http://www.youtube.com/v/22t6GcZJ3ew
In the video, Molecular Oncologist Professor Dr. Michael Giesing, the innovator of pharmacogenomic chemosensitivity testing, elaborates on a few important and often overlooked aspects of cancer. Firstly, he mentions that the general outcome of a cancer patient is not related to the original clinical tumor, but related to the tiny amounts of cells that are disseminated throughout their blood and bone marrow. These cells are occult micrometastases, better known today as Circulating Tumor Cells, or "CTC's". It is these cells that eventually cause relapses by spreading ti distant organs. Secondly, he points out that chemotherapies work in only about 20% of cases, and in the other 80% that don't work, 60% actually make the cancer more aggressive by provoking the cancer to develop resistance factors. These same resistance factors, assist in the cancer's ability to spread. Finally, he explains that research has shown that the genetic make up of these micrometastases or CTCs, is very different from the genetic makeup of the original tumor. Due to this genetic variation, pharmacogenomic chemosensitivity testing should be performed on the circulating tumor cells in order to pinpoint which drugs would work, since it is these cells that have the biggest impact on the patient's prognosis.
the first test "DETECTS tumor cells" $1000
the second test runs the sample against agents and gives a report $4000
Dr. Joachim Fluhrer, Acupuncture, Homeopathy
... now in the lucrative cancer business.
http://doctorfluhrer.com.au/doctorfluhrer.com.au_website/About_Me.html
Domain Name: doctorfluhrer.com.au
Registrant: NATURAL MEDICAL CENTRE PTY. LTD.
Registrant Contact Name: Julia Fluhrer
http://doctorfluhrer.com.au/doctorfluhrer.com.au_website/Welcome.html
Julia Fluhrer
jfluhrer@ozemail.com.au
$4000 test results
SAMPLE ANALYSIS
Chemotherapies Tested
Dear Dr,
we send you the results from the analysis made about patient (patient's name) suffering from prostate carcinoma stage IV . The sample that was sent to us for analysis was a sample of 45ml of whole blood that contained EDTA-Ca as anti-coagulant , all packed with water ice .
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 epithelial cell marker and negative selection using anti-CD45 particles .
· 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 gloutathion-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 alkyliating factors (products of platinum , cyclophosphamide and the products of it )
· We defined the mRNA of the thymidylate synthetase ( TS ) and the DHFR . (sensitivity in 5-FU, capecitabine and methotrexate )
· We defined the mRNA of the reductase of 5-CMP (sensitivity in gemcitabin)
· 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 nucleous protein kinases which are a target of the carbazin compounds .
· We defined the overexpression of TGFa and TGFb factors as targets for suramin sulfate.
· We defined the overexression 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 also confirmed by the cell cultures of the tumor and in the diagrams there is a development curve for each category of cytostatics.
| GENE NAME | GENE RELATED FUNCTION | RESULTS |
| CES1 &2 (carboxyesterase) | Resist to camptothecin | Normal |
| E2F1 | Transcr. Fact of TS & topoI | Normal |
| p180 | Tyrosin kinase growth f. | 40% over control |
| p27 | Cell arrest (G0) | 50% over control |
| DPD | Resist to 5FU | Normal |
| UP | Resist to 5FU | Normal |
| NP | Resist to pyrim. antagonist | Normal |
| TP | Resist to 5FU | Normal |
| Gamma GC | Resist to alkyliating drug | Normal |
| p53 | Cell cycle regulator | 30% over control |
| p16 | Apoptosis | 70% over control |
| VEGF | Angiogenesis | 65% over control |
| FGF | Angiogenesis | 50% over control |
| PDGF | Angiogenesis | 30% over control |
| COX2 | Tumour Growth | Normal |
| 5-LOX | Tumour Growth | Normal |
| MMP | Metastases | 50% over control |
| TS | Rapid cell cycle (THFA) | Normal |
| DHFR | Rapid cell cycle (THFA) | Normal |
| SHMT | Rapid cell cycle (THFA) | Normal |
| GARFT | Rapid cell cycle(THFA) | Normal |
| NFκB | Transcription fact | 45% over control |
| IκB (a,d,e) | Inhibitor of NFκB | 15% below control |
| Ribonucleoside reductase | DNA synthesis | Normal |
| DNA methyltransferase I | DNA methylation | Normal |
| DNA demethylase | DNA methylation | Normal |
| O6-methylguanin-DNA-tran. | DNA methylation | Normal |
| TGF-b | Tumour Growth | 75% over control |
| EGF | Tumour Growth | 40% over control |
| IGF | Tumour Growth | Normal |
| Bcl-2 | Anti-apoptotic gene | 55% over control |
| CypB1 | Xenobiotic metabolism | Normal |
| DNA Histone deacylase | DNA un-coiling | Normal |
| c-erb-B2 | Her/neu2 | 30% over control |
| c-erb-B1 | Her1 | Normal |
| Bcr-abl | Resist phenotype | Normal |
| h-TERT (Human telomerase) | M2 crisis-aggresive phen. | Normal |
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 stable in the low limits (no resistance to platinum compounds )
4. The activity of CES1 and CES2 is 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 (especially in docetaxel) and partial sensitivity noticed in alkaloids of Vinca.
8. Minimal sensitivity noticed in 5FC, in 5-FU, in UFT , in FUdR in capecitabine, in raltitrexed, in methotrexate, in pemetrexed and in gemcitabine.
9. Increased sensitivity in alkyliating factors (especially in cisplatin).
10. There is great overexpression of TGF b (75% over control), of NFkBeta (45% over control) and EGF-r (40%,<70%) growth factors and supression of expression of isoforms of IκB (a, d, e) (15% below control) .
11. It appears to have great sensitivity in the inhibitors of topoisomerase II a and II b (especially in mitoxantrone and etoposide).
12. There is no sensitivity in the inhibitors of topoisomerase I .
13. There is no overexpression of SS-r receptor , , of estrogen receptor mRNA , of 5-LOX mRNA , of COX-2 , of c-erb-B1 mRNA, but there is great overexpression in dihydrotestosterone receptor mRNA (15% over control) and of c-erb-B2 (30% over control).
14. We notice great neoangiogenetic ability (overexpression of VEGF-R – 65% over control sample).
15. Finally , there is no sensitivity in dacarbazine .
16. We notice that taurolidin cannot induce the apoptosis to the malignant cells (in IV route dosage).
17. We notice that taurolidin cannot induce the apoptosis to the malignant cells (in intraperitoneal route dosage).
18. We notice no down-regulation of HSP 27 (Heat schock proteins) 27 , HSP 90 and HSP 72 .
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 alkyliating agent cisplatin , in the inhibitor of topoisomerse II mitoxantron and etoposide and in the nucleus spindle stablelizer docetaxel .
-
Also you can use: suramine sulfate as inhibitor of TGF-b and PDGF, bortezomib as inhibitor of proteasome over-activity and indirectly the transcriptional activity of NFκB, goserelin for inhibition of androgen positive feedback and bevacizumab as inhibitor of angiogenesis.
INDEX: M0 : Abnormal p16 , normal p53 and hTERT ,
M1: Normal hTERT , abnormal p53 , p16 ,
M2 crisis : over-expression of hTERT , p53 , p16
6th Sample viability : <20% greater sensitivity , 65%-20% partial sensitivty , >65% no sensitivity
Alternative Holistic Substances & Biological Response Modifiers Tested
Dear Dr.,
we send you the results from the analysis made about a patient (patient's name) suffering from prostate carcinoma stage IV. The sample that was sent to us for analysis was a sample of 45ml of whole blood that contained EDTA-Ca as anti-coagulant , packed with water ice .
