Circulating Tumor Cell Testing & Analysis

Circulation

In order to help guide more individualized cancer care support we offer circulating tumor cell blood testing which can help determine the presence of cancer, response to therapy and risk of cancer return. Additionally, oncogenetic analysis of circulating cancer cells taken from two sample tubes of blood can help guide individualized medical oncology treatments as well as determine potential benefits of supportive nutrient therapies. The following sample tests are provided here for your review as well as for your oncologist.

Circulating Tumor Cell Testing

“We have been using Dr. Michael Giesing’s TIRNA (Tumor Induced RNA) blood test since 2005 . In this test, now done by RGCC labs in Greece, circulating tumor cells or micrometastatic cells are isolated from the patient’s blood, genetically fingerprinted and then pharmaco genetically tested to determine effectiveness of commonly used chemotherapies, targeted therapies and off label use of other pharmaceuticals. Additionally, over 40 nutrient based and herbal therapies are tested. Each effective therapy identifies targeted oncogenes and method of action. With this basic information patients and their doctors can more objectively personalize the treatment while reducing the risk of stimulating even more aggressive and resistant tumor expression.”

Onco trace

Dear Colleague,

We send you the results from the analysis on a patient Ms/Mr ________________ suffering from _______ carcinoma stage ____. The sample of 25ml of whole blood that contained EDTA-Ca as anti-coagulant, and packed with an ice pack. In our laboratory we made the following:

• We isolated the malignant cells using Oncoquick with a membrane that isolates malignant cells from normal cells after centrifugation and positive and negative selection using multiple cell markers.

The results during the isolation procedure are presented below :

Table of Markers

CD45 Positive Cells
(Hematologic origin cells)

Index of marker: CD45: Hematologic origin cell marker, BCR-ABL, CD30, CD15: hematologic malignancy marker, CD133, Sox-2, OKT-4, Nanog, CD44: tumor stem cell marker, CD19: lung cancer cell marker (NSCLC), CD31: endothelial cell membrane antigen , CD34: hematological stem cell and blast cell marker, epithelioid sarcoma marker, CD63: melanoma cell marker, CD99: sarcoma marker, EpCam: epithelial origin marker, MUC-1: lung cancer cell marker (SCLC), PSMA: prostate spesific cancer stem cell membrane antigen , VHL mut: renal carcinoma marker , c-MET: membrane antigen that regulates the mesenchymal to epithelial transition, panCK: epithelial origin cell marker.

Conclusion: We notice that after isolation procedure there are remaining malignant cells. The concentration of these cells was ___cells/ml, SD +/- 0.3cells.

Sincerely,
Ioannis Papasotiriou M.D., PhD
Head of molecular medicine dpt of
R.G.C.C.-RESEARCH GENETIC CANCER CENTRE LTD

Index of circulating cells number: (upper limit : progress of disease, lower than limit: beginning of disease or stable of disease when the patient is on treatment plan)

Breast cancer: 5cell/7.5ml , Prostate cancer 20cells/ml , Sarcoma: 15cells/6.5ml, Colon cancer: 5cells/ml, Lung cancer (Lc=0, r=0.99): 10cell/ml.

*This test will NOT DETECT cancers of the brain or other cancers that have been “encapsulated” by the body, not releasing circulating tumor or stem cells (CTC, CSC) into the blood stream or if any of these cells are dormant. We still recommend the use of biopsy, blood markers and/or various scans with this test when cancer is suspected or known to exist. No test is 100% accurate.

Ms/Mr _________________
ADDRESS : Florina-GR P.O. 53070
TEL : +30-24630-42264 , FAX: +30-24630-42265
Web site : www.rgcc-genlab.com E-mail :Papasotiriou.ioannis@rgcc-genlab.com

Chemo Agents

Dear colleague,

We send you the results from the analysis on a patient Ms/Mr ___________ suffering from ______ carcinoma stage ____. The sample that was sent to us for analysis was a sample of 25ml of whole blood that contained EDTA-Ca as anti-coagulant, and packed with an ice pack.

