing each of said expression levels with a marker gene specific threshold level;
(c) performing multiple sequential classification steps on the basis of the outcome of said comparison steps under (b) , thereby classifying said tumor into one of at least two distinct risk classes.
Classification on the basis of sequential classification steps using the specific marker genes of the present inven¬ tion allows for an efficient and accurate prognosis of cancer in a patient. At least two sequential classification steps are preferably performed so that a useful prognosis can be obtained .
The invention further relates to the methods described above, wherein all expression levels in said group of expression levels are determined.
Determination of all expression levels allows for a classifi¬ cation of any sample, irrespective of the actual expression levels determined therein. Determination of less than all expression levels may not allow classification of the sample in any case.
The invention further relates to the methods described above, wherein said prognosis is the determination of the risk of recurrence of cancer in said patient within 5 years.
The invention further relates to the methods described above, wherein said prognosis is a classification of said patient into one of three distinct classes, said classes correspond- ing to a "high risk of recurrence" class, an "intermediate risk of recurrence" class and a "low risk or recurrence" class .
The invention further relates to the methods described above, wherein said cancer is breast cancer or ovarian cancer.
The invention further relates to the methods described above, wherein said determination of expression levels is in a formalin-fixed paraffin embedded sample or in a fresh-frozen sample .
Methods of the invention are optimized for analysis of forma¬ lin-fixed paraffin embedded samples and for fresh-frozen sam¬ ples, however, they can be applied to different samples as well .
The invention further relates to the methods described above, wherein said tumor is classified "low risk", if the ex¬ pression level of MLPH is equal to or above a predetermined first threshold level and the expression level of TOP2A is equal to or below a predetermined second threshold level and the expression level of UBE2C is equal to or below a prede- termined third threshold level, and
wherein said tumor is classified "low risk", if the expression level of MLPH is equal to or above said predeter¬ mined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is above a predetermined fourth threshold level, and
wherein said tumor is classified "intermediate risk", if the expression level of MLPH is equal to or above said prede- termined first threshold level and the expression level of
TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is equal to or below said predetermined fourth threshold level and the expression level of MMP1 is below a predetermined fifth threshold level, and
wherein said tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter- mined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is equal to or below said predetermined fourth threshold level and the expression level of MMP1 is equal to or above said predeter¬ mined fifth threshold level, and
wherein said tumor is classified "intermediate risk", if the expression level of MLPH is equal to or above said prede- termined first threshold level, and the expression level of TOP2A is above said predetermined second threshold level or the expression level of UBE2C is above is above said prede¬ termined third threshold level, and the expression level of UBE2C is equal to or above said predetermined third threshold level, and the median-normalized immunoglobulin expression is above a predetermined sixth threshold level, and
wherein said tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter¬ mined first threshold level, and the expression level of TOP2A is above said predetermined second threshold level or the expression level of UBE2C is above is above said prede¬ termined third threshold level, and the expression level of UBE2C is equal to or above said predetermined third threshold level, and the median-normalized immunoglobulin expression is equal to or below said predetermined sixth threshold level, and
wherein said tumor is classified "intermediate risk", if the expression level of MLPH is below said predetermined first threshold level and the median-normalized immunoglobu- lin expression is above said predetermined sixth threshold level, and
wherein said tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter¬ mined first threshold level and the median-normalized immu- noglobulin expression is equal to or below said predetermined sixth threshold level.
Preferred methods of the invention thus follow the decision tree shown in Figure 1. Methods as defined in the previous paragraph contain a complete set of rules which allow for a classification of any sample, irrespective of the individual expression levels determined therein.
