DE102005056365A1 - Individualized prognosis, monitoring and aftercare of tumor patients, by determining changes in genomic or expression profiles over time - Google Patents

Abstract

Method for individualized prognosis, therapeutic monitoring and/or aftercare of patients with tumors based on analysis of genomic or expression profiles. Method for individualized prognosis; therapeutic monitoring and/or after care of patients with tumors based on analysis of genomic or expression profiles in which (a) a genomic or expression profile of a first sample, containing tumor tissue, is determined at the time the tumor is diagnosed, where the profile contains the genomic/expression status for a first multiplicity of human genes, selected for each particular patient, and the results are stored; (b) at some later time, a second such profile is taken on a second sample (containing lymph node tissue, bone marrow and/or body fluid); and (c) the two profiles are compared and, on the basis of this comparison, therapeutic recommendations and evaluations and/or prognosis are made.

Description

The The present invention relates to a method of individualizing Prognosis, therapy recommendation and / or tracking and / or aftercare a tumor disease of a patient. Such methods are needed to be precise Decision criteria for a certain therapy or the resumption of a therapy receive.

nowadays Are already used in the clinic molecular gene expression profiles, to decision criteria for a particular therapy, for example, for or against a cytostatic Therapy or radiotherapy of a carcinoma, especially one Breast cancer, to receive. The gene expression profile is there which genes are activated in a tumor and which are not. By means of such Genex pressionsprofile example, already a therapy recommendation for pronounced the chemotherapy drug Herceptin.

Of the Whole state of the art refers to consistently so-called tumor marker proteins, of which it is known that their increased expression with the appearance of a primary tumor correlated. Such tumor marker genes are, for example, the genes for the estrogen receptor, the Her-2 gene as well as genes regarding the profiling properties of the tumor. Other relevant genes are dihydrofolate reductase (DHFR) for methotrexate therapy, dihydropyrimidine dehydrogenase (DPYD) for Xeloda therapy and the multidrug resistance genes, such as MDR, ABCG2. The following is a table showing the correlation between the recommended therapeutic and the expression of various tumor marker genes indicates.

The Approach, however, is for all Patients or each tumor type identical, so that due to the expression Of a few predetermined tumor markers, it is uniform for all patients Prognosis, therapy recommendation and the like is pronounced.

This However, the approach will be diversity of the disease does not do justice and causes only on an experimental basis Ways can be determined whether the particular patient on the Respective proposed therapy also responds.

task It is therefore an object of the present invention to provide a method for individualizing Prognosis, therapy recommendation and / or tracking and / or aftercare to provide a tumor disease of a patient. This task is achieved by the method according to claim 1.

According to the invention will now a first expression profile or profile of the genomic DNA of a Tumor tissue sample created. Ideally, an overall profile will be about almost created the entire or the entire genome of the human. For this The inventors have an array as an examination system available, which it allowed the expression or genome of approximately 28,600 human To capture genes in which the function of the encoded protein is known.

In this way, a very complex profile of the biological properties of each tumor determined.

Out this variety of genome or expression information is now for one individualized therapy an individual selection of human Genes or their expression recorded.

On this way can for each individual patient an independent genome or expression profile not only tumor marker genes but also non-tumor marker genes may include. The profile can all or some of those genes or their expression from the first measured gene expression profile containing there gene expression have that over is a predetermined threshold. In particular, it is not on the conventional ones Tumor marker genes limited, but advantageously also contains some or predominantly Genes that are not considered tumor markers.

in the The following will now be for the prognosis, therapy recommendation and / or tracking and / or aftercare each containing or consisting of another sample of the patient bone marrow, lymph node tissue and / or body fluid, e.g. peripheral blood, urine, ascites, pleural effusion, the patient examined on the profile according to the above selection. When examining a blood sample, this step is always done a determination of the expression profile instead of the genomic profile DNA while at Examination of a tissue sample both profiling the genomic DNA as well as expression possible is. This profile may be with the snapshot of the biology of the Tumors at the time of initial diagnosis (i.e., the selection from the first created profile), thus enabling an individual meaningful and cost-effective Aftercare of the patient. Here, for example, indicates a missing further detection of gene expression of this gene combination in the peripheral Blood on a high probability of complete removal of the tumor. A decrease in gene expression under adjuvant Therapy speaks for a high probability of the response of the therapy. Becomes On the other hand, gene expression persists or after some time again for the selection of genes detected or a new with the tumor formation associated genomic mutation, so is highly likely from a recurrence or a therapy failure.

