Molecular Profiling in Clinical Decision Making

[Hopeful]

For anyone who has had (or is considering) molecular profiling (i.e., Oncotype Dx testing) to assist in tx decisions, this is eye-opening reading. We need to remember that the individuals developing these tests (and the labs that market them) are conducting not just research, but business. This well written article explains many of the potential biases involved in the development and performance of the various tests: http://www.theoncologist.com/cgi/content/full/12/3/301

For Oncotype Dx uses, pay specific attention to Figure 1 in the above referenced article.

Hopeful

[gdpawel]

In chemotherapy selection, molecular profiling examines a single process within the cell or a relatively small number of processes. The aim is to tell if there is a theoretical predisposition to drug response.

Functional profiling tests not only for the presence of the molecular profile but also for their functionality, for their interaction with other genes, proteins, and processes occurring within the cell, and for their response to anti-cancer drugs.

The goal of molecular testing is to look for patterns of normal and abnormal gene expression which could suggest that certain proteins might or might not be produced within a cell. However, just because a gene is present, it does not mean that an associated protein has been produced.

Protein testing goes one step further by testing to see if the relevant protein actually has been produced. However, even Protein testing cannot tell us if a protein is functional or how it will interact with other proteins in the presence of anti-cancer drugs.

Gene and protein testing involve the use of dead, formaldehyde preserved cells that are never exposed to chemotherapy drugs. Gene and protein tests cannot tells us anything about uptake of a certain drug into the cell or if the drug will be excluded before it can act or what changes will take place within the cell if the drug successfully enters the cell.

Gene and protein tests cannot discriminate among the activities of different drugs within the same class. Instead, gene and protein tests assume that all drugs within a class will produce precisely the same effect, even though from clinical experience, this is not the case. Nor can gene and protein tests tell us anything about drug combinations.

Functional tumor cell profiling tests living cancer cells. It assesses the net result of all cellular processes, including interactions, occurring in real time when cancer cells actually are exposed to specific anti-cancer drugs. It can discriminate differing anti-tumor effects of different drugs within the same class. It can also identify synergies in drug combinations.

Gene and protein tests are better suited for ruling out "inactive" drugs than for identifying "active" drugs. When considering a cancer drug which is believed to act only upon cancer cells that have a specific genetic defect, it is useful to know if a patient's cancer cells do or do not have precisely that defect.

Although presence of a targeted defect does not necessarily mean that a drug will be effective, absence of the targeted defect may rule out use of the drug. Of course, this assumes that the mechanism of drug activity is known beyond any doubt, which is not always the case.

Although gene and protein testing currently are limited in their reliability as clinical tools, the tests can be important in research settings such as in helping to identify rational targets for development of new anti-cancer drugs.

As you can see, just selecting the right test to perform in the right situation is a very important step on the road to personalizing cancer therapy.

http://cancerfocus.org/forum/showthread.php?t=734

[gdpawel]

Why hasn’t there been any progress at all in drug selection through the use of molecular diagnostics and biomarkers? Simply put, they do not work! Little progress has been made in identifying which therapeutic strategies are likely to be effective for individual patients by molecular prognostic and predictive markers.

It was hoped that any patient with cancer would have their tumor biopsied and profiled. The profile would then be displayed as a unique genetic signature, which would in turn predict which therapy would most likely work. However, gene-expression signatures are not ready for prime time.

Although molecular profiling of tumors has led to the identification of gene-expression (biological activity) patterns, a new review published in the March 16, 2010 JNCI has found little evidence that any of the signatures are ready for use in the clinical setting.

Then further analyses revealed evidence that the technologies for the prediction of response in individual patients could not be reproduced. The NCI concluded, it’s absolutely premature to use these prediction models to influence the therapeutic options open to cancer patients. The genomic methodology is not ready for clinical application.

What went wrong? The simple answer is that cancer isn’t simple. Cancer dynamics are not linear. Cancer biology does not conform to the dictates of molecular biologists. Once again, we are forced to confront the realization that genotype does not equal phenotype.

The particular sequence of DNA that an organism possess (genotype) does not determine what bodily or behaviorial form (phenotype) the organism will finally display. Among other things, environmental influences can cause the suppression of some gene functions and the activation of others. Out knowledge of genomic complexity tells us that genes and parts of genes interact with other genes, as do their protein products, and the whole system is constantly being affected by internal and external environmental factors.

The gene may not be central to the phenotype at all, or at least it shares the spotlight with other influences. Environmental tissue and cytoplasmic factors clearly dominate the phenotypic expression processes, which may in turn, be affected by a variety of unpredictable protein-interaction events. This view is not shared by all molecular biologists, who disagree about the precise roles of genes and other factors, but it signals many scientists discomfort with a strictly deterministic view of the role of genes in an organism’s functioning.

Until such time as cancer patients are selected for therapies predicated upon their own unique biology, we will confront one targeted drug after another. Our solution to this problem has been to investigate the targeting agents in each individual patient’s tissue culture, alone and in combination with other drugs, to gauge the likelihood that the targeting will favorably influence each patient’s outcome. Functional profiling results to date in patients with a multitude type of cancers suggest this to be a highly productive direction.