It is, however, also possible to classify a tumor with only some of the branches of the decision tree shown in Figure 1. In this case, the invention relates to the methods described above,
wherein said tumor is classified "low risk", if the expression level of MLPH is equal to or above a predetermined first threshold level and the expression level of TOP2A is equal to or below a predetermined second threshold level and the expression level of UBE2C is equal to or below a prede¬ termined third threshold level, or
wherein said tumor is classified "low risk", if the ex- pression level of MLPH is equal to or above said predeter¬ mined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is above a predetermined fourth threshold level, or
wherein said tumor is classified "intermediate risk", if the expression level of MLPH is equal to or above said prede¬ termined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is equal to or below said predetermined fourth threshold level and the expression level of MMP1 is below a predetermined fifth threshold level, or
wherein said tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter¬ mined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is equal to or below said predetermined fourth threshold level and the expression level of MMP1 is equal to or above said predeter¬ mined fifth threshold level, or wherein said tumor is classified "intermediate risk", if the expression level of MLPH is equal to or above said prede¬ termined first threshold level, and the expression level of TOP2A is above said predetermined second threshold level or the expression level of UBE2C is above is above said prede¬ termined third threshold level, and the expression level of UBE2C is equal to or above said predetermined third threshold level, and the median-normalized immunoglobulin expression is above a predetermined sixth threshold level, or
wherein said tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter¬ mined first threshold level, and the expression level of TOP2A is above said predetermined second threshold level or the expression level of UBE2C is above is above said prede- termined third threshold level, and the expression level of
UBE2C is equal to or above said predetermined third threshold level, and the median-normalized immunoglobulin expression is equal to or below said predetermined sixth threshold level, or
wherein said tumor is classified "intermediate risk", if the expression level of MLPH is below said predetermined first threshold level and the median-normalized immunoglobu¬ lin expression is above said predetermined sixth threshold level, or
wherein said tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter¬ mined first threshold level and the median-normalized immu¬ noglobulin expression is equal to or below said predetermined sixth threshold level.
When using only branches of the complete decision tree, how¬ ever, an exact classification may in some cases not be possi¬ ble, if the determined expression levels lie outside the se¬ lected branches of the tree.
The invention further relates to the methods described above, wherein said predetermined first threshold level is about 2000,
wherein said predetermined second threshold level is about 150, wherein said predetermined third threshold level is about 1000,
wherein said predetermined fourth threshold level is about 600,
wherein said predetermined fifth threshold level is about 200,
wherein said predetermined sixth threshold level is about 1.95.
The invention further relates to the methods described above, wherein said predetermined threshold levels are exactly the ones mentioned above.
While these specific threshold values are optimized threshold values for the purpose of the current method, and for the specific classification scheme shown in Figure 1, it is en- visaged that different threshold values can also be used.
The expression level of a co-regulated gene is equivalent to information provided by the original marker gene.
The invention further relates to the methods described above, wherein an expression level of a marker gene is substituted with the expression level of a substitute marker gene, said substitute marker gene being co-regulated with said marker gene .
The invention further relates to systems for the prognosis of cancer in a patient from samples taken from said patient, said system comprising
(a) means for determining in said tumor sample multiple expression levels selected from the group consisting of the expression level of MLPH, the expression level of TOP2A, the expression level of UBE2C, the expression level of CXCL13, the expression level of MMP1, and the median-normalized immu¬ noglobulin expression level;
(b) means for comparing each of said expression levels with a marker gene specific threshold level;
(c) means for performing multiple sequential classifi- cation steps on the basis of the outcome of said comparison steps under (b) , and for classifying said tumor into one of at least two distinct risk classes. Systems of the invention can comprise various features. They may comprise a gene-chip system for the determination of the expression levels of preferred marker genes, and a personal computer, adapted to perform a classification of a tumor sam- pie according to the present invention. Systems of the in¬ vention, however, may also not comprise means for determining the expression level of marker genes of the invention, but consist only of the means for comparing given expression levels of marker genes of the invention with marker-gene spe- cific threshold levels.
