Background information
The Testing
Overview
Appendices
Introduction to Molecular Tumor Diagnostics
Almost all human cells contain molecules called DNA (deoxyribonucleic acid). The DNA is harboring the hereditary information, the genes. The genes are controlling processes like growth, cell division, and programmed cell death (apoptosis). In tumors, the cells became abnormal by damages and alterations of the DNA, which results in uncontrolled growth and cell proliferation. These alterations are usually complex and manifold.
Initially, the tumor cells divide locally defined. But when the tumor gets larger and acquires more and more genetic alterations, the tumor may grow invasive beyond the local boundary and become metastatic.
During the process of metastasis, the primary tumor is shedding cancer cells into the lymphatic system and bloodstream. Thereby, the cancer cells are transported to distant body sites, where metastases can emerge.
Modern techniques of molecular biology have allowed the identification of numerous of the genetic alterations responsible for the emergence of cancer. This knowledge can be used for the detection of tumor cells for diagnostic purposes. Consequently, many molecular markers are available for the detection of circulating tumor cells in the bloodstream. From our wide panel of analytic parameters we select for the appropriate combination depending on type of tumor and each specific case.
Because carcinomas are of epithelial origin and epithelial cells are normally absent in blood, genes active in epithelial cells can be used for detection of circulating carcinoma cells. In this context, the cytokeratin genes (e.g. CK19, CK20) are suitable markers for many carcinomas. Depending of the origin of the tumor, genes coding for specific organ functions can be measured in blood. For example, the detection of cells expressing the prostate specific antigen (PSA) are indicative for circulating prostate carcinoma cells.
Typical cancerous aberrations are mutations in oncogenes (e.g. K-ras), allelic losses (i.e. LOH) or overexpression of particular genes (e.g. erbb2, c-myc). Genes are known to be silent in normal differentiated tissues but to be re-activated in malignant cells (e.g. telomerase, G250). Furthermore, enzymes with anti-oxidative functions (e.g. MnSOD or Thioredoxin-reductase) are frequently over-expressed in human tumors*. On the other hand, so called tumor suppressor genes keeping cell proliferation under control in normal tissues are almost always inactivated in tumors. Inactivation happens by mutation, genetic loss or predominantly by the mechanism of promotor hypermethylation which can also be diagnostically detected.
Determination of resistance factors and metabolic properties of the tumor cells can provide important information for selection of an appropriate therapy. For many anti-cancer drugs metabolic enzymes are known whose differential expression in tumor cells may influence the effect of therapy. The appended schedule in this brochure depicts the relations between genes and drugs response.
In particular newer drugs can be accurately directed against specific target molecules (e.g. Herceptin, Erlotinib or Sutent). These drugs are usually very effective and evoke rather less severe side effects than undirected drugs. A therapy with the specific inhibitor appears rational only if the molecular targets are present in the tumor cells. We use genetic analysis if these molecular drug targets are expressed in the isolated tumor cells and a therapy with these potent drugs is appropriate.
*supporting evidence from the academic literature:
Lincoln D. The Thioredoxin-Thioredoxin reductase system: over-expression in human cancer. Anticancer research.
2003; 23; 2425-2434.
Comment: Increased levels of thioredoxin (TRX) positively correlate with thioredoxin reductase (TR) erpression. Aggressive tumors over-express
both TRX and TR.
Principles of our Services
The discovery of specific genetic alterations in malignant tumors has opened up new directions in cancer research. Knowledge about function of oncogenes and tumor proteins has fundamentally contributed to the understanding of the molecular and cellular principles of malignant growth. Based on these principles, molecular analyses of genes involved in emergence and progression of human cancers are performed in our laboratory. We are focused on aspects of:
- early detection of cancer & detection of minimal residual disease
- sensitivity and resistance to anticancer therapy
It is possible to isolate tumor cells from the peripheral blood of patients. By genetic characterization of these cells, high-risk and low-risk patients can be identified. Moreover, resistance factors can be determined which provide important information for setting up an optimized therapy. This may influence decisions about:
- early initiation of therapy or supportive treatment
- targeted, patient specific therapy
- identification of drug resistance
Our services for the molecular detection of circulating cancer cells in the blood stream allows the identification of cancer patients probably at higher risk of relapse and metastasis. These patients may benefit from additional adjuvant therapies. We use special techniques to isolate micrometastases from peripheral blood.
For determination what therapies are most likely effective, we provide molecular analyses for the expression of drug targets and chemo-resistance markers in the tumor cells. We perform these analyses usually from a blood sample, but according to specific demands, tumor tissues, ascites or bone marrow could be used as well.
Early detection of cancerous cells is also possible in body fluids like urine, lavages or in sputum.
Isolation of tumor cells from blood
The isolation of tumor cells from the bloodstream can be performed by two different principles:
- with immuno-magnetic beads
- by filtration techniques
Isolation of tumor cells from the bloodstream by filtration
Carcinomas are tumors of epithelial origin. Since epithelial cells are usually larger than blood cells, the smaller blood cells can be separated based on these size difference from larger carcinoma cells. Moreover, disseminated cancer cells occur in the bloodstream frequently as clusters of several cells – so called micrometastases. Consequently, it is common practice to isolate the micrometastases from body fluids by filtration*. We use an optimized filtration technique which retains the clumpy micrometastases and washes most of the normal blood cells away. The pictures below show putative isolated tumor cells stained positive for cytokeratin (CK). The cells were isolated by our technique from the bloodstream of cancer patients.
After the isolation, the cells on the filter are analysed by molecular genetic profiling to characerize these cells to be of cancerous nature.