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 thirty eight cell cultures in a fetal calf serum media . In each culture of the well plate we added a biological modifier substance (H2O2, ascorbic acid , carnivora , misteltoe, quercetin , indol-3-carbinol , c-statin , ukrain , poly-MVA, co enzyme Q10, essiac tea, modified citrus pectin, IP6 , pancreatic enzymes, salvestrol, Uncaria Tomentosa, carctol, noni juice, annonaceous acetogenins, caesium chloride, reolysin, amygdalin-B17-, artesunate, maitake, lycopene, curcumin, green tee extract, melatonin, ellagic acid, L-methionine, N-acetyl-cystein, Niacin (Vit.B3), L-carnithine, Vitamin E (tocopherol), superoxide dismutase (SOD) , selenium, aloe vera, IFNa2) 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 it contains all substance we measure the apoptotic ability using the oncogen apoptosis kit
· In the culture that it contains the carnivora we measure the inhibition of tyrosin kinase catalytic ability from growth factors receptor (EGF-r, IGF-r,) and the production of cytokines.
· In the culture that it contains the Ukrain we measure the inhibition of tyrosin kinase catalytic ability from growth factors receptor (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 it contains the misteltoe we measure the inhibition of tyrosin kinase catalytic ability from growth factors receptor (EGF-r, IGF-r,) and the production of cytokines and the increase of PMBC
· In the culture that it contains the H2O2 we measure viability of the culture in 4 days of treatment.
· In the culture that it 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 it 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 it contains the artesunate we measure the catalytic activity of GSH and GSSG (redox reaction for free radical since artesunate bind free radicals with iron molecule ) , the inhibition of VEGF , FGF and PDGF (since it act to the angiogenesis cascade reactions) and the induction of cytochrome C (apoptosis).
RESULTS:
1. We notice that in culture that contains the ascorbic acid we have increase of the cascade of caspase (especially 3 and 9) and cytochrom-c by 30% .
2. We notice that in culture that contains the PolyMVA we have no increase of the cascade of caspase (especially 3 and 9) and cytochrom-c.
3. We notice that in culture that contains carnivora we have inhibition of EGF-r by less than 5% and for IGF-r by 5% and we notice no increase of cytokine production 5% .
4. We notice that in the culture that contains quercetin we have inhibition of EGF by % 35 and IGF by 30%
5. We notice that in the culture that contains indol-3-carbinol we have inhibition of VEGF by less than 5% , of FGF by 5% , and PDGF by 5%
6. We notice that in culture that contains misteltoe we have inhibition of EGF-r by less than 5% and for IGF-r by 5% and we notice no increase of cytokine production, and there is no increase of PMBC .
7. We notice that in culture that contains the c-statin we have increase of the cascade of caspase (especially 3 and 9) and cytochrom-c by 40%.
8. We notice that in culture that contains Ukrain we have inhibition of EGF-r by less than 5% and for IGF-r by 5% and we notice no increase of cytokine production, and there is no increase of PMBC.
9. We notice that in culture that contains the H2O2 we have increase of the cascade of caspase (especially 3 and 9) and cytochrom-c by less than 5% and the viability of the culture remain stable .
10. We notice that in culture that contains the co enzyme Q10 we have increase of the cascade of caspase (especially 3 and 9) and cytochrom-c by less than 5% and the viability of the culture remain stable .
11. We notice that in culture that contains the essiac tea we have increase of the cascade of caspase (especially 3 and 9) and cytochrom-c by less than 5% and the viability of the culture remain stable .
12. We notice that in culture that contains the modified citrus pectin we have increase of the cascade of caspase (especially 3 and 9) and cytochrom-c by less than 5% and the viability of the culture remain stable .
13. We notice that in culture that contains the IP6 we have increase of the cascade of caspase (especially 3 and 9) and cytochrom-c by less than 5% and the viability of the culture remain stable .
14. We notice that in culture that contains the pacreatic enzymes we have increase of the cascade of caspase (especially 3 and 9) and cytochrom-c by less than 5% and the viability of the culture remain stable .
15. We notice that in culture that contains the salvestrol we have increase of the cascade of caspase (especially 3 and 9) and cytochrom-c by 55% and the viability of the culture reduced by 30%.
16. We notice that in culture that contains the uncaria tomentosa we have increase of the cascade of caspase (especially 3 and 9) and cytochrom-c by less than 5% and the viability of the culture remain stable.
17. We notice that in culture that contains the caesium chloride we have no increase of the cascade of caspase (especially 3 and 9) and cytochrom-c and the viability of the culture remain stable.
18. We notice that in culture that contains the carctol we have increase of the cascade of caspase (especially 3 and 9) and cytochrom-c by less than 5% and the viability of the culture remain stable.
19. We notice that in culture that contains the noni juice we have increase of the cascade of caspase (especially 3 and 9) and cytoc
20. We notice that in culture that contains the annonaceous acetogenins we have increase of the cascade of caspase (especially 3 and 9) and cytochrom-c by less than 5% and the viability of the culture remain stable.
21. We notice that in culture that contains the reolysin we have increase of the cascade of caspase (especially 3 and 9) and cytochrom-c by less than 5% and the viability of the culture remain stable.
22. We notice that in culture that contains the amygdalin-B17- we have no increase of the cascade of caspase (especially 3 and 9) and cytochrom-c and the viability of the culture remain stable.
23. We notice that in culture that contains maitake we have inhibition of EGF-r by less than 5% and for IGF-r by 5% and we notice no increase of cytokine production , and there is no increase of PMBC.
24. We notice that in culture that contains the curcumin (turmeric) we have increase of the cascade of caspase (especially 3 and 9) and cytochrom-c by 65% and the viability of the culture reduced by 50%.
25. We notice that in culture that contains the lykopene we have no increase of the cascade of caspase (especially 3 and 9) and cytochrom-c and the viability of the culture remain stable.
26. We notice that in culture that contains the green tea extract we have no increase of the cascade of caspase (especially 3 and 9) and cytochrom-c and the viability of the culture remain stable
27. We notice that in culture that contains artesunate , there is inhibition of redox reaction and increase of intracellular free radicals , there is increase of cytochrome c (apoptosis) by 40% and the inhibition rate of VEGF is 45%, of FGF is 35% and of PDGF is 30%.
28. We notice that in culture that contains the melatonin we have no increase of the cascade of caspase (especially 3 and 9) and cytochrom-c and the viability of the culture remain stable.
29. We notice that in culture that contains the ellagic acid we have no increase of the cascade of caspase (especially 3 and 9) and cytochrom-c and the viability of the culture remain stable.
30. We notice that in culture that contains the L-methionine we have no increase of the cascade of caspase (especially 3 and 9) and cytochrom-c and the viability of the culture remain stable.
31. We notice that in culture that contains the N-acetyl-cystein we have no increase of the cascade of caspase (especially 3 and 9) and cytochrom-c and the viability of the culture remain stable.
32. We notice that in culture that contains the niacin we have no increase of the cascade of caspase (especially 3 and 9) and cytochrom-c and the viability of the culture remain stable.
33. We notice that in culture that contains the L-carnithine we have no increase of the cascade of caspase (especially 3 and 9) and cytochrom-c and the viability of the culture remain stable.
34. We notice that in culture that contains the vitamin E we have no increase of the cascade of caspase (especially 3 and 9) and cytochrom-c and the viability of the culture remain stable.