['lizbeth]

GDP,

I'm confused. I understand that Germany does chemosensitivity testing to see if a chemo will be effective.

Is this different from molecular profiling of tumors?

[gdpawel]

'lizbeth

Getting tumor cells from blood maybe feasible for solid tumors, though ususally only when the tumor is very advanced, and then only in small numbers.

It seems plausible that in Germany a procedure can get enough specimen from circulating tumor cells for solid tumors. It may be possible using PCR (Polymerase Chain Reaction) or similar technology like microchip arrays (gene chips). They are of great promise for molecular profiling, but not for functional profiling (which is only involved in "drug selection," not for any other purpose).

The endpoints of molecular (genetic) profiling are gene expression, examines a single process (pathway) within the cell or a relatively small number of processes (pathways), to determine only if there is evidence of a theoretical predisposition to drug susceptibility. Protein testing goes one step further by testing to see if the relevant protein actually has been produced, but cannot tell you if a protein is functional or how it will interact with other proteins in the presence of anti-cancer drugs.

The endpoints of functional profiling are expression of cell death, both tumor cell death and tumor associated endothelial (capillary) cell death, examines not only for the presence of the molecular profile but also for their functionality, for their interaction with other genes, proteins and processes occuring within the cell, and for their response to anti-cancer drugs.

Only minute quantities of DNA are necessary for PCR. DNA can be amplified from a single cell. PCR amplification techniques raise considerable concerns regarding contamination from one specimen to another, creating the potential for false positive results. Clinical interpretation of PCR results may also be challenging.

But, PCR may be useful when culture is difficult due to the low numbers of the organisms, for lengthly culture requirements, or when there is difficulty in collecting an appropriate sample. Don't know if the results would be indicative of what would happen inside the human body.

They ususally proliferate (grow) cancer cells from a small sample and subject those cells to chemo. Cells 'grown' in the lab will not behave the same way as the actual cancer cells do in your body's own environment. Because they test on subcultured cells (as opposed to fresh tumor cultures) and test the cells in monolayers (as opposed to three dimensional cell clusters), the cell grown in the lab will not behave the same way as the actual cancer cells do in your body's own environment.

Older technology assay tests failed because scientists looked to see which drugs inhibited the cancer cells' growth (cell-growth endpoint), not which chemotherapies actively killed the tumor cells (cell-death endpoint). Cancer wasn't growing faster than other cells, it's just dying slower. The newer assay testing technology connects drugs to patients by what 'kills' their cells, not by what 'slows' them down.

All of the work in the past almost twenty years in the cell culture field has been carried out largely on three dimensional clusters of cells (not monolayers). Work is done exclusively with three dimensional, floating, tumor spheroids. When you test the cells as three dimensional spheroids, they are many-fold resistant in vitro, just as they are in vivo (multicellular resistance). Even researchers at Johns Hopkins and Washington University in St. Louis discovered that 3D analysis is more accurate.

There is a problem with "growing" or "manipulating" tumor cells in any way. When looking for cell-death-related events, which mirror the effect of drugs on living tumors, cells are generally not "grown" or "amplified" in any way. The object is occurrence of programmed cell death in cells that come into contact with therapeutic agents.

How do you aggregate a sufficient number of cancer cells to make accurate determinations? Detectable tumor cells in the peripheral blood are present only in extremely small numbers. This precludes allowing a sufficient number of cells to incubate for a few days in the presence of chemotherapeutic agents.

Analysis of a relatively small number of isolated cancer cells cannot yield the same quality information as subjecting living cells to chemotherapeutic agents, begging the question of whether or not it can accurately predict which drugs will work and which will not.

CTCs are free-floating cancer cells that can remain in isolation from a tumor for over twenty years. What is the relationship of such long-lasting cells to the tumor cells that need to be attacked through tested substances?

Then there is the question of heterogeneity. Tumors in the body are genetically variable. What is the relationship between CTCs and primary tumors or their already established metastases? It has already been established that the gene expression profile of a metastatic lesion can be different compared to that of the primary. The status of the marker Her2/neu in CTCs sometimes differs from that of the original primary tumor, which would point to different prescriptions for Herceptin.

The number of cells discovered in the CTC technique has turned out to be a good prognosticator of how well empiric treatments are working, but less certain in the ability to use it for drug selection. The "problem" is in isolating and analyzing single cancer cells. The supposition is that common cancers can be detected and cured through analysis at a genetic level of a small number of cells or even a single wayward cell.

This does have a great potential, for drug selection, ten or twenty years down the road, and they should continue to try and make strides.

Greg

Question- my sister had a test from caris life science in hope of finding a chemo match for her stage 4 cholangiocarcinoma. my understanding is they perform a molecular biomarker test which tells the doctor which drugs wont work and some that may, but not definatively what is a match, just possible.

I am also looking at the rational therapeutic chemosensitivity tests as an option as we are running out of options,

can you tell me what the differences are in the two types of tests?
do you think the chemosensitivity test it better and more accurate?

