Gene-expression patterns in drug-resistant acute lymphoblastic leukemia cells

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Gene-expression patterns in drug-resistant acute lymphoblastic leukemia cells and response to treatment.

An international study published in the August 5, 2004 issue of the New England Journal of Medicine contains an article relating microarray gene expression patterns to clinical drug resistance. It reported that cell culture assay tests with a cell-death endpoint are effective in identifying gene expression patterns that correlate with clinical drug resistance. The study, titled "Gene Expression Patterns in Drug Resistant Acute Lymphoblastic Leukemia Cells and Response to Treatment" employed the cell-death assay to examine drug resistance at the molecular level.

The investigators exposed cells to drugs and cultured in a 96 hour suspension cell culture drug resistance assay (MTT) to define sensitivity and resistance. They used the data to define gene expression patterns associated with sensitivity and resistance to each of 4 drugs commonly used in the treatment of childhood leukemia. They were able to show that the gene expression definitions of sensitivity and resistance were significantly and independently associated with treatment outcome.

This work could not have been done without prior work in more than a thousand cell culture drug resistance test assays from children with leukemia to define sensitivity and resistance for each of the four drugs. Cell culture assays are the Rosetta Stone which allows for identification of clinically relevant gene expression patterns which correlate with clinical drug resistance for different drugs in specific diseases.

In an accompanying editorial, a review of the study findings indicated that the observed gene expression profiles represent fundamental biochemical features and suggests that gene expression profiles could be used to alter therapy instead of in vitro sensitivity testing. They go on to state that there is no single gene whose expression accurately predicts therapy outcome, emphasizing that cancer is a complex disease and needs to be attacked on many fronts.

A number of cell culture assay labs across the country have data from tens of thousands of fresh human tumor specimens, representing virtually all types of human solid and hematologic neoplasms, in which were tested a median of 17 drugs and/or drug combinations under very similar conditions to that of this acute lymphoblastic leukemia study. Cells were exposed to drugs and cultured in suspension for 96 hours and tested simultaneously with two different assays (MTT and DISC). What this means is that the these cell culture assay labs have the Rosetta Stone database necessary to define sensitivity and resistance for virtually all of the currently available drugs in virtually all types of human solid and hematologic neoplasms.

(N Engl J Med. 2004 Aug 5;351(6):533-42;601-3)

An atlas of fresh human tumors in short term suspension culture, illustrating examples of (1) the appearance of the fresh tumors immediately following scissor mincing and digestion with collagenase/DNAse, (2) appearance of “control” (cultured without drugs) tumor cells, following 96 hours of suspension culture, in anchorage-independent conditions, (3) appearance of cells when cultured with an “effective”/”active” drug, and (4) appearance of cells when cultured with an “ineffective”/”inactive” drug.

[url]http://weisenthal.org/Human_Tumor_Assay_Journal/Atlas.html

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N Engl J Med. 2004 Aug 5;351(6):533-42.

Gene-expression patterns in drug-resistant acute lymphoblastic leukemia cells and response to treatment.

Holleman A, Cheok MH, den Boer ML, Yang W, Veerman AJ, Kazemier KM, Pei D, Cheng C, Pui CH, Relling MV, Janka-Schaub GE, Pieters R, Evans WE.

Source: Division of Pediatric Oncology-Hematology, Erasmus University Medical Center, Sophia Children's Hospital, Rotterdam, The Netherlands.

Abstract

BACKGROUND: Childhood acute lymphoblastic leukemia (ALL) is curable with chemotherapy in approximately 80 percent of patients. However, the cause of treatment failure in the remaining 20 percent of patients is largely unknown.

METHODS: We tested leukemia cells from 173 children for sensitivity in vitro to prednisolone, vincristine, asparaginase, and daunorubicin. The cells were then subjected to an assessment of gene expression with the use of 14,500 probe sets to identify differentially expressed genes in drug-sensitive and drug-resistant ALL. Gene-expression patterns that differed according to sensitivity or resistance to the four drugs were compared with treatment outcome in the original 173 patients and an independent cohort of 98 children treated with the same drugs at another institution.