The invention thus further relates to systems for the progno¬ sis of cancer in a patient said system comprising
(a) means for obtaining multiple expression levels se¬ lected from the group consisting of the expression level of MLPH, the expression level of TOP2A, the expression level of UBE2C, the expression level of CXCL13, the expression level of MMP1, and the median-normalized immunoglobulin expression level in a tumor sample of said patient;
(b) means for comparing each of said expression levels with a marker gene specific threshold level;
(c) means for performing multiple sequential classifi¬ cation steps on the basis of the outcome of said comparison steps under (b) , and for classifying said tumor into one of at least two distinct risk classes.
Expression levels, according to this aspect of the invention, can be obtained by manual input e.g. on a keyboard, or by reading expression levels from a storage medium, such as a diskette, hard drive, a memory, etc.. Expression levels can also be transferred from the means for determining the ex- pression levels via a cable connection or a wireless connec¬ tion, or via the internet. Accordingly, means for obtaining multiple expression levels can be a computer keyboard, a touch-screen, a cable connection, a wireless connection or any other type of suitable interface.
The invention further relates to a system as described above, wherein said means for determining multiple expression levels is a gene-chip system, a real time PCR system. The invention further relates to a system as described above, wherein said means for performing multiple sequential classi¬ fication steps is a separate personal computer, or a computer integral with the remaining components of the system. Use of a separate personal computer simplifies the manufacturing process, whereas the use of a built-in computer, integral with the remaining hardware parts of the system provides a more compact, space-saving system for use in e.g. a labora¬ tory .
The invention further relates to a system as described above, wherein the computer receives expression level measurements directly from said means for determining the expression level .
The invention further relates to a system as described above, wherein said means for performing multiple sequential classi¬ fication steps is adapted to classify said tumor,
wherein said tumor is classified "low risk", if the expression level of MLPH is equal to or above a predetermined first threshold level and the expression level of TOP2A is equal to or below a predetermined second threshold level and the expression level of UBE2C is equal to or below a prede¬ termined third threshold level, and
wherein said tumor is classified "low risk", if the expression level of MLPH is equal to or above said predeter- mined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is above a predetermined fourth threshold level, and
wherein said tumor is classified "intermediate risk", if the expression level of MLPH is equal to or above said prede¬ termined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is equal to or below said predetermined fourth threshold level and the expression level of MMP1 is below a predetermined fifth threshold level, and wherein said tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter¬ mined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is equal to or below said predetermined fourth threshold level and the expression level of MMP1 is equal to or above said predeter¬ mined fifth threshold level, and
wherein said tumor is classified "intermediate risk", if the expression level of MLPH is equal to or above said prede¬ termined first threshold level, and the expression level of TOP2A is above said predetermined second threshold level or the expression level of UBE2C is above is above said prede- termined third threshold level, and the expression level of
UBE2C is equal to or above said predetermined third threshold level, and the median-normalized immunoglobulin expression is above a predetermined sixth threshold level, and
wherein said tumor is classified "high risk", if the ex- pression level of MLPH is equal to or above said predeter¬ mined first threshold level, and the expression level of TOP2A is above said predetermined second threshold level or the expression level of UBE2C is above is above said prede¬ termined third threshold level, and the expression level of UBE2C is equal to or above said predetermined third threshold level, and the median-normalized immunoglobulin expression is equal to or below said predetermined sixth threshold level, and