In our laboratory we made the following:

• We isolated the malignant cells using Oncoquick with a membrane that isolates malignant cells from normal cells after centrifugation and positive selection using anti-EpCam and negative selection using anti-CD45 particles (isolated 5.1cells/ml, SD +/- 0.3cells).
• Then we developed cell cultures in a fetal calf serum media and at the same time we developed colony cultures in soft agar. In each culture of the well plate we added a chemotherapeutic substance that is used in clinical application. Then we developed those cultures and we harvested a sample every 24 hours for 6 days and made the following assays.
• There was made an isolation of the genomic DNA using the kit Invisorb of INVITEK.
• We isolated mRNA using the mRNA Magprep blood isolation kit of NOVAGEN.
• We traced the mRNA and the genes of MDR1 (multi drug resistant 1), MRP and LRP using the technique of Northern Blot (resistance in drugs used in chemotherapies).
• We tracked the mRNA and the gene of topoisomerase I and II a & b using the technique of Northern Blot (sensitivity in cytostatic inhibitors of topoisomerase).
• We tracked the quantity of the mRNA of the tubulin using the RT-PCR(sensitivity in cytostatics of the kind of taxanes and the products of the alkaloids of Vinca).
• We defined the activity of the enzyme complex of the glutathione-S-transferases (GST kit of NOVAGEN) (resistance in drugs used in chemotherapies-especially in platinum compounds).
• We defined the DNA methyl transferase which is a target of the alkylating factors (products of platinum, cyclophosphamide and the products of it).
• We defined the mRNA of the Thymidylatesynthetase (TS) and the DHFR(sensitivity in 5-FU, capecitabine and methotrexate).
• We defined the mRNA ofthe reductase of 5-CMP (sensitivity in gemcitabine).
  We defined the receptors of the MMP and the receptors of laminin (invasive ability of the tumor).
• We defined the expression of protein p27 that is responsible for cell arrest in G0 stage.
  We defined the VEGF (neoangiogenetic factor) and the induction of the apoptotic pathway using ONCOGENE kit from NOVAGEN.
• We defined the ability of acting of the nucleus protein kinases which are a target of the Carbazinecompounds.
• We defined the overexpression of TGFa and TGFb factors as targets for Suraminsulfate.
• We defined the overexpression of somatostatin receptor (SS-R) , of COX-2 and 5-LOX , of c-erb-B2 (Her/Neu2) , c-erb-B1, and androgen, estrogen and progesterone receptors.

The above conclusions were confirmed by the cell cultures of the tumor (or circulating tumor cells and the results are displayed in the bar graph on the next pages.

INTERPRETATION: The numbers above the bars indicate % of cancer cell DEATH caused by the drug tested. This equates the % SENSITIVITY to that drug. Therefore, the drugs with the highest numbers are the most effective drugs at inducing cancer cell death for the patient tested. The numbers below or beside the bars refer to the drugs tested, asindicated in the diagrams in pages 2 to 7.

Growth factors proliferation stimuli

Self Repair – Resistance

Angiogenesis

Cell Cycle Regulation & Immortalization/Apoptosis

Drug Metabolisms & Targets

Markers

From the investigation above we concluded to the following:

1. From the whole neoplasmic population we have an expression of MDR1 in a percentage of 40% over control sample (positive in the check of resistance).
2. The activity of GST is stable in the low limits ( no resistance to platinum compounds ).
3. The activity of GammaGC is in normal range (no resistance to platinum compounds).
4. The activity of CES1 and CES2 is in normal range (no resistance to camptothecin compounds).
5. The concentration of p180 is in high range.
6. Increased activity of the Laminin and the MMP (increased invasive ability).
7. There is great sensitivity in taxanes ( Docetaxel ).
8. There is partial sensitivity in alkaloids of vinca.
9. There is partial sensitivity in Eribulin.
10. Partial sensitivity noticed in MTX, in Gemcitabine, in Capecitabine, in Fudr, in UFT, in Raltitrexed, in Pemetrexed, no sensitivity noticed in Cytarabine, in Fludarabine but there is great sensitivity in (5FU).
11. There is partial sensitivity in Epothilones.
12. Decreased sensitivity in alkylating factors.
13. There is great overexpression of EGF (55% over control), TGF-b (20% over control), there is normal expression of NFkB, but there is suppression of expression of IkB(a,b,c) (15% below control).
14. It appears to have great sensitivity in the inhibitors of topoisomerase II ( Doxorubicin ).
15. There is partial sensitivity in the inhibitors of Topoisomerase I.
16. There is great over-expression of C-erb-B1 (25% over control), Estrogen-Receptor (15% over control) but there is normal expression of 5-LOX, SS-r, COX2, C-erb-B2 and Progesterone-Receptor.
17. We notice great neoangiogenetic ability (overexpression of VEGF-R 55% over control sample).
18. Finally, there is no sensitivity in Dacarbazine.
19.  We notice that taurolidine cannot induce the apoptosis to the malignant cells (in IV route dosage).
20. We notice that taurolidine can induce the apoptosis to the malignant cells (in intraperitoneal route dosage).
21. We notice no down-regulation of HSP27 ( Heat Shock Protein ), HSP72 ( Heat Shock Protein ) and HSP90 ( Heat Shock Protein ).
22. There is over-expression of ANG 1 at 25% over control, ANG 2 at 10% over control, IGF-r 1 at 15% over control, IGF-r 2 at 15% over control, but we notice no down-regulation of ALK, EML-4-ALK, C-MET, NPM-ALK, CD 117 (c-kit), HDAC, HAT, NR3C4-A and NR3C4-B.

Conclusion:

• The specific tumor appears to have resisting populations because of the MDR1 overexpression that can be reversed by the use of verapamil combined with imidazole compounds (ketoconazole).
• The neoplasmatic cells have the greatest sensitivity in the inhibitors of Topoisomerase II ( Doxorubicin), in the nucleous spindle stabilizer ( Docetaxel) and in the antagonist ( 5FU).
• Also can be used Erlotinib as inhibitor of EGFr, Bevacizumab as inhibitor of neo-angiogenesis and Tamoxifen as inhibitor of estrogen positive feedback.

Sincerely,

Ioannis Papasotiriou MD., PhD
Head of molecular medicine dpt. of
R.G.C.C.-RESEARCH GENETIC CANCER CENTRE LTD

INDEX: M0 : Abnormal p16 , normal p53 and hTERT ,
M1: Normal hTERT , abnormal p53 , p16 ,
M2 crisis : over-expression of hTERT , p53 , p16
Sample viability :<35% no sensitivity , 35%-80% partial sensitivity , >80% great sensitivity

*Be advised that any nutritional program suggested is not intended as a treatment for any disease. The intent of any nutritional recommendation is to support the physiological and biochemical processes of the human body, and not to diagnose, treat, cure, prevent any disease or condition. Always work with a qualified healthcare provider before making changes to your diet, prescription medication, lifestyle or exercise activities.

Natural Substances

Dear Colleague,

We send you the results from the analysis on a patient Ms/Mr __________ suffering from __________ carcinoma stage ____. The sample that was sent to us for analysis was a sample of 25ml of whole blood that contained EDTA-Ca as anti-coagulant, and packed with an ice pack.

In our laboratory we made the following:

• We isolated the malignant cells using Oncoquick with a membrane that isolates malignant cells from normal cells. Then we centrifuged at 350g for 10 min and we collected the supernatant with the malignant cells. Then we proceed to isolation of malignant cells from mononuclear cells by negative selection.
• Then we developed fifty cell cultures in a fetal calf serum media. In each culture of the well plate we added a biological modifier substance [ Quercetin, Acemannan, Super Artemisinin, Oncoplex ES, Poly-MVA, C-statin, Ascorbic Acid, Pancreas Pork, Ellagic Acid, Superoxide Dismutase, Unique E, Maitake, Ukrain, Bio-Ae-Mulsion Forte, Bio-D-Mulsion NuMedica Micellized D3, Ezzeac Plus Cat’s Claw, Mangosteen, Se-100 Bio-Tech, Polysaccharide Ganoderma, Curcumin, Vitanox, Mistletoe, AHCC Active Hexose Correlated Compound, Amygdalin- (B17), Thymex, Burdock Complex, Salvestrol, Virxcan, Immune Plus (fermented soy extract), Agaricus Phalloides, DCA (dichloroacetate), genistein, Avemar Pulvis, PME, new PME, Sodium Bicarbonate, OPC, Intenzyme Forte, Cruciferous, CV247, Larrea (chaparral), Lycopene, Green Tea extract, Paw-Paw, Indol-3-Carbinol, Melatonin, Naltrexone, Resveratrol, Mesenchymal Factor, oleander extract] that is used in clinical application. Then we developed those cultures and we harvested a sample every 24 hours and made the following assays.
• In the culture that contains all the substances we measure the apoptotic ability using the oncogen apoptosis kit.
• In the culture that contains the ukrain we measure the inhibition of tyrosine kinase catalytic ability from the growth factor receptors (EGF-r, IGF-r) and the production of cytokines PMBC.
• In the culture that contains quercetin we measure the inhibition of EGF and IGF.
  In the culture that contains indol-3-carbinol we measure the inhibition of VEGF and FGF and PDGF.
• In the culture that contains the mistletoe we measure the inhibition of tyrosine kinase catalytic ability from the growth factor receptors (EGF-r, IGF-r) and the production of cytokines and the increase of PMBC.
• In the culture that contains the ascorbic acid we measure the catalytic activity of GSH and GSSG (redox reaction) and the induction of cytochrome C (apoptosis).
• In the culture that contains the PolyMVA we measure the catalytic activity of GSH and GSSG (redox reaction) and the induction of cytochrome C (apoptosis).
• In the culture that contains the super artemisinin we measure the catalytic activity of GSH and GSSG (redox reaction for free radical since super artemisinin binds free radicals with the iron molecule), the inhibition of VEGF, FGF and PDGF (since it acts to the angiogenesis cascade reactions) and the induction of cytochrome C (apoptosis).

CONCLUSION: It seems that this specific population of malignant cell have greater sensitivity in Super Artemisinin , in Ascorbic acid , in CV247 , in Bio D Mulsion NuMedica Micellized D3 , in DCA(dichloroacetate) , in Genistein , in New PME , in PME , in Quercetin , in Salvestrol , in Curcumin (turmeric) and less in Larrea (chaparral) , in Cruciferous complete , in Oncoplex ES , in Intenzyme Forte , in OPC , in Thymex , in Sodium bicarbonate , in Avemar pulvis , in Superoxide dismutase , in Polysaccharide Ganoderma , in Ellagic acid , in Green tea extract , in Lycopene , in Maitake , in Amygdalin-(B17) , in Poly-MVA , in Ukrain , in C-statin , in Burdock complex , in Vitanox , in Bio-Ae-Mulsion Forte , in Ezzeac Plus Cat Claw , in Mangosteen Rx , in SE 100 Bio Tech , in Virxcan , in
Unique E , in Immune Plus (fermented soy extract) , in Agaricus phalloides , in Oleander Extract , in Pancreas Pork , in AHCC-Active Hexose Correlated Compound , in Acemannan , in Mistletoe , in Melatonin , in Naltrexone , in Resveratrol , in Mesenchymal Factor , in Indol 3 Carbinol , in Paw-Paw.

Sincerely,

Ioannis Papasotiriou MD., PhD
Head of molecular medicine dpt. of
R.G.C.C.-RESEARCH GENETIC CANCER CENTRE LTD

*Be advised that any nutritional program suggested is not intended as a treatment for any disease. The intent of any nutritional
recommendation is to support the physiological and biochemical processes of the human body, and not to diagnose, treat, cure, prevent any disease or condition. Always work with a qualified health care provider before making changes to your diet prescription medication, and lifestyle or exercise activities.

Mr/Ms ______________
ADDRESS :Florina-GR P.O. 53070
TEL : +30-24630-42264 , FAX: +30-24630-42265 Website : www.rgcc-genlab.com E-mail :papasotiriou.ioannis@rgcc-genlab.com