35. We notice that in culture that contains the superoxide dismutase we have no increase of the cascade of caspase (especially 3 and 9) and cytochrom-c and the viability of the culture remain stable.
36. We notice that in culture that contains the aloe vera extract we have no increase of the cascade of caspase (especially 3 and 9) and cytochrom-c and the viability of the culture remain stable.
37. We notice that in culture that contains selenium we have inhibition of EGF-r by less than 5% and for IGF-r by 5% and we notice no increase of cytokine production , and there is no increase of PBMC and NK .
38. We notice that in culture that contains IFNa2 we have inhibition of EGF-r by less than 5% and for IGF-r by 5% and we notice no increase of cytokine production , and there is no increase of PMBC .
CONCLUSION: It seems that this specific population of malignant cell have greater sensitivity in quercetin, in c-statin, in artesunate, in curcumin (turmeric), in salvestrols, and in ascorbic acid and less in hydrogen peroxide (H2O2), co enzyme Q10, essiac tea, modified citrus pectin, IP6 , N-acetyl-cystein, pancreatic enzymes, caesium chloride, in carnivora, ellagic acid, in indol-3-carbinol, in L-carnitine, in L-methionine, amygdalin-(B17), vitamin E (tocopherol), maitake, in Poly-MVA, in ukrain, in IFNa2, in superoxide dismutase, uncaria tomentosa (samento), in melatonin, in selenium, carctol, noni juice, niacin, aloe vera extract, in misteltoe, annonaceous acetogenins (paw paw), reolysin (reovirus), lycopene and in green tea extract .
Thalidomide Analysis
Dear Dr.
we send you the results from the analysis made about a patient (patient's name) suffering from breast carcinoma stage IV. The sample that was sent to us for analysis was a sample of 25ml of whole blood that contained EDTA-Ca as anti-coagulant, all packed with water ice . In our laboratory we made the following:
The results are presented below:
Malignant cell cultures compared analysis
Conclusion: We notice that the thalidomide can inhibit the neovascularization and it can induce the apoptosis to the cancer cell becoming from the patient above, but it cannot inhibit the invasion activity of the cancer cells.
New York residents may reach us locally at:
Tel - 347-878-7422
Fax – 212-504-2682
Email – info@oncogenetic.com
SOURCEwww.oncogenetic.com/sample-test-results
the following is SOURCEwww.rgcc-genlab.com/CST-1.pdf
A brief information letter from Dr.Papasotiriou Ioannis
Dear colleague ,
I am glad to inform you about our assays that we follow on order to perform
the chemosensitivity testing in cancer types using micro-array techniques. Until
now the assays that have been in use are the clonogenic assays , the ATP-TCA,
the MTT assay and the SRB assay. All those assays use tissue sample for
beginning the whole process- or they can use (on MTT) the tumor effusion
material. Also, they are based only in the quantification analysis of living cells
using indirect parameters. For example, the SRB assay uses sulforhodamine B which
can bond with the total proteins of the cells and by colorimetric technique we
can find the number of living cells on a cancer cell culture (where in the growth
medium we put a chemotherapeutic drug) . As for MTT , is based on the
tetrazolium assay that depends on the NADP and the NADPH quantity . Finally,
as for the ATP-TCA assay, it measures the production of ATP by fluorometric
and photometric technique that measure the catalysis of luciferin by the
enzyme luciferase , and this reaction needs mitochondrial ATP to be done.
Unfortunately, these assays have major disadvantages. Therefore, they do not
have extended clinical use. Specifically, the MTT assay is very much effected
from the intracellular concentration of glucose and from the pH . Additionally,
this measurement is time depended and the time point must be precise . As for
the SRB , this assay is much more easier than MTT and it is not time
depended , but it is based on the fact that sulforhodamine B binds with the
intracellular proteins to the amino-end point edge and that is an indirect factor
of the cells viability. Also SRB, as all other chemosensitivity assays cannot give
you information quickly and easy about the development of resistant
mechanisms or about the metastatic ability of the tumor through neoangiogenesis.
Also all those assays cannot predict the sensitivity or the
resistance in a short time (for example the clonogenic assay needs 20 days). All
these assays give only few information about the malignant phenotype of the
cancer and they cannot give solutions about avoiding or reversing the
resistance mechanisms . Additionally, all these assays (SRB , MTT ,Disc etc)
cannot predict toxicities from chemotherapeutic drugs , such as 5-FU . Finally all
those assays cannot give any information about drugs for example of the category
of biological modifiers .
All the above disadvantages can be overcomed by the use of microarray
chemosensitivity test (CST) . But before that, we must mention that we can
complete our analysis not only using tissue sample from tumor or tumor
effusion material , but also from peripheral (whole) blood –in cases on advanced
stage IV. From the whole blood we isolate the circulating cancer cells using
gradient solutions and membranes with pores. After that we proceed to negative
selection using cytospin with anti CD+45 (those antibodies bond to haematologic
cells and they separate them from epithelial cells-malignant) and after that we
proceed to positive selection using EpCam antibody that bond and isolate
trough cytospin the epithelial origin cell (cancer cell) . Then we identify that we
have actually isolated the malignant cells using the immortalization detection kit
from Oncogen. After that we develop many cell cultures in a 96 well plate and
in each slot we add in the culture medium a different chemotherapeutic drug .
Then every 24 hours we take from each culture a small sample and we isolate
the mRNA. Then with the RT-PCR we produce the cDNA. Then we follow the
microarray hybridization and the colorimetric microarray analysis (pict.1) using
probes for the genes bellow :
TS ,DHFR , Tubulin a and b , transcription factor about tubulin a , Topoisomerase I
and II (a &b) , SHMT , DPD , TP, p27 , p53 , DNA methyltransferase , O6
acyltransferase , DNA deaminase , MRP (I & II) , LRP , GST , VEGF , PDGF , EGF ,
TGFb, IGF , MMP9 , nucleotide reductase, COX-2 , 5-LOX , SS-r, c-erb-B2 .
To be more precise the thymidylate synthase and DHFR are genes that
produce enzymes very crucial for the folate pathway. As you already know
this pathway is the only biochemical way for the cells to produce thymidine
nucleotide which is necessary for the DNA duplication and cell division. When
cancer cells are resisting to antifolates such as methotrexate or 5 fluoruracil
,they over-express those genes so the amount of enzymes becomes so big that
the inhibitor ahs to be in very high concentration and the toxicities are very
severe. Also the mRNA of those genes bounds with the enzymes and it cannot
allow the inhibitor to bind with them. Additionally to that we must mention
that especially for 5 FU we test the expression of DPD and TP that catalyze
the transformation of 5-FU to 5-FUMP, the active product . If the activity of
these genes is not regular the 5FU cannot transform properly and so, the
toxicities are very severe also.
As for the DNA crosslinking rearranging enzymes topoisomerase I and II with
all their isophorms, they produce enzymes which become tartgets for the
anthracyclines, camptothecins , epipodophylins and adriamycin compounds. These
enzymes are crucial on S phase of the cell cycle (in DNA doublication) and on
DNA repair mechanisms. Unfortunately. the resistance mechanisms for the
above inhibitors depend only on the over-expression of the p glycoprotein
pumps MDR1, MRP and LRP in lung's tissue.