[gdpawel]

Some molecular tests (like Caris) do utilize living cells, but generally of individual cancer cells in suspension, sometimes derived from tumors and sometimes derived from CTCs. This was tried with the human clonogenic assay, which had been discredited long ago. A technology that hasn't been used by any private sector laboratory for more than twenty years.

The endpoints (point of termination) of molecular profiling are gene expression, examining a single process (pathway) within the cell or a relatively small number of processes (pathways), to test for "theoretical" candidates for targeted therapy.

The endpoints of functional profiling (Rational Therapeutics) are expression of cell death, both tumor cell death and tumor associated endothelial (capillary) cell death, and examines not only for the presence of the molecular profile but also for their functinality, for their interaction with other genes, proteins and processes occuring within the cell, and for their "actual" response to anti-cancer drugs.

The core understanding is the cell, composed of hundreds of complex molecules that regulate the pathways necessary for vital cellular functions. If a targeted drug could perturb any of these pathways, it is important to examine the effects of drug combinations within the context of the cell. Both genomics and proeomics can identify potential therapeutic targets, but these targets require the determination of cellular endpoints.

Cell culture work (as mentioned above) is in three dimensional (3D) tumor cell clusters. Three dimensional clusters of cells do not circulate. It is useful in analyzing circulating endothelial cells (the tumor associated blood vessel cells). It tests for a lot more than just a few mutations.

What research scientists in universities and cancer centers have been doing for the past ten years is to try and figure out a way to use this mutation testing to look for patterns of gene expression which correlate with and predict for the activity of anticancer drugs. However, genes do not operate alone within the cell but in an intricate network of interactions.

Since the new millenium there has been the increasing acceptance of the concept that cancer is a very heterogenous disease and that it would be a good thing to try and "individualize' treatment. The cell is a system, an integrated, intereacting network of genes, proteins and other cellular constituents that produce functions. One needs to analyze the systems' response to drug treatments, not just one or a few targets (pathways/mechanisms).

Despite its allure, the genetic path is not all that personalized. Treatment based on genetic testing is still a guessing game. A gene test is simply incapable of capturing the complexities associated with human tumor biology. Examing a patient's DNA can give physicians a lot of information, but as mentioned above, the NCI has concluded (J Natl Cancer Inst. March 16, 2010), it cannot determine treatment plans for patients. It cannot test sensitivity to any of the targeted therapies. They just test for "theoretical" candidates for targeted therapy.

Cancer dynamics are not linear, Cancer biology does not conform to the dictates of molecular biology. Once again, we are forced to confront the realization that genotype does not equal phenotype. Cancer cells utilize cross talk and redundancy to circumvent therapies. They back up, zig-zag and move in reverse, regardless of what the sign posts say. The building blocks of human biology are carefully construed into the complexities that we recognize as human beings. However appealing gene profiling may appear to those engaged in this field, it will be years, perhaps decades, before these profiles can approximate the vagaries of human cancer.

Functional profiling analyses, which measure biological signals rather than DNA indicators, will continue to provide clinically validated information and play an important role in cancer drug selection. The data that support functional profiling analyses is demonstrably greater and more compelling than any data currently generated from DNA analyses. Functional profiling remains the most validated technique for selecting effective therapies for cancer patients.

Functional profiling assesses the activity of a drug upon combined effect of all cellular processes, using several metabolic (cell metabolism) and morphologic (structure) endpoints, at the cell "population" level, rather than at the "single cell" level, measuring the interaction of the entire genome.

The original Human Genome Project dealth with a homogeneous population of normal diploid cells. This is different from primary tumors, which are heterogeneous and have a genomic signature unique to each and every patient. Functional profiling is a biomarker of heterogeneous cancer cells and genomic signatures unique to every individual patient.

That is the major differences between the two types of technology. A chemosensitivity test is too limited, to generic. There has been cutting-edge techniques that have made cell culture assays with "functional profiling" mimic what will happen in the human body. Cancer is already in 3D (three dimensional) conformation. Cell-based functional profiling cultures "fresh" live tumor cells in 3D conformation and profiles the "function" of cancer cells (is the whole cell being killed regardless of the targeted mechanism or pathway). It distinguishes between susceptibility of cancer cells to different drugs in the same class and the susceptibility to combinations.

And the reason for at least a "tru-cut" biopsied tumor specimen is that "real life" 3D analysis makes functional profiling indicative of what will happen in the body. It tests fresh "live" cells in their three dimensional (3D), floating clusters (in their natural state). As researchers at Johns Hopkins and Washington University at St. Louis have recently found out, our body is 3D, not 2D in form. Older assay technology (human clonogenic assay) was in 2D form. Traditionally, in-vitro (in lab) cell-lines have been studied in 2 dimensions (2D) which has inherent limitations in applicability to real life 3D in-vivo (in body) states. Recently, other researchers have pointed to the limitations of 2D cell line study and chemotherapy to more correctly reflect the human body.

I do not promote any particular lab or laboratories. I advocate a technology which I firmly believe in because it works: cell function analysis. In other words, which combinations are best and in what sequence would they be most effective - targeted and/or conventional.