wherein said tumor is classified "intermediate risk", if the expression level of MLPH is below said predetermined first threshold level and the median-normalized immunoglobu¬ lin expression is above said predetermined sixth threshold level, and
wherein said tumor is classified "high risk", if the ex- pression level of MLPH is equal to or above said predeter¬ mined first threshold level and the median-normalized immu¬ noglobulin expression is equal to or below said predetermined sixth threshold level. The invention further relates to a system as described above, wherein said means for performing multiple sequential classi¬ fication steps is adapted to classify said tumor,
wherein said tumor is classified "low risk", if the ex- pression level of MLPH is equal to or above a predetermined first threshold level and the expression level of TOP2A is equal to or below a predetermined second threshold level and the expression level of UBE2C is equal to or below a prede¬ termined third threshold level, or
wherein said tumor is classified "low risk", if the ex¬ pression level of MLPH is equal to or above said predeter¬ mined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is above a predetermined fourth threshold level, or
wherein said tumor is classified "intermediate risk", if the expression level of MLPH is equal to or above said prede¬ termined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is equal to or below said predetermined fourth threshold level and the expression level of MMP1 is below a predetermined fifth threshold level, or
wherein said tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter¬ mined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is equal to or below said predetermined fourth threshold level and the expression level of MMP1 is equal to or above said predeter¬ mined fifth threshold level, or
wherein said tumor is classified "intermediate risk", if the expression level of MLPH is equal to or above said prede¬ termined first threshold level, and the expression level of TOP2A is above said predetermined second threshold level or the expression level of UBE2C is above is above said prede¬ termined third threshold level, and the expression level of UBE2C is equal to or above said predetermined third threshold level, and the median-normalized immunoglobulin expression is above a predetermined sixth threshold level, or
wherein said tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter¬ mined first threshold level, and the expression level of TOP2A is above said predetermined second threshold level or the expression level of UBE2C is above is above said prede¬ termined third threshold level, and the expression level of UBE2C is equal to or above said predetermined third threshold level, and the median-normalized immunoglobulin expression is equal to or below said predetermined sixth threshold level, or
wherein said tumor is classified "intermediate risk", if the expression level of MLPH is below said predetermined first threshold level and the median-normalized immunoglobu¬ lin expression is above said predetermined sixth threshold level, or
wherein said tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter¬ mined first threshold level and the median-normalized immu¬ noglobulin expression is equal to or below said predetermined sixth threshold level.
The invention further relates to systems as described above, wherein said predetermined first threshold level is about 2000,
wherein said predetermined second threshold level is about 150,
wherein said predetermined third threshold level is about 1000,
wherein said predetermined fourth threshold level is about 600,
wherein said predetermined fifth threshold level is about 200, and
wherein said predetermined sixth threshold level is about 1.95. Marker genes
The following marker genes are used in methods of the inven¬ tion. They are identified by their respective entries in the UniProtKB/Swiss-Prot database (see e.g.
http://www.expasy.org/sprot/) and Affymetrix Probeset ID, be¬ low .
MLPH :
Entry name MELPH_HUMAN
Primary accession number Q9BV36
Secondary accession number Q9HA71
Integrated into Swiss-Prot on June 16, 2003
Sequence was last modified on June 1, 2001 (Sequence version 1)
Annotations were last modified on November 14, 2006 (Entry version 45)
Protein name Melanophilin
Synonyms Exophilin-3