By the possibility, the profile of a variety of genes (for example, by means of a High-density microarrays with approximately 36,500 DNA probes), is it now possible an individualized prognosis, an individualized therapy recommendation and / or tracking and / or performing individual aftercare. One Such overall profile of a tumor or a sample of a patient At the same time, it also represents a knowledge base for the patient, who is responsible for the planning and application of new therapies in the future or at a later date to disposal stand. Last but not least, the method according to the invention is suitable also for target identification of new experimental therapies.

The Capture of genomic DNA profile or gene expression profile takes place according to conventional, in the prior art described methods, for example for the determination the expression profile of the combination of a reverse transcriptase and subsequent Polymerase chain reaction after digestion of the sample. Also the proof single mRNA or c-DNA thereof by means of a DNA probe array Well known in the art.

in the The following are some examples of the method according to the invention on concrete patient data Darge presents. Show it

the 1 to 4 the results of a selected example;

5 Results of the same example as in 4 ;

6 a findings report and a resulting individualized therapy recommendation as well as

7 the comparison of several measurements according to the present invention with each other.

The by the 1A to 4B constructed table shows experimental measurement results to illustrate the invention. Here are the 1A . 2A . 3A and 4A arranged side by side, as well as the 1B . 2 B . 3B and 4B , The 1B . 2 B . 3B and 4B put in each case the columns of 1A . 2A . 3A and 4A go down. The assignment of the individual lines is indicated by the reference numerals 1 to 4 in the respective figures.

According to the invention, a total of 93 genes were examined with regard to their expression in tumor tissue using the DNA array. The columns 1 to 3 in the 1A and 1B and the next column (column 4) in the 2A and 2 B indicate in this order the systematic name, the identification number of the gene under investigation, the symbol of the gene and the respective gene product. The following three columns (the first three columns in the 3A and 3B ) report measured values for the expression of these genes in samples from a total of 23 mammary carcinomas. The first column in the 3A and 3B gives the mean value for the expression of each of the genes, these mean values having already been normalized with respect to the different signal sizes in different DNA arrays used (array-normalized signal, determined according to the manufacturer's instructions). They therefore represent values that can be compared across different arrays and also between different genes. The following two columns in the 3A and 3B give the respective limits between the first and the second third of the samples (second column in the 3A and 3B ) and the limit between the second third and the third third of the samples (column 3 in 3A and 3B ) at. All measured values here and also for the following figures were determined in each case according to the manufacturer-specific protocol for the named DNA array.

The following four columns (last four columns in the 3A and 3B ) are measured values on tissue samples of a patient. The first of these four columns (sample E11709) shows the corresponding measurements of a breast cancer specimen of the female patient from the primary tumor. The dark shaded values are striking. The composition of these dark-shaded markers then serves as a patient-specific expression profile in the following measurements. Above this column is the diagnostic classification of the patient.

Following this measurement of the sample from the primary tumor, the patient was treated in the conventional manner with the TAC scheme. At time t2 (third last column in the 3A and 3B ), another sample was examined. The corresponding measured values can be found again in the third to last column in the 3A and 3B , It is striking that certain marker proteins, such as ErbB2 were increasingly expressed. After this measurement, Herceptin-Xeloda therapy was switched. At a later time t3 sample material of the patient was again examined, for the result of the investigation in the second last column in the 3A and 3B place. It can be seen that the Herceptin-Xeloda therapy did not lead to the desired success (see, for example, the further increase in the expression of the ErbB2 gene) and therefore was also discontinued after this measurement. Thereafter, a therapy with the bifunctional EGFR / Her2 inhibitor was changed, but no therapeutic success (see, for example, the expression of the ErbB2 gene) could be observed more, as shown in the last column in the 3A and 3B can be seen at a time t4 measured values.