The genes that produce tubulin a and b in all their isophormes are crucial for
the formation of the nucleus spindle. The alkaloids of Vinka bond with tubulin
B ,they inhibit the formation of the spindle and they spot the cell cycle in
metaphasis stage. Also the taxanes bond with the dimmers of tubulin a and b
and make the nucleus spindle so stable that it is impossible to depolymerize .
With this mechanism taxanes stop the cell cycle in telophasis stage.
Unfortunately, both Vinka alkaloids and taxanes become targets for membrane
pumps MDR1, MRP1 and LRP or the mutations especially on tubulin b genes,
make the molecule unable to bond with the inhibitors.
Additionally, DNA methyltransferase , O6 acyltransferase , and demethylase of
DNA are genes that are involved in the DNA methylation and they regulate
the coiling and uncoiling of genes. With these mechanisms the above genes are
regulating other genes' expression to cells. Especially in cancer cells the DNA
hypermethylation is one of the mechanisms of resistance and regulation of
cancer phenotype and behavior. Also those genes are involved in alkyliating
drugs inhibition mechanism.
Finally, the VEGF, FGF and PDGF genes are involved to the angiogenesis
procedure. As you already know angiogenesis is necessary for the metastatic
procedure . Additionally to that the genes of TGF , of EGF , of IGF and
somatostatin (somatomedins) are overexpressed in cancer cells and it triggers
again and again the proper membrane receptors -that are also over-expressedand
they create a positive feed back mechanism of cell growth and mitosis
without ending –the start of immortalization. Also the ecosanoids are involved as
growth factors or transcriptional factors in carcinogenesis and in cancer
phenotype of the cells. Particularly the overexpression of cycloxygenase 2 and 5
lipoxygenase are crucial for the above procedure. Studies have already shown
that inhibition of these enzymes (by coxibs or analogues of triens) leads to
inhibition of cancer cell growth.
The pharmaceutical substances that are used and are inserted in the culture medium
are:
Oxaliplatin, cisplatin, cyclophosphamide, ifoshpamide, trophosphamide, treosulphan,
melphalan, CCNU, BCNU, ACNU, mitomycin, procarbazine, dacarbazine,
bleomycin, temozolomide, doxorubicin, epirubicin, daunorubicin, dactinomycin,
irinotecan, topotecan, idarubicin, etoposide, mitoxandrone, paclitaxel, docetaxel,
vincristin, vinblastin, vinorelbin , taurolidin, 5fluoruracil, uracil-tegafur, raltitrexed,
floxuridine, capecitabine.
We also check the bio-availability of the cells using a colouring substance –Trypan
Blue 0.4% - taking micro-photos. The Trypan Blue is the most reliable method to
measure the viability of a cell culture . As the specialists know, Trypan Blue is
a unique analogue of aminopterin and it can participate to the folate cycle –a
chemical pathway that is crucial and overactive in cancer cells. But this
aminopterin cannot pass the cell membrane therefore it paints it in blue color
but without painting the inner cellular area. In Debris cells (dead cells) it can pass
through the broken membrane and it can paint the total cell body in deep
blue(pict.2). From the 6 samples that we take every in 6 days we develop a
viability diagram for each chemotherapeutic drug (pict.3)
This procedure is made once every 24 hours for 6 days.
The micro-array data as well as the micro-photos of each case are sent to our labs and
our computers where they are analyzed and evaluated.
With all the above-mentioned data for each case we have a safe profile –genetic &
cytological- of the neoplasmic cells of each case.
In this way we can conclude to which pharmaceutical substances the cells have
sensitivity and to which they are resistant, quickly and safely.
The truth of the previous sentence is justified by the clinical data that we
collect from our clients clinical doctors and we have organized those information
in DATA TABLES OF CANCER PATIENTS' RESPONSE TO SUGGESTED
SCHEMES THROUGH CHEMO-SENSITIVITY – CHEMO-RESISTANCE
TESTING (see the related analysis with the same title as the capital letters)
TABLES
Table of response in all cases of cancer
Response Number Percent
Complete Regression 31 / 254 12,2 %
Partial Regression 103 / 254 40,6%
Minor Response 33 / 254 12,9 %
Stable Disease 34 / 254 13,4 %
Progression 26 / 254 10,2 %
Treatment not started 27 / 254 10,7 %
Table : Response in colorectal cancer patients
Response Percent
Complete Regression 8,7 %
Partial Regression 21,2 %
Minor Response 12,3 %
Stable Disease 15,3 %
Progression 30,3 %
Treatment not started 12,2 %
Table : Response in bronchial carcinoma patients
Response Percent
Complete Regression 10,3 %
Partial Regression 65,8 %
Minor Response 6,2 %
Stable Disease 5,3 %
Progression 3,1%
Treatment not started 9,3 %
Table : Response in pancreatic carcinoma patients
Response Percent
Complete Regression 17,3 %
Partial Regression 31,4 %
Minor Response 11,2 %
Stable Disease 17,9 %
Progression 8,7 %
Treatment not started 13,5 %
Table : Response in ovarian carcinoma patients
Response Percent
Complete Regression 10,9 %
Partial Regression 41,3 %
Minor Response 10,7 %
Stable Disease 17,8 %
Progression 5,8 %
Treatment not started 13,5 %
Table : Response in breast cancer patients
Response Percent
Complete Regression 13,8 %
Partial Regression 48,2 %
Minor Response 13,6 %
Stable Disease 10,4 %
Progression 9,2
Treatment not started 4,8 %
LITERATURE
1. Comparison of the sulforhodamine B protein (SRB) and tetrazolium
(MTT) assays for in vitro chemosensitivity testing, Keepers YP,Pizao
PE,Peters GJ,van Ark-Otte J, Winograd B, Pine HM,Eur J Cancer
1991;27(12):1717
2. Profiling Novel Sulfonamide antitumor agents with cell-based phenotypic
screens and array based gene expression analysis, Akira Y,Kuromitsu
J,Kawai T,Nagasu T, Hata Sugi N. Yoshimatsu K, Yoshino H , Owa T,
Mol cancer therapeutics, vol1,275-286, Febr 2002
3. Prediction of sensitivity of esophageal tumors to adjuvant chemotherapy
by cDNA microarray analysis of gene expression profiles, Kihara C,
Tsonuda T, Tanaka T, Yamana R, Hirata K, Takagi T, Nakamo Y, Cancer
Res.2001 Sep 1;61(17):6474-9
4. Tetrazolium based asaays for cellular viability: a critical examination of
selected parameters affecting formazan production, DT Vistica, P Skehan,
D Scudiero , A Monks , A Pittman , BR Boyd, Cancer Res Vol51, issue
10;2515-2520
5. Identification of molecular targets associated with selenium-induced
growth inhibition in human breast cells using cDNA microarrays, Y
Dong,H E Ganther, C Stewart, C Ip, Cancer Res, Feb 1,2002;62(3):708-
714
6. Comparison of the sulforhodamine B assay and the clonogenic assay for
the in vitro chemoradiation studies , Cancer chemotherapy
pharmacology.2003 Mar.;51(3):221-6
7. Furukawa, T., Kubota, T., Tanino, H., Oura, S., Yuasa, S., Murate, H.,
Morita, K., Kozakai, K., Yano, T., and Hoffman, R.M. Chemosensitivity
of breast cancer lymph node metastasis compared to the primary tumor
from individual patients tested in the histoculture drug response assay.
Anticancer Research 20, 3657-3658, 2000.
8. Hoffman, R.M. The clinical benefit of the histoculture drug response
assay. Jpn. J. Cancer Chemother 27, Supplement 11, 321-322, 2000.