*supporting evidence from the academic literature:
Hirte HW, et al. A rapid and simple method for the purification of tumor cells from ascitic fluid of ovarian carcinoma. Gynecol Oncol. 1992 Mar;44(3):223-6.
Rostagno P, et al. Detection of rare circulating breast cancer cells by filtration cytometry and identification by DNA content: sensitivity in an experimental model. Anticancer Res. 1997 Jul-Aug;17(4A):2481-5.
Molecular detection of circulating tumor cells (CTCs)
After the putative micrometastases are isolated from the blood stream, the identification of these cells as cancerous cells is done by genetic analysis. We use real-time quantitative PCR to measure the expression of genes which are typical for cancerous cells.
By our own experience, CTCs isolated from tumor patients frequently show genetic alterations typical for cancer cells. According to academic research, the presence of tumor cells in the bloodstream is a risk-factor for relapse and occurrence of metastases*. Such high-risk patients may profit from adjuvant therapies to prevent further progression of the disease.
*supporting evidence from the academic literature:
Koch M, et al. Detection of hematogenous tumor cell dissemination predicts tumor relapse in patients undergoing
surgical resection of colorectal liver metastases. Ann Surg. 2005 Feb;241(2):199-205.
Comment: This is the first study demonstrating that detection of hematogenous tumor cell dissemination during hepatic rescection of colorectal
cancer metastases predicts tumor relapse.
Pantel K, Cote RJ, Fodstad O. Detection and clinical importance of micrometastatic disease. J Natl Cancer Inst. 1999 Jul 7;91(13):1113-24. Review.
Testing for chemotherapy resistance and chemosensitivity
A fundamental treatment option for cancer is chemotherapy. For each type of tumor, different chemotherapy regimes (drugs or drug combinations) exist. The efficacy of these regimes is usually determined by clinical trials and those regimes showing statistically the best success-rates are classified the most effective. However, an individual patient may respond differently to each therapy and it is a difficult task for the physician to choose the most appropriate from the available treatment regimes. Chemosensitivity testing can aid to select those drugs more likely to be effective than others for an individual patient.
Our chemosensitivity testing is based on genetic profiling of drug-metabolizing genes and cellular drug-target genes in the isolated tumor cells. The expression of these genes is determined by accurate technique of real-time quantitative PCR. From the academic literature it is known that the expression of drug metabolizing genes in tumor cells is influencing the action of the drug*1,2. Some drugs act directly on specific cellular molecules, so called drug targets. An effective response of such drugs requires the presence of these drug targets in the cells, which we can assess by measuring the gene expression of the drug target gene.
Several chemo-drugs (e.g. cyclophosphamide, capecitabine, irinotecan, dacarbazine) are virtually inactive in its parent form and need prior activation in the body, e.g. by liver enzymes. Such drugs are rather problematic to test ex-vivo on tumor-tissues with the widely used cell-viability chemosensitivity assays. In contrast, chemosensitivity testing by our genetic profiling does not depend on prior activation of the test drugs by liver enzymes.
The aim of adjuvant therapies is the elimination of residual cancer cells (micrometastases) which are responsible for relapse or metastasis after the primary tumor has been removed. Our philosophy is therefore to use isolated micrometastases for the resistance analyses, since these residula tumor cells should be actually killed by the therapy. It is well known from the literature that tumors are very heterogeneous. They do not consist of identical cancer cells, but are rather composed of groups of cells which may differ in regard of their genetic alterations. The cells shed in the blood stream forming micrometastases may consequently be not genetically identical with the average primary tumor, which has recently been proven by academic work*3.
Therefore, a targeted therapy against the disseminated cancer cells may be a more straightforward way to prevent the emergence of relapse and metastases.
Mechanisms of drug resistance and susceptibility
Tumors may become resistant to multiple drugs by activation of functions like MDR or GST, leading to increased detoxification and efflux of chemotherapeutic drugs. We can measure the levels of these multidrug-resistance conferring functions in the tumor cells.
Some drugs need metabolic activation within the tumor cells by enzymatic functions. A reduced expression of these enzymes in the tumor cells may lead to diminished sensitivitiy to those drugs.
It has also been observed that tumor might better respond to certain drugs, if the molecules which are inhibited by these drugs are increased (overexpressed) in the tumor cells. Such molecules are called drug-targets. The main drug-target for anthracycline drugs (e.g. doxorubicine) is topoisomerase II. Tumors with higher level of topoisomerase II generally show better sensitivity to anthracyclines.
Her2 (ERBB2) is the drug-target for Herceptin, an antibody specifically directed against ERBB2. Besides breast cancer, Her2 can be detected in many different types of malignancies. Usually, effective treatment of breast cancer with Herceptin requires overexpression of Her2 in the tumor cells, which can be determined by genetic testing. Discordance of Her2 status between primary tumor and metastases can occur by acquiring Her2 activation during tumor progression. In breast cancer patients, circulating tumor cells have shown to overexpress Her2 despite Her2negative primary tumor. Importantly, after such cases had been treated with Herceptin, clinical responses could have been achieved*4.
Especially in hematological malignancies, genetic malfunction induced by chromosomal rearrangements leads to the uncontrolled production of molecules which are targets for specifically designed anti-cancer agents. Leukemia positive for the bcr-abl chromosomal rearrangement can be efficiently treated with Gleevec. Whereas most cases of chronic myeloid leukemia (CML) are positive for bcr-abl and have a good prognosis, acute myeloid leukemia (AML) positive for bcr-abl is a rare disease, characterized by a poor prognosis, with resistance to induction chemotherapy and frequent relapses in responsive patients. Genetic testing for the presence of bcr-abl can identify those patients which profit from Gleevec treatment.