4A and 4B indicate for each of these markers the respective clinical significance.

It is shown by this example that by a specific selection of marker proteins or their expression a statement about the Prognosis, the success of treatment or generally a follow-up too performed a therapy can be. Since this is a study on the principle suitability the method according to the invention All measurements (t1 to t4) were performed with the mentioned DNA arrays. In However, the diagnostic application in practice, only the Genes of the expression profile (itself the first time on a DNA array is determined) as described above, for example by means of multiplex PCR, examined. this leads to at tremendous cost savings without the benefit of a patient-specific Abandon profile.

5 shows the in the 1 to 4 data shown in a selection, wherein in each case the expression of a gene in the individual A to D over time via the measurements t1 to t4 is shown.

For the topoisomerase 2-alpha gene, it can be seen that TAC therapy actually reduced its expression. However, this does not apply to c-erbB-2, thymidine synthetase or c-myc (see p. 5B . 5C and 5D ). Shown in these four figures are in each case the average values over a normal collective as well as the values of the patient sample.

It can be concluded that the original therapy actually treated certain tumor clones. However, other clones were selected out, as can be seen in the course of the c-erbB-2 curve. It can also be seen that the Herceptin therapy had no further success between the times t2 and t3. Although the switch to the bifunctional EGFR-Her2 inhibitor had the effect of suppressing the clone of the tumor expressing c-erbB-2, the expression of the Thymidine synthetase can be observed in increasing amounts until time t4. This also applies to the gene c-myc. As a result, the tumor was resistant to all conventional therapies at time t4.

6 shows an example of how a finding report and a therapy recommendation can be determined from the determination of the gene expression status. So was in the examples in 6 hormone receptor status, the status of progression markers, the status of oncogenes such as Her-2, EGFR, c-myc, etc. as well as markers for chemoresistance or sensitivity to certain therapeutics. A definite pattern of expression is followed by a final recommendation regarding the therapy to be included, as described under point 3 in 6 is shown.

7 shows that the values determined by the method according to the invention, in particular the values determined via the abovementioned DNA chip, are very reliable. In 7 the same sample was measured with respect to a total of 10 genes (PLCG1 ... FOS) with 6 DNA arrays each. It turns out that after normalization to the signal intensity of the respective array (so-called array-normalized signal), the scattering of the measured values of the individual arrays is extremely low. In 7 the two show the mean of the measurement result over the 6 arrays, and the dashes the standard deviation of each measured value from that mean. This shows that extremely reliable results can be achieved with the method according to the invention.

Around To use the DNA array, it is necessary to use either genomic Prepare DNA accordingly. However, this can be done according to conventional Standard laboratory methods take place. On the other hand, provided an expression profile is to be determined, it is necessary to rewrite the mRNA to cDNA by means of a reverse transcription. Again, this can be done by default according to textbook procedures respectively.

Becomes as a sample with a body fluid it works for an expression profile that is tailored to the individualized Patient profile belonging previously to amplify mRNA (RT-PCR). This procedure is also in textbooks sufficiently described. The evaluation of the DNA arrays used or the evaluation a conventional thus described RT-PCR is device-specific according to the manufacturer or also standardized according to Textbook requirements.

in the Following is an example of a follow-up concept will be described in detail.

After this in a first step from primary tissue by means of a DNA microarray a set of marker protein (gene profile or expression profile) determined in the follow-up approach to be examined are in the context of the patient's follow-up after therapy at regular intervals (e.g. every three months) a few milliliters of blood taken to a possibly Occurrence of tumor cells in the blood and, if necessary their change capture. The blood is taken directly into vessels with a RNA stabilizing lysing reagent (Tempus Blood RNA Tube, Applied Biosystems). In these vessels is the RNA then frozen several days at room temperature, several weeks transportable and durable.