9. Hoffman, R.M. Taking chemotherapy from random to rational with
the histoculture drug response assay. Jpn. J. Cancer Chemother. 24, 206-
229, 1997
10. Furukawa, T., Kubota, T., Hoffman, R.M. Clinical applications of the
histoculture drug response assay. Clinical Cancer Research 1, 305-311,
1995.
11. Robbins, T., Connors, K.M., Storniolo, A.M., Hanchett, C., and Hoffman,
R.M. Sponge-gel supported histoculture drug-response assay for head and
neck cancer: Correlations with clinical response to cisplatin. Arch. Otol.
Head & Neck Surg. 120, 288-292, 1994. Bosanquet AG. Correlations
between therapeutic response of leukaemias and an in-vitro drugsensitivity
assay. Lancet 1991; 337: 711-714.
12. Hinkley HJ, Bosanquet AG. The in vitro radiosensitivity of lymphocytes
from chronic lymphocytic leukaemia using the differential staining
cytotoxicity (DiSC) assay.I--Investigation of the method. International
Journal of Radiation Biology 1992; 61: 103-110.
13. Hinkley HJ, Bosanquet AG. The in vitro radiosensitivity of lymphocytes
from chronic lymphocytic leukaemia using the differential staining
cytotoxicity (DiSC) assay. II--Results on 40 patients. International Journal
of Radiation Biology 1992; 61: 111-121.
14. Bosanquet AG. The DiSC assay 10 years and 2000 tests further on. In:
Kaspers GJL, Pieters R, Twentyman PR, Weisenthal LM, Veerman AJP
(Eds) "Drug resistance in leukemia and lymphoma. The clinical value of
laboratory studies". London: Harwood 1993; 373-384.
15. Bosanquet AG, Bell PB. Handling requirements to achieve active drugs in
in_vitro drug sensitivity and resistance assays. In: Kaspers GJL, Pieters R,
Twentyman PR,Weisenthal LM, Veerman AJP (Eds) "Drug resistance in
leukemia and lymphoma. The clinical value of laboratory studies."
London: Harwood 1993; 227-255.
16. Bosanquet AG. In vitro drug sensitivity testing for the individual patient -
an ideal adjunct to current methods of treatment choice. Clinical Oncology
1993; 5: 195-197. (Editorial)
17. Fruehauf JP, Bosanquet AG. In vitro determination of drug response: A
discussion of clinical applications. In: Cancer, Principals and Practice of
Oncology, PPO updates.Eds. DeVita VT, Hellman S, Rosenberg SA.
Philadelphia: Lippincott 1993; 7(12): 1-16. (Review)
18. Bosanquet AG. Short-term in vitro drug sensitivity tests for cancer
chemotherapy. A summary of correlations of test result with both patient
response and survival. Forum Trends Experimental and Clinical Medicine
1994; 4: 179-195. (Review)
19. Bosanquet AG, McCann SR, Crotty GM, Mills MJ, Catovsky D.
Methylprednisolone in advanced chronic lymphocytic leukaemia: rationale
for, and effectiveness of treatment suggested by DiSC assay. Acta
Haematologica 1995; 93: 73-79.
20. Bosanquet AG, Bell PB. Enhanced ex vivo drug sensitivity testing of
chronic lymphocytic leukaemia using refined DiSC assay methodology.
Leukemia Research 1996; 20: 143-153.
21. Bosanquet AG, Bell PB. Novel ex vivo analysis of nonclassical,
pleiotropic drug resistance and collateral sensitivity induced by therapy
provides a rationale for treatment strategies in CLL. Blood 1996; 87: 1962-
1971.
22. Bosanquet AG. Ex vivo Phase II trials and cross resistance with lithium
gamma-linolenic acid. Annals of Oncology 1996; 7 (Suppl 1): 33.
(Abstract)
23. Bosanquet AG, Bell PB, Burlton AR, Amos AS. Correlation of bcl-2 with
P-glycoprotein expression in chronic lymphocytic leukaemia and other
haematological neoplasms but of neither marker with ex_vivo
chemosensitivity or patient survival. Leukemia and Lymphoma 1996; 24:
141-147.
24. Bosanquet AG, Burlton AR, Bell PB, Harris AL. Ex vivo cytotoxic drug
evaluation by DiSC assay to expedite identification of clinical targets:
results with 8-chloro-cAMP. British Journal of Cancer 1997; 76: 511-518.
25. Bosanquet AG. Fludarabine: phosphate or no phosphate, that is the
confusion. Clinical Cancer Research 1999; 5: 475-476 (letter).
26. Bell PB, Rooney N, Bosanquet AG. CD79a detected by ZL7.4 separating
chronic lymphocytic leukemia from mantle cell lymphoma in leukemic
phase. Communications in Clinical Cytometry 1999; 38: 102-105
27. Mason JM, Drummond MF, Bosanquet AG, Sheldon TA. The DiSC assay:
a cost-effective guide to treatment for chronic lymphocytic leukaemia?
International Journal of Technology and Assessment in Health Care 1999;
15: 173-184.
28. Bosanquet AG, Johnson SA, Richards SM. Prognosis for fludarabine
therapy of chronic lymphocytic leukaemia based on ex vivo drug response
by DiSC assay. British Journal of Haematology. 1999; 106: 71-77.
29. Bosanquet AG, Copplestone JA, Johnson SAN, Smith AG, Povey SJ,
Gillingham R, Oscier DG. Response to cladribine in previously treated
patients with chronic lymphocytic leukaemia identified by ex vivo
assessment of drug sensitivity by DiSC assay. British Journal of
Haematology 1999; 106: 474-476.
30. Bosanquet AG, Bell PB, Rooney N. Effect of interleukin-2 on CD95
(Fas/APO-1) expression in fresh chronic lymphocytic leukaemia and
mantle cell lymphoma cells: relationship to ex vivo chemoresponse.
Anticancer Res 1999; 19: 5329-5334.
31. Bosanquet AG, Bosanquet MI. Ex vivo assessment of drug response by
differential staining cytotoxicity (DiSC) assay suggests a biological basis
for equality of chemotherapy irrespective of age for patients with chronic
lymphocytic leukaemia. Leukemia 2000; 14: 712-715.
32. Bosanquet AG, Burlton AR, Bell PB. Parameters affecting the ex vivo
cytotoxic drug sensitivity of normal human cells. J Exp Ther Oncol 2002;
2: 53-63.
CIRCULATING TUMOUR CELLS (CTCs)
A SUMMARY
Authors: Fluhrer, JG MBBS,FACNEM; Fluhrer, JE BSc (cand ) Biochemistry and Molecular Biology
Email: joachim@doctorfluhrer.com.au.
Circulating Tumour Cells are emerging as a vital tool in the diagnosis and monitoring of malignant disease [1-3].
"It is undeniable that CTCs have enormous research potential for individualised medicine in the future." [3]
The purpose of this paper is to summarise the literature currently available about Circulating Tumour Cells (CTCs). Topics covered are as follows: the definition of CTCs and their unequivocal role in malignant disease and how the analysis of CTCs may assist in individualising the diagnosis, aid in therapeutic choices and enable monitoring of the progression of malignant disease.