As ever more specifically designed anticancer agents are developed and clinically used, the determination of the target molecules in the tumor cells gets more and more important to stratify patients for the appropriate use of expensive therapies. Examples of recently applied drugs are Avastin, Sutent or Bortezomib, all known to attack certain molecules frequently altered in many different tumors.
The treatment success of our clinical partners, who guide therapies according to our analytical findings convinces us that our methods are sensible. Evidence from the academic literature is supporting the principles of our strategy *.
Beside the metabolic properties of the tumor cells, there are several other critical factors why the chemosensitivity tests cannot always predict that the tumor will respond as supposed by the results of our analyses:
- The drug administered systemic must reach all the tumor cells
- Patient-to-patient differences may exist how the drug is metabolized and excreted from the body
- Subsets of disseminated tumor cells may be present which behave differently
- Some cells may have yet unknown genetic mechanisms which render them resistant.
- During therapy, tumor cells accumulate further mutations which may result in the selection of resistant clones.
Consequently, partial responses may occur and not necessarily a complete remission despite the results of in vitro testing has not indicated resistance. Since in vitro testing can not be absolutely accurate because of the mentioned reasons, our assay results should not be used as a reason to refuse giving chemotherapy. If a proven effective therapy has not yet been tried, the assay results should not determine that this therapy should not be used. However, the tests can help to choose drugs from the available therapy options that might be more effective than others . If resistance occurs to a drug, the tests can help to identify another agent that might work better.
*supporting evidence from the academic literature:
Adlard JW, et al.: Prediction of the response of colorectal cancer to systemic therapy. Lancet Oncol. 2002 Feb;3(2):75-82. Review.
2 Holland-Frei Cancer Medicine 6th edition (April 2003): by Donald W., Md Kufe, Raphael E., Md Pollock, Ralph R., Md Weichselbaum, Robert C., Jr., Md Bast, Ted S., MD Gansler By BC Decker. Section 12: Chemotherapeutic agents.
3 Schmidt-Kittler O, et al. From latent disseminated cells to overt metastasis: genetic analysis of systemic breast cancer progression. Proc Natl Acad Sci U S A. 2003 Jun 24;100(13):7737-42.
4 Meng S, et al. HER-2 gene amplification can be acquired as breast cancer progresses. Proc Natl Acad Sci U S A. 2004 Jun 22;101(25):9393-8.
What types of tumors can be tested?
Most types of tumors can be tested, including tumors originating from lung, colon, breast, ovary, cervix, prostate, skin, intestine (stomach, gastric), esophagus, liver, kidney. In addition to these most prominent tumor diseases, other less frequent tumors can also be tested. Please inquire for special cases.
Hematological tumors can be assayed for typical rearrangements of hematological tumor cells to permit detection of minimal residual leukemia cells in the bloodstream and to monitor the success of treatment. If the therapy is effective, residual leukemia cells should disappear from the bloodstream.
Alternative Treatment
The concept of integrative medicine claims higher success rates if conventional anti-cancer therapy (e.g. chemotherapy) is combined with alternative treatment. Conventional therapy may be limited by toxicity to the patients or tumor resistance. Many agents from natural sources have shown to have anti-tumor activity. Moreover, some of these agents are able to modulate the cellular enzymatic functions causative for chemotherapy resistance in the tumor cells, which could render the chemoresistant tumor cells again sensitive to chemotherapy.
Drug-transporter molecules (e.g. MDR or MRP) may be induced in tumor cells by chemotherapy. As a consequence, increased efflux of drugs leads to multi-drug resistance of the tumor cells which is a serious problem in cancer treatment. Natural agents like Curcumin or Haelan 951 (an fermented soy-extract) are known modulators of MDR, possibly supporting chemotherapy of tumors showing the MDR-phenotype.
Another mechanism for multidrug resistance is induction of conjugating enzymes leading to increased detoxification of the chemo-drugs. In this context, elevated expression of GST/GCS in tumor cells play an important role. Ellagic acid, an inhibitor of GST may here used to modulate GST-related resistance.
Heat-shock proteins (HSP) may render tumor cells thermoresistant (see below “Hyperthermia”) and resistant to some chemo-drugs. Quercetin is an inhibitor of the heat-shock protein HSP27 and may be used for treatment if tumors show high levels of HSP27.
Testing the use of alternative agents in conjunction with chemotherapy:
For many natural anti-cancer agents, mechanisms of action are known and genetic testing can provide useful advise for appropriate application. Beside the conventional drugs of chemotherapy, alternative agents are also useful in treatment of cancer. Those alternative agents may have anti-cancer activity by directly killing tumor cells, e.g. by induction of apoptosis (programmed cell death). Alternative agents are reported to modulate cellular functions conferring chemo-resistance to the tumor cells. In such cases, supportive therapy with appropriate alternative agents may have synergistic action with conventional chemotherapy.
Our testing determines which alternative agents are likely to be effective or which may have a beneficial use especially in conjunction with chemotherapy.