The Isolation of the RNA is carried out with a 6100 workstation (Applied Biosystems) after for this device specified standard protocol. The thus isolated mRNA is determined by reverse transcriptase into cDNA and transcribed into a Taq-Man Real-time PCR amplified.

The at the original Microarray analysis selected Genes (for example 4 to 6 genes) are those which are to be examined in the Patient in the primary tissue were found particularly strongly expressed. It does not have to be necessarily or necessarily exclusively as tumor marker genes / proteins known marker genes / proteins act.

Become the genes mentioned (which together form a profile) or their Expression in the blood of the patient during the individual sampling stronger as expressed in the blood of healthy controls, so suggests this affects the spread of tumor cells and affects that further therapy.

For the blood collection and RNA isolation will be a suitable working protocol below stated:

Blood removal:

• 1) Blood collection (3 ml) directly into the tense Blood RNA Tubes (contain lysis buffer),
• 2) strong mix for 20 sec,
• 3) store or transport at room temperature for up to 5 days or at -20 ° C for several Store for weeks.

RNA isolation

6100 workstation protocol "Tempus Blood RNA "

• 1) Mix blood with lysis buffer 1: 1 with PBS in 50 ml tubes,
• 2) Vortex for 30 sec,
• 3) "Large Volume RNA Prep Filters "and Put 20 ml reservoirs on the adapter plate and place on the "waste Position of the workstation place,
• 4) diluted Add blood to the reservoirs and apply 80% vacuum for 500 sec,
• 5) Place fresh 5 ml reservoirs and add 1 to 4 ml wash solution 80% vacuum wash,
• 6) with 4.5 ml washing solution 2 to 80% vacuum wash,
• 7) Repeat steps 5 + 6 until all filters are washed clean are,
• 8) Place a new 5 ml reservoir and mix with 350 μl of Absolute RNA wash solution Incubate for 15 min,
• 9) with washing solution 2 for 5 incubate min,
• 10) wash at 80% vacuum for 120 sec,
• 11) two more times with 3 ml of washing solution 2 at 80% vacuum for 120 sec to wash,
• 12) Remove 5 ml reservoir and dry at 90% vacuum for 400 sec,
• 13) Filter plate in the "Collection Position "bring,
• 14) with 500 μl elution Incubate for 120 sec,
• 15) at 60% vacuum for Elute 120 sec in 2 ml reaction vessels,
• 16) optionally concentrate RNA and concentration photometrically measure up.

Claims (8)

A method for individualized prognosis, therapy tracking and / or aftercare of a tumor disease of a patient, characterized in that a first genome profile or expression profile of a first sample containing tumor tissue is detected at the time of diagnosis of the tumor containing the genome status or expression status of a first plurality of human genes, for the individual patient for an individual selection of human genes from the plurality of detected human genes the genome status or expression status is stored, at a later time for the genes of the individual selection a further genome profile or expression profile of a second sample containing lymph node tissue, bone marrow and / or a body fluid is detected and compared with the first genome profile or expression profile, and based on this comparison, a therapy recommendation, a prognosis assessment or a therapy assessment is performed. Method according to the preceding claim, characterized characterized in that the first genome profile or expression profile the genome status or expression status of more than 8,000 genes, advantageously more than 28,000 genes, advantageously more as having 36,000 genes. Method according to one of the preceding claims, characterized characterized in that the first selection is tumor marker genes, not tumor marker genes or has both tumor marker genes and genes that do not have tumor marker genes are. Method according to one of the preceding claims, characterized characterized in that the comparison is qualitative and / or quantitative carried out becomes. Method according to one of the preceding claims, characterized characterized in that the first genome profile or expression profile of tissue of a primary tumor or determined from a sample before therapy of the tumor disease becomes. Method according to one of the preceding claims, characterized in that the first selection contains or consists of those genes or a subset thereof, which according to the first Ge Nom profile or expression profile are strongly expressed. Method according to one of the preceding claims, characterized characterized in that the body fluid contains peripheral blood, urine, ascites or pleural effusion or it consists. Method according to one of the preceding claims, characterized characterized in that the overall profile the presence and / or Absence and / or the allelic status of each gene in the genome profile included.