Research Method: The phrase 'Circulating Tumour Cells' produces well over a thousand search results in the databases of PubMed, Cinahl, Proquest, and Google Scholar. Based on the topics mentioned above, 350 research articles published predominantly in the last seven years were extrapolated from the search. Of these 150 are review articles. 65 of the review articles on Circulating Tumour Cells (CTCs) have been published in the last two years (2008-2010). Thirty of these articles have been cited in a comprehensive document that is currently being reviewed and edited. The other half of the 2008-2010 reviews are yet to be included in the article. This paper is a summary of the document, and is intended for medical practitioners interested in gaining an understanding of Circulating Tumour Cells. The estimated time of final copy is July 2010.
CTCs and their role in malignant disease
Circulating Tumour Cells (CTCs) sparked scientific interest over fifty years ago and their detection and analysis is proving to be an invaluable tool in the individualisation of cancer diagnosis and treatment [3].
It is very well established that Circulating Tumour Cells are absolutely essential for the establishment of metastases: they function as the single haematological route of malignant neoplasias and metastases cannot occur without them [1]. In fact, 'metastatic insufficiency' is officially defined as the elimination of CTCs [4]. Regardless of their critical role in the metastatic cascade and despite the need for their detection and analysis as a widespread tool used in cancer management [5-7], a definition of CTCs has yet to enter a medical dictionary.
CTCs are a subpopulation of tumour cells derived from the primary cancer site that have:
•detached from the primary tumour mass [8]
•adopted genetic mutations that enabled migration through the basement membrane (if the tumour is of epithelial origin) and the extracellular matrix [4, 9],
•de-differentiated or undergone Epithelial-Mesenchymal Transition (carcinoma derived cells only) [1, 4],
•entered into the peripheral blood stream where they circulate as tumour cells with metastatic potential – this is the point at which they are termed 'Circulating Tumour Cells.' [1, 10],
•have the potential to disseminate and proliferate as a metastatic lesion[1, 4],
•Can stimulate angiogenesis . [1, 10],
•have stem-cell like properties (see below) [1, 2, 11].
Survival of CTCs in the circulation requires evasion of anoikis and of the immune system. There are complex mechanisms present in CTCs that allow for this prevarication to occur [9, 12, 13]. When the intercellular signaling is appropriate, CTCs extravase from the circulation, disseminate in a tissue foreign to that of the primary lesion, and proliferate in the 'permissive' organ [1, 3, 5, 10, 14]. This proliferating mass forms a secondary cancer at a site foreign to that of the primary cancer [1].
Stephen Paget's well recognised 'seed and soil' hypothesis states that metastases exhibit tropism, i.e. the organ site wherein they disseminate and form a secondary tumour is not random [5, 14]. The organ site of a metastasis is the 'soil' which is absolutely biologically the ideal place for a specific 'seed' (CTC) to grow [1]. Both the CTC (seed) and the organ site (soil), which will harbour the metastases, have biomarkers that specifically recognise and interact with each other [1] . Together they facilitate the development of the environment necessary for a metastatic lesion to develop and thrive [1, 3, 5, 10, 14]. CTCs have adopted genetic mutations that equip them to respond to local growth factors and stimulate neovascularisation in the microenvironment of new site. [5, 14] [4]. These biological markers on CTCs may differ entirely from the markers of the bulk of primary cancer cells [1, 11].
Heterogenicity of Tumour Cells: Understanding the heterogeneous nature of tumour cells is necessary in order to fully appreciate the critical role CTCs play in the formation of metastases [15]. A vast number of the CTC characteristics are yet to be determined, however, it is known that CTCs are likely to have heterogeneous biomarkers to that of the parent tissue and other subpopulations of the primary tumour [1, 16]. Common CTC properties that identify them as heterogeneous to other primary cancer cells are their increased invasiveness, their heightened resistance to threat, and their biological likeness to stem or progenitor cells [1, 4, 6]. .
The heterogeneous nature of tumour has the following consequences:
•Classification and morphological analysis of tumour cells from a surgical biopsy may differ to the character of the tumour's CTCs [1, 11].
•The majority of the cells of the biopsy will not have initiating capacity and therefore may be less relevant in terms of diagnosis and treatment [1].
•CTCs have the potential to behave totally differently to the original primary cancer cells and respond to entirely different treatments [1, 11]
CTCs and Tumour Initiating Cells (TICs)
( *N.B There is confusing terminology existing in the literature about tumour stem cells. Tumour cells that have progenitor/stem cell characteristics and are responsible for tumour progression are called Tumour Initiating Cells (TICs) [15]. They are known colloquially as 'Cancer Stem Cells' (CSCs) [17])
CTCs share similar genotypic and phenotypic characteristics with Tumour Initiating Cells (TICs) [1, 6]. CTCs have the capacity to self-renew, to divide asymmetrically, for genetic adaptation, to accumulate mutations [4]. They have the ability to sustain tumour genesis and growth, and to initiate tumours with multiple descendent lines. [2, 11, 18]. CTCs may circulate as non-proliferating tumour cells, potentiating their resistance to chemotherapy [19, 20]. They can transition from this non-proliferating pluripotent-progenitor cell phenotype into a proliferating cell upon dissemination [21].
The similarities that CTCs have to cancer stem cells may explain the eventual relapse of disease in a patient previously considered to be in remission following primary therapy [6, 15, 22].
The sub-population of neoplastic cells that have stem cell properties are known to:
•be responsible for tumour progression [15]
•have unique biomarkers that may correspond to radio- and chemotherapy resistant mechanisms [23]
•derived from and regulated by both genetic and epigenetic programs [24]
If therapy is to be targeted toward cells responsible for tumour progression, these epigenetic determinants of mutations need to be considered [24].
Cancer treatments may be unsuccessful if they fail to target the specific minority subpopulation of tumour cells that have capacity for invasion and tumour initiation [15]. These populations are an absolutely essential target for therapy and if metastatic disease is to be prevented. [15, 25]
What can CTCs tell us about the patient's malignancy?
"CTCs have a wealth of clinical information in the evaluation of tumour progression, prediction of long term prognosis, identification of patients who are likely to respond to treatment of curative intent, and assessment of likelihood of recurrence" [4]
The identification and analysis of CTCs is emerging as an essential clinical tool in the diagnosis of malignancy, and in the monitoring of disease progression and effect of cancer treatment [1, 3, 26, 27].
CTC detection and analysis is a valuable tool in the management of cancer because it enables the following information to be realised:
1.Evaluation of tumour progression in real-time.
Analysis of CTCs enriched from the peripheral blood of patients with advanced or metastasising cancer represents the real-time biopsy that has been up until this point impossible without surgical intervention [18][11]. Detection of CTCs in the peripheral circulation of cancer patients indicates the presence of metastatic disease [1, 4, 11]. Due to the ease of sample collection, it is possible to monitor tumour progression and stage, and assist in determining the success of cancer treatment [5].
CTC count in the peripheral blood of a patient is indicative of tumour stage, tumour progression, and success of treatment [1]. The difference in CTC count between two samples, taken prior to and following surgery or cancer therapy, can inform the practitioner of the success of the treatment [5]. CTC count falls significantly with the regressing of disease, and similarly CTC count rises with the advancement of the malignancy [4, 5].
The CTC count is indicative of tumour stage [3]. The numerical value which determines how advanced the cancer is will differ across the various types of malignant neoplasias, and their comparative averages have already been determined [4]. For example, more than 5 CTCs per 7.5ml of peripheral blood is considered to be a progressive disease.
2.Prediction of long-term prognosis
The presence of CTCs in the peripheral circulation has been confirmed as an independent prognostic indicator [1, 5]. CTC detection is predictive of clinical outcome and overall survival rate in multiple malignancies. [1, 5] The prognostic significance of CTCs relates to time to disease progression and to the prediction of recurrence, even after therapy of curative intent [1, 4, 14].