The testing covers the following alternative agents:
| Agent |
Functional Description |
| Quercetin |
Quercetin is an inhibitor of heat shock proteins (HSP). HSPs can confer thermo- and chemoresistance to tumor cells. A therapy with Quercetin may be considered if high HSP27 levels were measured in the tumor cells. |
| IP6 (Inositol-6P) |
IP6 is an inhibitor of telomerase. Telomerase is often overproduced in tumor cells. A therapy with IP6 may be considered if high levels of telomerase were measured in the tumor cells. |
| C-statin |
C-statin is an angiogenesis-inhibitor. Angiogenesis, i.e. the formation of new blood vessels in the tumor tissue is promoted by the factors bFGF and VEGF. Therapy with C-statin would be rational if tumor has high levels of bFGF or VEGF. |
| Dammarane sapogenins |
Dammarane sapogenins can arrest cancer cell growth and induce programmed cell death (apoptosis). Tumor cell may become resistant to multiple chemotherapy drugs by production of high levels of MDR. Dammarane can efficiently disable MDR which allows higher levels of chemotherapy drugs to accumulate in the cancer cells to enhance efficacy. Especially if high levels of MDR had been measured, a therapy with Dammarane may be considered. |
| Acetogenin Graviola ?GRAVIZON? |
Acetogenin (an ingredient of the formulation Graviola) can inhibit MDR. Tumor cell may become resistant to multiple chemotherapy drugs by production of high levels of MDR. |
| Haelan951 fermented soy-extract |
Haelan 951 can induce apoptosis and stop the growth of tumor cells Ingredients of Haelan 951 bind to estrogen-receptor (ER) beta, conducting growth inhibiting signals in the cell. Measuring the levels of ER-beta allows to estimate the efficacy of a therapy with Haelan 951. Haelan 951 itself can also stimulate the production of ER-beta, raising the levels of ER-beta in tumor cells. Moreover, Haelan 951 is a potent inhibitor of the multidrug resistance proteins MDR and MRP. If in tumor cells high levels of MRP or MDR had been measured, Haelan 951 may be used to disable these functions. |
| Curcumin |
Curcumin can inhibit several functions which may be overproduced in tumor cells, like MDR, Cox2 or NF-kB (p65). If the levels of these factors are elevated in the tumor cells, treatment with Curcumin could be especially helpful. |
| Ellagic Acid |
Another function capable to induce multidrug resistance in cancer cells is GSTpi. If tumors produce high levels of GSTpi, the GST-inhibitor ellagic acid may be administered to counteract GST mediated drug resistance. |
| Arglabin |
Arglabin can inhibit farnesyltransferase (FNTR), an important function for growth promoting signaling in tumor cells. If tumor cells show high levels of FNTR, Arglabin may be of especial use for therapy. |
| Artemsinin and derivatives (Artensunate, Artemeter) ?ARTEMIS? |
Artemisinin and its derivatives can inhibit tumor growth. This anti-tumor effects are, at least in part, exerted by inhibition of angiogenesis. Moreover, Artemisinin derivatives are inhibitors of multidrug resistance inducing functions MRP and GST. Measurement of MRP, GST, and the angiogenesis factor bFGF are therefore rational parameters for deciding a therapy with Artemisinin. |
| Amygdalin B17 (Laetrile) |
Cox2 may be overexpressed in cancer and is therefore a target for anticancer therapy with Cox2-inhibitors like Celebrex. Amygdalin B17 is capable to downregulate the expression of Cox2. Tumors of high Cox2 expression may therefore be candidates to be treated with Amygdalin B17. |
Testing for Hyperthermia
Hyperthermia (heat therapy) has shown to enhance the efficacy of chemotherapy or radiation treatment. Hyperthermia is successfully used in combination of chemotherapy or radiation in the treatment of malignant tumors.
A limiting factor for the efficiency of hyperthermia treatment is the induction of thermoresistance in the tumor cells. Responsible for thermo-resistance is the elevated expression of heat-shock proteins (HSP) in the tumor cells.
By measuring the gene expression of HSP, we can estimate the usability of hyperthermia. Hyperthermia is covered by the testing for chemosensitivity and alternative agents.
Immune function testing by the Cellular NK-Test
Beside chemotherapeutic or hormonal therapies that are directed against the malignant cells, certain alternative treatment options have the aim to stimulate the body’s immune cells and to augment the immune system in fighting the tumor. Numerous immuno-activating agents are available, e.g.:
- Botanicals (e.g. Mistletoe, Biobran, etc.)
- Thymus extracts: (e.g. Thymosin, Thymoject)
- Cytokines (e.g. interleukin 2)
All these agents have a potent propensity to stimulate and activate Natural Killer (NK) cells. NK-cells belong to the lymphocytes and play a very important role in the early defense of the immune system against viruses, bacteria and tumor cells. Activation of the NK-cells is regulated by a subtle balance between positive and negative signals. Surface receptors on the NK-cells transmit either activating or inhibiting signals into the cell.
In tumor patients, NK-cells are often diminished in numbers and restricted in functionality. The extent of this impaired immune function correlates with disease progression and time of survival. Enhancement if the immune function is therefore a important therapeutic goal.
The mere number of NK cells is not sufficient to assess the functionality of this important defense system. An in vitro assay has been established in our lab to measure the capability of a patient’s immune cells to destroy tumor cells. For this, lymphocytes are isolated from a patients blood sample and incubated together with dye-labeled tumor-cells. Lysis of the tumor cells by the immune cells liberates the dye and can be quantified in the culture supernatant.
The amount of liberated dye reflects the actual, spontaneous NK-cell activity (see Figure below). Beside the determination of the spontaneous NK-cell activity, we can determine if the immune cells are susceptible to be stimulated by immuno-acitvation therapy. The percentage increase of tumor cell lysis compared to the spontaneous NK-cell activity is a degree of capability of the NK-cells to be activated. This allows the estimation how promising a immune-stimulative therapy will be. During the course of such a therapy, treatment success could thereby be monitored with the assay.