Synaptotagmin-like protein 2a Sip homolog lacking C2 domains a
Gene name Name: MLPH
Synonyms: SLAC2A
From Homo sapiens (Human) [TaxID:
9606]
CXCL13
Entry name Q53X90_HUMAN
Primary accession number Q53X90
Secondary accession numbers None
Integrated into TrEMBL on May 24, 2005
Sequence was last modified on May 24, 2005 (Sequence version 1) Annotations were last modified on October 31, 2006 try version 11)
Protein name CXCL13 protein [Fragment]
Synonym Hypothetical protein CXCL13
Gene name Name : CXCL13
From Homo sapiens (Human) [TaxID
9606]
MMP1
Entry name MMP1_HUMAN
Primary accession number P03956
Secondary accession number P08156
Integrated into Swiss-Prot on October 23, 1986
Sequence was last modified on December 1, 1992 (Sequence version 3)
Annotations were last modified on November 28, 2006 (Entry version 93)
Name and origin of the protein Protein name Interstitial collagenase [Precursor]
Synonyms EC 3.4.24.7
Matrix metalloproteinase-1
MMP-1
Fibroblast collagenase
Contains 22 kDa interstitial col- lagenase
27 kDa interstitial collagenase
Gene name Name MMP1
Synonyms : CLG
From Homo sapiens (Human) [TaxID: 9606]
TOP2A:
Entry name T0P2A_HUMAN
Primary accession number P11388 Secondary accession numbers Q71UN1 Q71UQ5 Q9HB24 Q9HB25
Q9HB26 Q9UP44 Q9UQP9
Integrated into Swiss-Prot on July 1, 1989
Sequence was last modified on May 4, 2001 (Sequence ver¬ sion 3)
Annotations were last modified on November 14, 2006
(Entry version 92)
Protein name DNA topoisomerase 2-alpha
Synonyms EC 5.99.1.3
DNA topoisomerase II, alpha lsozyme
Gene name Name: TOP2A
Synonyms: TOP2
From Homo sapiens (Human) [TaxID: 9606]
UBE2C: Entry name UBE2C_HUMAN
Primary accession number 000762
Secondary accession numbers None
Integrated into Swiss-Prot on December 15, 1998
Sequence was last modified on July 1, 1997 (Sequence version 1)
Annotations were last modified on October 31, 2006 (En¬ try version 67)
Protein name Ubiquitin-conj ugating enzyme E2 C
Synonyms EC 6.3.2.19
Ubiquitin-protein ligase C
Ubiquitin carrier protein C
UbcHIO
Gene name Name: UBE2C
Synonyms: UBCHIO
FFrroomm Homo sapiens (Human) [TaxID: 9606] Examples :
Prognosis Example 1 In a tumor sample of a patient the expression levels of MLPH, TOP2A, UBE2C, CXCL13, MMP1, and a median-normalized expres¬ sion value of IG was determined.
The determined expression levels were 2523 for MLPH, 188 for TOP2A, 576 for UBE2C, 840 for CXCL13, and 1.8 for IG.
Following the decision tree shown in Fig. 1, the sample was classified as "low risk" of recurrence. Prognosis Example 2
Another tumor sample was analyzed. The expression levels of MLPH, TOP2A, UBE2C, CXCL13, MMP1, and a median-normalized ex¬ pression value of IG were determined.
The following expression levels were obtained: 2015 for MLPH, 166 for TOP2A, 1432 for UBE2C, 795 for CXCL13, and 1.5 for IG. Following the decision tree shown in Fig. 1, the sample was classified as "high risk" of recurrence.

Claims

Claims :
1. Method for the prognosis of cancer in a patient, from a tumor sample of said patient, said method comprising:
(a) determining in said tumor sample multiple expres¬ sion levels selected from the group consisting of the expres¬ sion level of MLPH, the expression level of TOP2A, the expression level of UBE2C, the expression level of CXCL13, the expression level of MMP1, and the median-normalized immu¬ noglobulin expression level;
(b) comparing each of said expression levels with a marker gene specific threshold level;
(c) performing multiple sequential classification steps on the basis of the outcome of said comparison steps under
(b) , thereby classifying said tumor into one of at least two distinct risk classes.
2. Method of claim 1, wherein all expression levels in said group of expression levels are determined.
3. Method of claim 1 or 2 wherein said prognosis is the de¬ termination of the risk of recurrence of cancer in said pa¬ tient within 5 years.
4. Method of any one of the preceding claims, wherein said prognosis is a classification of said patient into one of three distinct classes, said classes corresponding to a "high risk of recurrence" class, an "intermediate risk of recur- rence" class and a "low risk or recurrence" class.
5. Method of any one of the preceding claims, wherein said cancer is breast cancer or ovarian cancer.
6. Method of any one of the preceding claims, wherein said determination of expression levels is in a formalin-fixed pa¬ raffin embedded sample or in a fresh-frozen sample.