CTC detection in the blood may override the standard prognostic indicators [2, 4]. Specifically, detection and analysis of CTCs may be a more accurate predictor of clinical outcome in terms of Overall Survival than standard prognostic indicators [2]. Multivariate analysis has shown that CTC count is an independent prognostic indicator irrespective of other variables [1, 5, 6].
The presence of CTCs at time of diagnosis is an indicator of whether adjuvant chemotherapy is needed in early stage cancer patients [3]. Due to the similarity between Cancer Stem Cells and CTCs, (i.e their characteristics of longevity, ability of tumour initiation, self renewing and proliferative capacity), the presence of CTCs at the time of diagnosis and treatment, may explain the eventual relapse of disease in patients who have previously been 'in remission' after primary therapy [6].
3.Identification of patients who are likely to respond to treatment of curative intent
It is difficult to predict the biological fate of the cancer from biopsies obtained from the primary cancer [28]. A significant number of patients experience metastatic disease following primary therapy due to the treatment's inability to target the more aggressive metastasising population [3].
The biological fate of malignancies is determinable through the detection and bio-characterisation of CTCs [18, 28]. Chemosensitivity testing on the isolated CTC population can identify treatments that are likely to instigate the apoptosis of metastasising cells [3]. It follows that the benefits of CTC analysis and testing will have implications in clinical decision-making, making it possible to individualise diagnosis and treatment plans [3, 6, 29].
4.Assessment of likelihood of recurrence.
CTC detection and analysis makes it possible to assess the risk of disease recurrence after therapy of curative intent [1, 4]. CTC count in the peripheral circulation before both surgery and chemotherapy or other treatment is the marker that can independently predict the early recurrence in patients with cancer [14]. Novel enrichment and molecular analytic techniques have made it possible to detect metastasising disease that is undetectable using conventional imaging techniques. [4]
The detection and isolation of CTCs
Circulating Tumour Cells (CTCs) are rare events in the peripheral circulation of cancer patients with malignant disease [7]. They can be reliably detected, isolated, cultured and analysed using immunocytochemical and biomolecular techniques [1, 2, 7, 16].
When used in isolation, each of the available detection methods have their advantages and pitfalls [1]. There is yet to be a standardised CTC-enrichment technique, however, the FDA has approved the Veridex (Johnson & Johnson) CellSearch device for CTC detection in breast cancer patients only [1,4]. CellSearch is known for it's high specificity, but poor sensitivity [4]. Numerous studies indicate that using a combination of the more recent physical and immunochemical techniques overcomes the disadvantages each method may have when used on their own [1, 2, 6, 7, 16].
CTCs either express or, (given certain conditions), have the potential to express renegade proteins that are associated with the robustness of malignant tumours [30]. The earlier that cells with tumour initiation capacity are detected and analysed, the sooner an individualised treatment design is possible. [15, 31] The identification of both surface and intracellular markers that indicate metastatic progression are they key to detecting the CTCs in the blood.
As yet it is too complex to detect and isolate tumour stem-cells within a tumour mass due to the lack of identifiable stem cell markers [15]. The detection of CTCs however overcomes this problem: their significance lies in their similarity to tumour stem cells, and they are easily isolated from the peripheral circulation [1, 3].
Methods of enrichment may involve one or a combination of the following:
Note: a comprehensive explanation of each of these methods, including their advantages and disadvantages is available upon request: joachim@doctorfluhrer.com.au.
PHYSICAL:
•Centrifugation: isolating CTCs based on their gradient-density [1, 4, 16]
•ISET: 'Isolation by Size of Epithelial Tumour cell' [1, 3, 4, 6, 29]
•Isolation by other morphological characteristics unique to CTCs [4].
IMMUNOCHEMICAL
•ICE – 'Immunomagnetic Cell Enrichment' enriches CTCs via either positive or negative selection. ICE involves antibodies bound to magnetic beads that are selective to CTC markers. Isolating the antibody-selected cell complex from the blood occurs due to exposure to a magnetic field [1, 4]. CellSearch isolates CTCs via positive selection by utilising ICE and histological staining of EPCAM markers [4].
Biomolecular analysis of CTCs
RT-PCR:
•Reverse Transcriptidase Polymerase Chain Reaction detects genetic mutations in the DNA of CTCs. Primers or probes are designed which base-pair with the specific gene or chromosomal sequence (mutation) of interest, thereby identifying their presence. Multiple sequences (in fact the entire genomic sequence) can be analysed simultaneously [1].
DNA MICROARRAY:
•DNA microarrays enable the identification of genes, determines the active expression of genomic sequences, and detects oncogenic mutations/polymorphisms present in the nucleic acids of any cell. [32] The process makes biochemical calculations of the mRNA that is expressed in cells, hence revealing the cell's molecular biology. DNA-microarrays can analyse multiple genes simultaneously and have revolutionary diagnostic potential. [31, 33]
FLOW CYTOMETRY:
•Flow Cytomertry examines the biomolecular footprint of cells. In a nutshell, a cell is tagged for specific constituents and exposed to a laser beam of light. The presence of proteins and sub-cellular molecules in/on the cell will cause the light to fragment in a pattern that, in turn, identifies their existence. The patterns created by the scattering of light can be detected and analysed [34-38].
Note: a comprehensive explanation of these biomolecular methods is available upon request: joachim@doctorfluhrer.com.au
Chemosensitivity testing of CTCs
Chemosensitivity testing of CTCs is a diagnostic tool that enables individualised tailoring of cancer treatment [3]. Chemosensitivity testing involves exposing monocultures of enriched CTCs to each available cancer therapy agent and then analysing the cell's resistance or sensitivity to the treatment. The pathologist can observe sensitivity to treatment by calculating the extent to which a chemotherapy agent induces apoptosis of cultured cells [25]. The pathologist can also observe resistant mechanisms present in the culture of CTCs by observing the extent to which the cells maintain an active cycle despite exposure to the agent [25]. Recent studies that clarify the prospect of individualised cancer treatment through chemosensitivity testing all affirm the importance of testing resistant mechanisms in cells of tumour initiating capacity (TICs and CTCs) [3, 25]. These biomolecular markers on TICs and CTCs may be responsible for the failure of primary therapy and therefore are a promising target of individualised anti-cancer therapy [3, 17, 25, 29].
The significance of CTCs in terms of diagnosis and treatment
"It is undeniable that CTCs have enormous research potential for
individualised medicine in the future." [3]
Molecular diagnostics hold great promise for individualised diagnosis of cancer [15]. It is possible not only to detect defunct proteins that regulate the cell cycle but also to scan the entire genome of metastasising cells and detect the genes associated with cancer progression prior to them even being transcribed or expressed [2, 29]. Molecular technology also allows the detection and testing of resistant or chemosensitive mechanisms existing or dormant within the tumour cell [39]. Such knowledge of the biology of a patient's caner allows clinicians to select effective targeted therapies, to monitor the effects of treatment in real-time, and to adapt treatment according to new mutations or protein expression that may have arisen [2, 29]. Detection of these mechanisms is highly valuable in effective cancer management. [3, 18].
A major factor contributing to the possibility of individualised diagnosis through detection and analysis of CTCs is simply the ease of sample collection and accessibility of the cells [3]. Traditionally, clinicians have had to obtain a tissue sample that needs preservation in formalin and fixing in paraffin in order to analyse cancer cells [1]. Analysis of cancer cells isolated from the peripheral circulation overcomes this hassle as well as providing a continuous source of DNA, being free of selection bias, being instantaneous, less expensive and far less invasive than a biopsy surgically removed from a solid tumour [4, 15].