Principle of the Cellular NK-Test
Tumor cells are stained by uptake of dye
Patients immune cells may attack tumor cells
Agents which can be tested in the Cellular NK-Test:
Basically, every substance claimed to activate the NK-cells could be used in the assay. We routinely use thymus extracts, mistletoe extracts and interleukin 2.
Other agents could be tested upon request. In such instances, please provide a sample of the agent to be tested.
| Agent |
Function |
| Standard agents |
|
| Thymus-extract Thymoject |
NK-activator |
| Mistletoe (e.g. Helixor, Lektinol or Iscador) |
NK-activator |
| IL2 |
NK-activator |
| Other agents |
|
| Biobran |
NK-activator |
| Maitake Grifola frondosa |
NK-activator |
| any other |
claimed NK-activator |
Early detection of cancer
The main reason for the high mortality caused by cancer is too late detection of the disease. Higher cure rates are achieved if the disease was detected in the beginnings of an early stage. To increase the chance of surviving cancer, it is therefore important to detect the very first signs of a neoplasia.
Usually, cancer screening and foresight is done by imaging techniques. Standard imaging techniques may have the drawbacks of low sensitivity or to be uncomfortable (e.g. colonscopy). As a consequence, foresight by imaging techniques may be consulted too infrequently or the disease is perhaps detected in a progressed stage. To reduce overall cancer morality, diagnostic methods are required which are sensitive and well tolerated. Acceptance for foresight examinations may be raised if the test is non-invasive, i.e. it can be performed with body fluids like blood, urine, or stool which are easily accessible.
Molecular techniques allow the detection of genetic alterations typically observed in tumors. Cancer is a multi-factorial genetic disease, i.e., multiple genetic alterations have to occur in combination so that a malignant cancer cell can emerge. The very first genetic alterations during the evolution of a cancer cell can be detected in pre-cancerous lesions as well as in the tumors which arose thereof. The presence of such early signs in a patient must not inevitably lead to cancer, but can be considered as a risk factor. As a consequence of the detection of risk factors, changes in nutritional and environmental behavior may be applicable as well as enhanced vigilance to monitor for further progression.
We provide several assays for the detection of cancer risk factors from different body fluids:
--> From blood
This diagnostic test is done on an individual because of clinical suspicion of disease. Because tumor cells can be disseminated to the blood stream early, a cancer screening test can be done with peripheral blood. Improvements in technology made it possible to detect even a few tumor cells. Moreover, detection of disseminated cells can be used to monitor the efficiency of treatment. If the therapy is effective, minimal residual cancer cells should disappear from the bloodstream. A limitation of this assay is that it cannot be specified from which body site these tumor cells are scattered into the blood stream. Furthermore, the neoplasia must also have already progressed and gained access to the circulation. To overcome this limitations, we established detection assays using organ-confined specimens (urine, sputum) allowing a closer localisation of the source of the neoplastic cells.
*supporting evidence from the academic literature:
Fehm T., et al. Cytogenetic evidence that circulating epithelial cells in patients with carcinoma are malignant.
Clinical Cancer Research 2002 Vol. 8. 2073-2004.
Comment: Numerous studies of circulating epithelial cells have been described in cancer patients. The vast majority of these circulating
epithelial cells in breast, kidney, prostate, and colon cancer patients are aneusomic and are derived from the primary tumor.
--> From urine
Pre-malignant and malignant cells of prostate cancer or bladder cancer are shed in the urine. We have established molecular tests for the detection of genetic alterations typical seen in such tumors. Urinary detection of these alterations can be indicative for these cancers or pre-malignancies.
*supporting evidence from the academic literature:
Hoque MO, et al.: Quantitative methylation-specific polymerase chain reaction gene patterns in urine sediment distinguish prostate cancer patients from control subjects. J Clin Oncol. 2005 Sep 20;23(27):6569-75.
--> From sputum
Smokers are at higher risk for developing lung cancer. However, only about 20 % of smokers are affected. It is therefore important to identify those individuals which already progressed to a higher risk state. Pre-malignant and malignant cells of lung-cancer can be detected in sputum. We have established molecular tests for the detection of genetic alterations typical seen in such tumors. Detection of these alterations can be indicative for such cancers or pre-malignancies.
Diagnostic Assays for Hematological Malignancies
Typical chromosomal rearrangements like translocations are often associated with a certain type of hematological cancer. For example, the well known Philadelphia chromosome, which is the result of a translocation between the chromosomes 9 and 22 occurs in about 90% of CML cases.
Leukemia is categorized in subtypes which may differ regarding prognosis and the appropriate treatment modalities. Many subtypes are associated with distinct chromosomal abnormalities.
The sensitive detection of these rearrangements can be used to monitor minimal residual disease and consequently the success of an ongoing therapy.
We have established assays for the diagnosis of various chromosomal rearrangements occurring in leukemia. By identification of subtype associated translocations, prognosis or an appropriate therapy can be estimated.