7. Method of any one of the preceding claims,
wherein said tumor is classified "low risk", if the expression level of MLPH is equal to or above a predetermined first threshold level and the expression level of TOP2A is equal to or below a predetermined second threshold level and the expression level of UBE2C is equal to or below a prede¬ termined third threshold level, and
wherein said tumor is classified "low risk", if the expression level of MLPH is equal to or above said predeter- mined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is above a predetermined fourth threshold level, and
wherein said tumor is classified "intermediate risk", if the expression level of MLPH is equal to or above said prede¬ termined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is equal to or below said predetermined fourth threshold level and the expression level of MMP1 is below a predetermined fifth threshold level, and
wherein said tumor is classified "high risk", if the ex- pression level of MLPH is equal to or above said predeter¬ mined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is equal to or below said predetermined fourth threshold level and the expression level of MMP1 is equal to or above said predeter¬ mined fifth threshold level, and
wherein said tumor is classified "intermediate risk", if the expression level of MLPH is equal to or above said prede- termined first threshold level, and the expression level of TOP2A is above said predetermined second threshold level or the expression level of UBE2C is above is above said prede¬ termined third threshold level, and the expression level of UBE2C is equal to or above said predetermined third threshold level, and the median-normalized immunoglobulin expression is above a predetermined sixth threshold level, and
wherein said tumor is classified "high risk", if the ex- pression level of MLPH is equal to or above said predeter¬ mined first threshold level, and the expression level of TOP2A is above said predetermined second threshold level or the expression level of UBE2C is above is above said prede¬ termined third threshold level, and the expression level of UBE2C is equal to or above said predetermined third threshold level, and the median-normalized immunoglobulin expression is equal to or below said predetermined sixth threshold level, and
wherein said tumor is classified "intermediate risk", if the expression level of MLPH is below said predetermined first threshold level and the median-normalized immunoglobu¬ lin expression is above said predetermined sixth threshold level, and
wherein said tumor is classified "high risk", if the ex- pression level of MLPH is equal to or above said predeter¬ mined first threshold level and the median-normalized immu¬ noglobulin expression is equal to or below said predetermined sixth threshold level.
8. Method of any one of claims 1-6,
wherein said tumor is classified "low risk", if the expression level of MLPH is equal to or above a predetermined first threshold level and the expression level of TOP2A is equal to or below a predetermined second threshold level and the expression level of UBE2C is equal to or below a prede¬ termined third threshold level, or
wherein said tumor is classified "low risk", if the expression level of MLPH is equal to or above said predeter¬ mined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is above a predetermined fourth threshold level, or wherein said tumor is classified "intermediate risk", if the expression level of MLPH is equal to or above said prede¬ termined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is equal to or below said predetermined fourth threshold level and the expression level of MMP1 is below a predetermined fifth threshold level, or
wherein said tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter¬ mined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is equal to or below said predetermined fourth threshold level and the expression level of MMP1 is equal to or above said predeter¬ mined fifth threshold level, or
wherein said tumor is classified "intermediate risk", if the expression level of MLPH is equal to or above said prede¬ termined first threshold level, and the expression level of TOP2A is above said predetermined second threshold level or the expression level of UBE2C is above is above said prede¬ termined third threshold level, and the expression level of UBE2C is equal to or above said predetermined third threshold level, and the median-normalized immunoglobulin expression is above a predetermined sixth threshold level, or
wherein said tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter- mined first threshold level, and the expression level of
TOP2A is above said predetermined second threshold level or the expression level of UBE2C is above is above said prede¬ termined third threshold level, and the expression level of UBE2C is equal to or above said predetermined third threshold level, and the median-normalized immunoglobulin expression is equal to or below said predetermined sixth threshold level, or wherein said tumor is classified "intermediate risk", if the expression level of MLPH is below said predetermined first threshold level and the median-normalized immunoglobu¬ lin expression is above said predetermined sixth threshold level, or
wherein said tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter¬ mined first threshold level and the median-normalized immu¬ noglobulin expression is equal to or below said predetermined sixth threshold level.