Individualised treatment arises from the possibility of assessing treatment efficacy, assessing completeness of surgery, and monitoring the changing molecular biology of heterogeneous subpopulations of cancer cells [40]. Heterogeneous mutations and protein expression can be detected through highly sensitive methods of analysis of CTCs, deeming CTCs potentially central to the tailoring of cancer therapy [3, 4]
© Copyright Fluhrer, JE and Fluhrer JG, April 2011References:
1.Georg, L., et al., Circulating Tumor Cells in Gastrointestinal Malignancies: Current Techniques and Clinical Implications. Journal of Oncology, 2009. 2010.
2.Pantel, K. and S. Riethdorf, Pathology: are circulating tumor cells predictive of overall survival? Nat Rev Clin Oncol, 2009. 6(4): p. 190-1.
3.Yu, S.R., et al., Circulating tumor cells and individualized chemotherapy. Chin J Cancer, 2009. 28(11): p. 1225-32.
4.Panteleakou, Z., et al., Detection of circulating tumor cells in prostate cancer patients: methodological pitfalls and clinical relevance. Mol Med, 2009. 15(3-4): p. 101-14.
5.Olmos, et al., Circulating tumour cell (CTC) counts as intermediate end points in castration-resistant prostate cancer (CRPC): a single-centre experience. Annals of Oncology, 2009. 20(1): p. 27.
6.Ross, J.S. and E.A. Slodkowska, Circulating and disseminated tumor cells in the management of breast cancer. Am J Clin Pathol, 2009. 132(2): p. 237-45.
7.Mostert, B., et al., Circulating tumor cells (CTCs): detection methods and their clinical relevance in breast cancer. Cancer Treat Rev, 2009. 35(5): p. 463-74.
8.Pienta, K.J. and R. Loberg, The "emigration, migration, and immigration"of prostate cancer. Clin Prostate Cancer, 2005. 4(1): p. 24-30.
9.Gieseler, F., et al., Resistance mechanisms of gastrointestinal cancers: why does conventional chemotherapy fail? Int J Colorectal Dis, 2003. 18(6): p. 470-80.
10.Evans, R.A., The "seed and soil" hypothesis and the decline of radical surgery: a surgeon's opinion. Tex Med, 1990. 86(9): p. 85-9.
11.Mukai, M., Occult neoplastic cells and malignant micro-aggregates in lymph node sinuses: review and hypothesis. Oncol Rep, 2005. 14(1): p. 173-5.
12.Loberg, R.D., et al., Detection and isolation of circulating tumor cells in urologic cancers: a review. Neoplasia (New York, NY), 2004. 6(4): p. 302.
13.Alix-Panabieres, C., S. Riethdorf, and K. Pantel, Circulating tumor cells and bone marrow micrometastasis. Clin Cancer Res, 2008. 14(16): p. 5013-21.
14.Pierga, J.Y., et al., Circulating tumor cell detection predicts early metastatic relapse after neoadjuvant chemotherapy in large operable and locally advanced breast cancer in a phase II randomized trial. Clin Cancer Res, 2008. 14(21): p. 7004-10.
15.NCI. Executive Summary of the Tumour Stem Cell & Self-Renewal Genes Think Tank. 2009 [cited 12 December 2009]; Available from: http://dcb.nci.nih.gov/thinktank/Executive_Summary_of_the_Tumor_Stem_Cell.cfm.
16.Ross, J.S., et al., The HER-2 receptor and breast cancer: ten years of targeted anti-HER-2 therapy and personalized medicine. Oncologist, 2009. 14(4): p. 320-68.
17.Zhou, B.B., et al., Tumour-initiating cells: challenges and opportunities for anticancer drug discovery. Nat Rev Drug Discov, 2009. 8(10): p. 806-23.
18.Pestrin, M., et al., Correlation of HER2 status between primary tumors and corresponding circulating tumor cells in advanced breast cancer patients. Breast Cancer Research and Treatment, 2009. 118(3): p. 523.
19.Muller, V., D.F. Hayes, and K. Pantel, Recent translational research: circulating tumor cells in breast cancer patients. Breast Cancer Res, 2006. 8(5): p. 110.
20.Riethdorf, S., H. Wikman, and K. Pantel, Review: Biological relevance of disseminated tumor cells in cancer patients. Int J Cancer, 2008. 123(9): p. 1991-2006.
21.Tomaskovic-Crook, E., E. Thompson, and J. Thiery, Epithelial to mesenchymal transition and breast cancer. Breast Cancer Research, 2009. 11(6): p. 213.
22.Crea, F., et al., Targeting prostate cancer stem cells. Anticancer Agents Med Chem, 2009. 9(10): p. 1105-13.
23.Neuzil, J., et al., Tumour-initiating cells vs. cancer 'stem' cells and CD133: what's in the name? Biochem Biophys Res Commun, 2007. 355(4): p. 855-9.
24.Zhong, Y., et al., Cancer stem cells sustaining the growth of mouse melanoma are not rare. Cancer Lett, 2009: p. aheadofprint.
25.Morrison, B.J., et al., Future use of mitocans against tumour-initiating cells? Mol Nutr Food Res, 2009. 53(1): p. 147-53.
26.Tanaka, F., et al., Circulating tumor cell as a diagnostic marker in primary lung cancer. Clin Cancer Res, 2009. 15(22): p. 6980-6.
27.Scher, H.I., et al., Circulating tumour cells as prognostic markers in progressive, castration-resistant prostate cancer: a reanalysis of IMMC38 trial data. Lancet Oncol, 2009. 10(3): p. 233-9.
28.Mukai, M., et al., Local recurrence and occult neoplastic cells in the extranodal fat of dissected lymph nodes in patients with curatively resected primary colorectal cancer. Oncol Rep, 2007. 17(6): p. 1365-9.
29.Ross, J.S., Breast cancer biomarkers and HER2 testing after 10 years of anti-HER2 therapy. Drug News Perspect, 2009. 22(2): p. 93-106.
30.Kari, L., et al., Classification and prediction of survival in patients with the leukemic phase of cutaneous T cell lymphoma. J Exp Med, 2003. 197(11): p. 1477-88.
31.NCI. Molecular Diagnostics. 2006 [cited; Available from: http://www.cancer.gov/cancertopics/understandingcancer/moleculardiagnostics.
32.DNA microarray, in Collins Dictionary of Biology. 2005, Collins.
33.DNA microarray, in Collins Dictionary of Medicine. 2005, Collins.
34.flow cytometry, in Mosby's Dictionary of Medicine, Nursing, & Health Professions. 2009, Elsevier Health Sciences.
35.flow cytometry, in Collins Dictionary of Biology. 2005, Collins.
36.flow cytometry, in Mosby's Dictionary of Complementary and Alternative Medicine. 2005, Elsevier Health Sciences.
37.flow cytometry, in Webster's New World™ Medical Dictionary. 2003, Wiley.
38.flow cytometry, in Collins Dictionary of Medicine. 2005, Collins.
39.Pantel, K., C. Alix-Panabieres, and S. Riethdorf, Cancer micrometastases. Nat Rev Clin Oncol, 2009. 6(6): p. 339-51.
SOURCEgenostics.com.au/www.genostics.com.au_website/CTCs_-_Info_for_Doctors.html