Panel of translocation which can be determined at Biofocus:
| Leukemia |
Translocation |
Subtype |
Prognosis of Translocation |
| CML |
t(9;22) |
|
90 % of CML have t(9;22), which is associated with a better prognosis |
|
| t(9;22) |
M1 |
poor prognosis |
|
| t(6;11) |
M4, M5 |
poor prognosis |
| AML |
t(8;21) (q22;q22) |
M2 (50%), seldom M4 |
good prognosis in adults, sensitive to ara-C, in children poor prognosis |
|
| t(9;11) (p22;q23) |
M5a (30%), seldom M2, M4 |
rather good prognosis |
|
| t(15;17) |
M3 (APL) |
very good prognosis, responsive to retinoic acid treatment |
| CLL |
t(11;14) (q13;q32) |
B-CLL |
poor prognosis |
| ALL |
t(1;19) |
L1, L2, prä B (25 %) |
poor prognosis |
| t(9;22) |
L1, L2, 0 |
very poor prognosis |
| NHL |
t(11;14) (q13;q32) |
centrocytic NHL mantle cell lmyph. |
associated with poor prognosis |
| t(14;18) (q32,q21) |
follicular NHL (80%) diffuse large cell NHL (20%) |
good prognosis |
CML= chronic myeloid leukemia. AML = acute myeloid leukemia. CLL = chronic lymphocytic leukemia. ALL = acute lymphocytic leukemia. NHL = non-Hodgkin lymphoma.
Additionally, detection of minimal residual lymphoma can be done by analysis of immune-receptor rearrangements. The detection of individual, prominent VDJ-receptor rearrangements in the blood is conclusive for a clonal proliferation of lymphocytes, especially B-lymphocytes. VDJ-receptor rearrangements are found in 95 % of B-cell ALLs, but also in 14 % of T-cell ALLs.
Clonal expansion of T-cells can be diagnosed by detection of VJ-receptor rearrangements, which are found in 91 % of T-cell ALLs, but also in 55 % of B-cell ALLs.
|
VDJ receptor rearrangement |
ALL (B-cell) |
95 % of B-cell ALLs, 14 % of T-cell ALLs |
|
VJ receptor rearrangement |
ALL (T-cell) |
91 % of T-cell ALLs, 55 % of B-cell ALLs |
Overview of our assays and required specimens
Molecular Detection of Circulating Tumor Cells (CTCs)
By this assay, we only check if CTCs are present in the blood. Further assays like chemosensitivity testing or testing for use of alternative agents could be done afterwards without sending a new specimen.
Specimen required: 20 ml heparinized blood, freshly drawn as described in Appendix 2
Molecular detection of CTCs plus testing for use of chemotherapeutic drugs and alternative agents
This is the most comprehensive testing. It includes molecular detection of CTCs. If CTCs could have been detected, molecular markers for drug resistance and drug targets are analysed by genetic profiling. Markers for chemotherapeutic drugs as well as alternative agents are analysed. The testing allows an estimation, which drugs are more appropriate or less useful for therapy.
Specimen required: 20 ml heparinized blood, freshly drawn as described in Appendix 2
Detection and testing of CTCs for use of alternative agents in conjunction with standard chemotherapy
This assay is a subset of the test described in the former paragraph. CTCs in the blood are analysed for drug targets for alternative agents but not for chemotherapeutic drugs. In particular, with this analysis it is determined which alternative agents are suitable to be used supportive to chemotherapy
Specimen required: 20 ml heparinized blood, freshly drawn as described in Appendix 2
Immune function testing by the Cellular NK-Test
The immune cells isolated from a patients blood sample are tested how efficiently tumor cells are destroyed. Immune-stimulative additives can be used in the assay to check if the efficacy of tumor cell lysis could be enhanced.
Specimen required: 30 ml heparinized blood (50 ml if requested together with one of the other blood tests), freshly drawn as described in Appendix 2
Early detection assays
from sputum for lung cancer risk (e.g. smokers):
20 ml heparinized blood, freshly drawn in glass Vacutainers or comparable glass vessels. Please see Appendix 2 how blood samples should be handled.
Testable Chemotherapeutic Drugs and Their Clinical Application
|
Cyclophosphamide (Cytoxan) |
Advanced breast carcinoma |
| |
Advanced ovarial carcinoma |
| |
Rhabdomyosarcoma, Ewing- |
| |
sarcomas |
| |
Small cell lung carcinoma |
| |
Morbus Hodgkin |
| |
Leukemia (NHL, ALL adults) |
|
Nitrogen- |
Ifosfamide (Ifex) |
Lung carcinoma Ovarial carcinoma |
|
mustrads |
Trofosphamide (Ixoten) |
Testicular tumor |
| |
|
Ewing-sarcoma |
|
Base-alkylation at |
|
Zervix carcinoma |
|
N7-position |
|
Breast carcinoma |
| |
|
Pancreatic carcinoma |
| |
|
Malignant lymphoma |
| |
Melphalan (Alkeran) |
Multiple Myeloma (Plasmocytoma) |
| |
|
Ovarial carcinoma |
| |
Chlorambucil (Leukeran) |
Leukemia (CML, NHL, MH) |
| |
|
Advanced ovarial carcinoma |
| |
|
Breast carcinoma |
| |
Carmustine BCNU |
Primary brain tumors |