9. Method of claim 7 or 8
wherein said predetermined first threshold level is about 2000,
wherein said predetermined second threshold level is about 150,
wherein said predetermined third threshold level is about 1000,
wherein said predetermined fourth threshold level is about 600,
wherein said predetermined fifth threshold level is about 200, and
wherein said predetermined sixth threshold level is about 1.95.
10. Method of any one of the preceding claims, wherein an expression level of a marker gene is substituted with the ex¬ pression level of a substitute marker gene, said substitute marker gene being co-regulated with said marker gene.
11. Method of any one of claims 1-9, wherein an expression level of a marker gene is substituted with the expression le¬ vel of a substitute marker gene, said substitute marker gene being selected from Table 1.
12. A system for the prognosis of cancer in a patient, said system comprising (a) means for obtaining multiple expression levels in tumor sample of said patient, said multiple expression levels being selected from the group consisting of the expression level of MLPH, the expression level of TOP2A, the expression level of UBE2C, the expression level of CXCL13, the expres¬ sion level of MMP1, and the median-normalized immunoglobulin expression level;
(b) means for comparing each of said expression levels with a marker gene specific threshold level;
(c) means for performing multiple sequential classifi¬ cation steps on the basis of the outcome of said comparison steps under (b) , and for classifying said tumor into one of at least two distinct risk classes.
13. System of claim 12, wherein said means for determining multiple expression levels is a gene-chip system, a real time PCR system.
14. System of claim 12 or 13, wherein said means for per- forming multiple sequential classification steps is a sepa¬ rate personal computer, or a computer integral with the re¬ maining components of the system.
15. System of claim 14, wherein the computer receives ex- pression level measurements directly from said means for de¬ termining the expression level.
16. System of any of claims 12-15, wherein said means for performing multiple sequential classification steps is adap- ted to classify said tumor,
wherein said tumor is classified "low risk", if the expression level of MLPH is equal to or above a predetermined first threshold level and the expression level of TOP2A is equal to or below a predetermined second threshold level and the expression level of UBE2C is equal to or below a prede¬ termined third threshold level, and
wherein said tumor is classified "low risk", if the expression level of MLPH is equal to or above said predeter- mined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is above a predetermined fourth threshold level, and
wherein said tumor is classified "intermediate risk", if the expression level of MLPH is equal to or above said prede¬ termined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is equal to or below said predetermined fourth threshold level and the expression level of MMP1 is below a predetermined fifth threshold level, and
wherein said tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter¬ mined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is equal to or below said predetermined fourth threshold level and the expression level of MMP1 is equal to or above said predeter¬ mined fifth threshold level, and
wherein said tumor is classified "intermediate risk", if the expression level of MLPH is equal to or above said prede¬ termined first threshold level, and the expression level of TOP2A is above said predetermined second threshold level or the expression level of UBE2C is above is above said prede¬ termined third threshold level, and the expression level of UBE2C is equal to or above said predetermined third threshold level, and the median-normalized immunoglobulin expression is above a predetermined sixth threshold level, and
wherein said tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter- mined first threshold level, and the expression level of
TOP2A is above said predetermined second threshold level or the expression level of UBE2C is above is above said prede¬ termined third threshold level, and the expression level of UBE2C is equal to or above said predetermined third threshold level, and the median-normalized immunoglobulin expression is equal to or below said predetermined sixth threshold level, and
wherein said tumor is classified "intermediate risk", if the expression level of MLPH is below said predetermined first threshold level and the median-normalized immunoglobu¬ lin expression is above said predetermined sixth threshold level, and
wherein said tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter¬ mined first threshold level and the median-normalized immu¬ noglobulin expression is equal to or below said predetermined sixth threshold level.
17. System of any of claims 12-15, wherein said means for performing multiple sequential classification steps is adap¬ ted to classify said tumor,
wherein said tumor is classified "low risk", if the ex- pression level of MLPH is equal to or above a predetermined first threshold level and the expression level of TOP2A is equal to or below a predetermined second threshold level and the expression level of UBE2C is equal to or below a prede¬ termined third threshold level, or
wherein said tumor is classified "low risk", if the ex¬ pression level of MLPH is equal to or above said predeter¬ mined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is above a predetermined fourth threshold level, or
wherein said tumor is classified "intermediate risk", if the expression level of MLPH is equal to or above said prede¬ termined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is equal to or below said predetermined fourth threshold level and the expression level of MMP1 is below a predetermined fifth threshold level, or
wherein said tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter- mined first threshold level and the expression level of TOP2A is above said predetermined second threshold level and the expression level of UBE2C is below said predetermined third threshold level and the expression level of CXCL13 is equal to or below said predetermined fourth threshold level and the expression level of MMP1 is equal to or above said predeter¬ mined fifth threshold level, or
wherein said tumor is classified "intermediate risk", if the expression level of MLPH is equal to or above said prede¬ termined first threshold level, and the expression level of TOP2A is above said predetermined second threshold level or the expression level of UBE2C is above is above said prede¬ termined third threshold level, and the expression level of UBE2C is equal to or above said predetermined third threshold level, and the median-normalized immunoglobulin expression is above a predetermined sixth threshold level, or
wherein said tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter¬ mined first threshold level, and the expression level of TOP2A is above said predetermined second threshold level or the expression level of UBE2C is above is above said prede¬ termined third threshold level, and the expression level of UBE2C is equal to or above said predetermined third threshold level, and the median-normalized immunoglobulin expression is equal to or below said predetermined sixth threshold level, or
wherein said tumor is classified "intermediate risk", if the expression level of MLPH is below said predetermined first threshold level and the median-normalized immunoglobu¬ lin expression is above said predetermined sixth threshold level, or
wherein said tumor is classified "high risk", if the expression level of MLPH is equal to or above said predeter¬ mined first threshold level and the median-normalized immu- noglobulin expression is equal to or below said predetermined sixth threshold level.
18. System of claim 16 or 17,
wherein said predetermined first threshold level is about 2000,
wherein said predetermined second threshold level is about 150,
wherein said predetermined third threshold level is about 1000,
wherein said predetermined fourth threshold level is about 600,
wherein said predetermined fifth threshold level is about 200, and
wherein said predetermined sixth threshold level is about 1.95.
PCT/EP2009/050464 2008-01-28 2009-01-16 Methods and systems for breast cancer prognosis Ceased WO2011137912A1 (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003060470A2 (en) * 2001-12-21 2003-07-24 Arcturus Engineering, Inc. Breast cancer expression profiling
WO2004079014A2 (en) * 2003-03-04 2004-09-16 Arcturus Bioscience, Inc. Signatures of er status in breast cancer
WO2006133923A2 (en) * 2005-06-16 2006-12-21 Bayer Healthcare Ag Diagnosis, prognosis and prediction of recurrence of breast cancer
WO2006135886A2 (en) * 2005-06-13 2006-12-21 The Regents Of The University Of Michigan Compositions and methods for treating and diagnosing cancer

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003060470A2 (en) * 2001-12-21 2003-07-24 Arcturus Engineering, Inc. Breast cancer expression profiling
WO2004079014A2 (en) * 2003-03-04 2004-09-16 Arcturus Bioscience, Inc. Signatures of er status in breast cancer
WO2006135886A2 (en) * 2005-06-13 2006-12-21 The Regents Of The University Of Michigan Compositions and methods for treating and diagnosing cancer
WO2006133923A2 (en) * 2005-06-16 2006-12-21 Bayer Healthcare Ag Diagnosis, prognosis and prediction of recurrence of breast cancer

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