| |
|
Multiple myeloma |
| |
|
Leukemia (malignant Lymphoma, |
| |
|
Morb. Hodgkin) |
| |
|
Lymphosarcoma |
| |
|
Advanced gastrointestinal carcinoma |
|
Nitrosoureas |
Lomustine CCNU (CeeBU) |
Morbus Hodgkin Tumors o.t. central nervous system |
| |
|
Metast. malignant melanoma |
|
Base-alkylation at O6-position |
Nimustine ACNU |
Lung carcinoma Malignant glioma |
| |
|
Small cell lung carcinoma (brain |
| |
|
metas.) |
| |
|
Colorectal carcinoma |
| |
|
Advanced stomach carcinoma |
| |
|
Leukemia (CML, NHL, MH) |
|
Hydrazines |
Dacarbazine (DTIC-Dome) |
Metast. malignant melanoma |
|
Base-alkylation at |
|
|
|
O6-position |
|
|
| |
Thiotepa (Thioplex) |
localized apl.: |
| |
|
Bladder-papilloma and carcinoma |
| |
|
systemic apl.: |
|
Aziridines |
|
Breast carcinoma Ovarial carcinoma |
| |
|
Chron. Leukemia |
| |
|
Morbus Hodgkin |
|
Mitomycin C (Mutamycin, Mitozytex) |
Bladder tumor |
| |
Stomach carcinoma |
| |
Lung carcinoma |
| |
Pancreatic carcinoma |
| |
Colorectal carcinoma |
| |
Breast carcinoma |
| |
Liver carcinoma |
| |
Zervix carcinoma |
| |
Esophagal carcinoma |
| |
Head-neck carcinoma |
| |
CML |
| |
Osteosarcoma |
|
Alkyl |
Busulfan |
|
|
Sulfonates |
Treosulfan |
|
|
Cisplatin (Platinol) |
Lung carcinoma (NSCLC, SCLC) Bladder carcinoma Endometrial carcinoma Testicular tumor Head-neck carcinoma Melanoma Ovarial carcinoma Prostate carcinoma Sarcoma Cervical carcinoma |
|
Carboplatin (Paraplatin) |
Small cell lung carcinoma Head-neck carcinoma Zervical carcinoma Epithelial ovarial carcinoma |
|
Oxaliplatin (Eloxatin) |
Metast. colorectal carcinoma |
|
Methotrexate MTX |
Chorion epithelioma |
| |
Breast carcinoma |
| |
Head-neck carcinoma |
| |
Leukemia (NHL, ALL) |
| |
Osteosarcoma |
| |
Small cell lung carcinoma |
|
Pemetrexed |
NSCLC |
| |
Mesothelioma |
|
5 Fluoruracil (Adrucil, Ribofluor, |
Colorectal carcinoma |
|
Verrumal) |
Stomach carcinoma |
| |
Pancreatic carcinoma |
| |
Metast. breast carcinoma |
|
Capecitabine (Xeloda) |
Colorectal carcinoma |
| |
Metast. breast carcinoma |
|
Gemcitabine (Gemzar) |
Bladder carcinoma |
| |
Non-small cell lung carcinoma |
| |
Pancreatic adeno carcinoma |
|
Cytarabine araC (Cytosar-U) |
Leukemia (AML, ALL, CML) |
Anthrazyclines (intercalating)
Anthrazyclines (intercalating)
Podophyllotoxine derivates
(non-intercalating) Doxorubicin (adriamycin, Rubex, Doxil)
Epirubicin (Ellence)
Daunoruicin (Cerubidine) Mitoxantrone (Novantrone)
Etoposide VP16 (Vepesid)
Small cell lung carcinoma Endometrial carcinoma Ewing-Sarcom Bladder carcinoma Hodgkin-lymphoma Stomach carcinoma Breast carcinoma Neuroblastoma, Osteosarcoma Ovarial carcinoma Breast carcinoma Ovarial carcinoma Stomach carcinoma Lung carcinoma Soft tissue sarcoma ALL AML Breast carcinoma Leukemia (malignant Lymphome) Primary liver carcinoma Ovarial carcinoma Small cell Lung carcinoma Non-small cell lung carcinoma Leukemia (NHL, MH, AML) Testicular tumor Chorion carcinoma Ovarial carcinomae
Taxanes
Vinca alkaloides
Paclitaxel (Taxol)
Docetaxel (Taxotere) Vincristine (Oncovin)
Vinblastine (Velban)
Vinorelbine (Navelbine) Estramustine
Ovarial carcinoma Breast carcinoma Non-small cell lung carcinoma Breast carcinoma Non-small cell lung carcinoma Leukemia (akute Leukemia, ALL, MH) Small cell lung carcinoma Breast carcinoma Wilms-tumor Rhabdomyosarcoma Ewing-sarcoma Neuroblastoma Leukemia (NHL, MH) Testicular tumor Kaposi-sarcoma Breast carcinoma Non-small cell lung carcinoma Breast carcinoma Advanced prostate carcinoma
|
Special inhibitors |
|
DrugTarget |
|
Antibodies |
Cetuximab (Erbitux) Erlotinib (Tarceva) |
Colorectal carcinoma |
EGF-R |
|
Bevacizumab (Avastin) |
Colorectal carcinoma |
VEGF |
|
Trastuzumab (Herceptin) |
Breast carcinoma |
ERB-B2 |
|
Tyrosinkinase inhibitors |
Imatinib (Gleevec) |
Leukemia (CML) gastrointest. Tumors (GIST) |
bcr-abl c-Kit PDGFR a / b |
|
Gefitinib (Iressa) |
Non-small cell lung carcinoma |
EGF-R |
|
Farnesyltransferase-inhibitors |
Arglabin |
Colorectal carcinoma Pankreatic carcinoma |
FNTB |
|
Lonafarnib Tipifarnib |
Non small cell lung carcinoma (trials) |
|
Aromataseinhibitors |
Exemestane Formestane Anastrozole Letrozole |
Breast carcinoma |
Aromatase |
|
FGF-inhibitors |
Suramin |
|
bFGF |
|
COX-inhib. |
Celecoxib |
Colorectal carcinoma |
cox2 |
|
Proteasome inhibitors |
Bortezomib (Velcade) |
Multiple myeloma |
p65 NF-kB |
|
Anti-Adrogens |
flutamide, nilutamide, bicalutamide, cyproterone acetate |
Prostate CA |
AR |
|
Anti-Adrogens |
flutamide, nilutamide, bicalutamide, cyproterone acetate |
Prostate CA |
AR |
|
Bcl2-Inhibitors |
Oblimersen |
|
Bcl2 |
Appendix 1 : Molecular tumor markers and therapy-related gene
|
AFP |
Levels of alpha-Feto-Protein (AFP) are increased in Hepatoma and Teratoma (liver, pancreas, prostate). AFP has structural and functional similarities to albumin and is normally decreased in adults. Gibbs et al. Structure, polymorphism and novel repeated DNA elements revealed by a complete sequence of the human alpha-fetoprotein gene. Biochemistry 26: 1332 ?1343, 1987 |
|
Albumin |
Specific product of hepatocytes. Detection of gene expression is specific for the diagnosis of hepatocellular carcinoma. |
|
APC |
The adenomatosis poliposis coli (APC) gene is a tumor suppressor. Germ line mutations in APC cause an inherited form of colorectal cancer (adenomatosis poliposis coli). Defects in the APC gene (mutations, LOH, methylation) are also frequently found in spontaneous colorectal carcinomas and other tumor types. Polakis P.: The adenomatous polyposis coli (APC) tumor suppressor. Biochimica Biophysica Acta 1332, F127-48, (1997) |
|
Aromatase |
Aromatase is catalyzing the formation of C18 estrogens from C19 androgens. Inhibitory anti-cancer drugs of aromatase prevent the production of estrogens and consequently the growth of estrogen dependent tumors. Expression of aromatase in tumors is may be considered as a prerequisite for a rational therapy with aromatase inhibitors. Johnston SR, Dowsett M Aromatase inhibitors for breast cancer: lessons from the laboratory. Nat Rev Cancer. 2003 Nov;3(11):821-31. |
|
BAX |
BAX is a pro-apoptotic mitochondrial membrane protein. It inhibits Bcl2 and accelerates the programmed cell death (apoptosis). Reduced expression of BAX in relation to Bcl2 correlates with non-response to 5-fluorouracil, epirubicin and cyclophosphamide. Le Blanc H. et al. Tumor-cell resistance to death-receptor induced apoptosis through mutational inactivation of the proapoptotic Bcl 2 homolog BAX. Nat Med, 2002. 8(3):p. 274-81 Krajewski S. et al. Prognostic significance of apoptosis regulators in breast cancer.Endocr Relat Cancer, 1999. 6(1):p.29-40 |
|
Bcl 2 |
Bcl2 is coding for an anti-apoptotic mitochondrial membrane protein. Bcl 2 is overexpressed in many tumors and consequently causes resistance to apoptosis-inducing drugs (e.g. intercalating agents, alkylating agents, platinum compounds). Ikeguchi M.S. et al. Quantitative analysis of expression levels of BAX, Bcl 2 and survivin in cancer cells during cisplatin treatment. Oncol Rep, 2002. 9(5):p. 1121-6 |
|
CEA |
Carcinoembryonic antigen (CEA) is found in gastrointestinal and colorectal tumors. Measuremnt of expression in blood is used for diagnosis of circulating cancer cells, since expression of CEA is usually absent in blood cells. Tremblay F.: Breast cancer masquerading as a primary gastric carcinoma. J Gastrointest Surg 2002 Jul ? Aug; 6(4):614-6 |
|
c-KIT |
c-kit (= CD117) is the receptor of the stem cell growth factor. The receptor type is a tyrosine kinase. In some special small-cell lung cancers and gastrointestinal tumors, c-kit is overexpressed. Overexpression is indicative for considering therapy with the tyrosine kinase-inhibitor Gleevec (STI571). Potti A et al. CD117 (c-KIT) overexpression in patients with extensive-stage small-cell lung carcinoma. Ann Oncol. 2003 Jun;14(6):894-7. Allander SV et al.: Gastrointestinal stromal tumors with KIT mutations exhibit a remarkably homogeneous gene expression profile. Cancer Res. 2001 Dec 15;61(24):8624-8. |
|
Cytokeratin CK19 CK20 CK7 |
Cytokeratins (CK) are expressed in epithelial cells and usually not in mononuclear blood cells. Therefore, they are suitable for the detection of circulating tumor cells of epithelial origin. Which cytokeratins are used as detection markers depend on the tumor type: CK19: tumors of breast, lung and prostate CK20: gastrointestinal tumors CK7: tumors of ovaries, uterus, breast and stomach Burchill et al.: Detection of epithelial cancer cells in peripheral blood by reverse transcriptase PCR. British Journal of Cancer 71:278-281, 1995 |
|
c-myc |
The gene for the transcription factor c-myc is amplified (DNA) or overexpressed (RNA) in advanced, aggressive tumors. Zajac-Kaye M.: Myc oncogene: a key component in cell cycle regulation and its implication for lung cancer. Lung Cancer 2001 Dec;34 Suppl 2:S43-6 |
|
Cox 2 |
Cyclooxigenase 2 (Cox2) is overexpressed in colorectal adenomas and tumors. These tumors can be treated with specific Cox-2 inhibitors, since high expression levels confer susceptibility to these drugs. Adlard et al.: Prediction of the response of colorectal cancer to systemic therapy. Lancet Oncology 3, 75-82 (2002) |
|
DCC |
DCC (Deleted in Colorectal Carcinoma) is a tumor suppressor, which is frequently altered by deletion or LOH in colorectal but also other tumors. |
Updated (07/2007)
|