RESULTS: We identified sets of differentially expressed genes in B-lineage ALL that were sensitive or resistant to prednisolone (33 genes), vincristine (40 genes), asparaginase (35 genes), or daunorubicin (20 genes). A combined gene-expression score of resistance to the four drugs, as compared with sensitivity to the four, was significantly and independently related to treatment outcome in a multivariate analysis (hazard ratio for relapse, 3.0; P=0.027). Results were confirmed in an independent population of patients treated with the same medications (hazard ratio for relapse, 11.85; P=0.019). Of the 124 genes identified, 121 have not previously been associated with resistance to the four drugs we tested.

CONCLUSIONS: Differential expression of a relatively small number of genes is associated with drug resistance and treatment outcome in childhood ALL.

[url]http://www.nejm.org/doi/full/10.1056/NEJMoa033513

A meta-analysis in human hematologic malignancies, correlated the outcome in 1929 patients who received chemotherapy with the results of cell-based functional profiling analysis of drug induced programmed cell death. The objective response rate for patients who received “assay-sensitive” drugs was 84.6 percent, compared with patients who received “assay-resistant” drugs of 28.3 percent (p<0.0001).

Source: Andrew G. Boasanquet, Gertjan J. Kapers, Rolf Larsson, Robert A. Nagourney, Peter Nygren, Rob Pieters, Peter Staib, Ulf Tidefelt, C Michael Zwaan and Larry Weisenthal. Individualized Tumor Response (ITR) Profiling for Drug Selection in Tailored Therapy: Meta-Analysis of 1929 Cases of Leukemia and Lymphoma. Blood (ASH Annual Meeting Abstracts) 110:3471, 2007.

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By Dr. Robert Nagourney
Medical and Laboratory Director of Rational Therapeutics (Long Beach, Calif.)

In some ways, the slow adoption of these techniques - compared with Europe and Asia - does reflect the relative conservatism of American medicine. We have been slow to adopt acupuncture and incorporate diet and lifestyle changes into medical therapy, despite their manifest importance. We are often slower to improve drugs, even when they establish clinical utility in well-conducted foreign trials. So, there may indeed be a component of late adoption and conservatism.

However, The re-importation of technologies is not only seen in the medical community. The early adoption of transistor technology by the Japanese despite their development by American inventors; the late adoption of robotics and fuzzy logic by Americans; and our tardiness in adopting smaller, more fuel-efficient automobiles all illustrate this point. But, the most vexing hurdle of all has been the dismissal by mostly university-based investigators who have weighed in against the adoption of human tissue tests for the prediction of response to chemotherapeutics.

These investigators - who, in aggregate, provide care to less than 10 percent of the cancer patients in need - have an inordinate amount of influence upon the application of novel technologies. In what can only be viewed as a sour grapes phenomenon, many of these physicians even tried to apply early forms of human tumor study in their own labs and medical centers.

The utter failure of the clonogenic assay in the 70s and 80s and related growth-based technologies, poisoned these academics and closed their minds to newer developments based on the modern discoveries of apoptosis and other forms of programmed call death. When we, and our colleagues, reported discoveries using these more modern endpoints, the academic community turned a deaf ear. As our data improved, they dug in their heels. And when the data rose to the level of the best peer reviewed journals in the field, the critics became ever more vocal.

We can now thank these "scientists" for putting the United States behind Europe and Asia in the adoption of these important methodologies. While patients in America must struggle with their physicians to get ex-vivo analyses conducted, children in Europe with leukemia have immediate access to these tests. Adults in England with leukemia can all request these assays, German patients regularly take advantage of assay methodologies. And the Japanese often apply related techniques for the treatment of their solid tumors.

Not unlike robotics, total quality management and fuel-efficient automobiles, the Americans (who invented in vitro chemosensitivity testing) will again be importing the technology that they are responsible for developing."

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There are a family of assays based on the concept of total cell kill, or cell-death occurring in the entire popluation of tumor cells.

A fresh specimen is obtained from a viable neoplasm. The specimen is most often a surgical specimen from a viable solid tumor. Less often, it is a malignant effusion, bone marrow, or peripheral blood specimen containing "tumor" cells. These cells are isolated and then cultured in the continuous presence or absence of drugs, most often for 3 to 7 days. At the end of the culture period, a measurement is made of cell injury, which correlates directly with cell-death. There is evidence that the majority of available anticancer drugs may work through a mechanism of causing sufficient damage to trigger so-called programmed cell-death or apoptosis.

Some patients may not have easily-accessible tumors (needle biopsies do not gather enough specimen), making it difficult to harvest a large enough sample (200mg or 10mm in size). The tests are most reliable before a tumor has been exposed to chemotherapy. However, after a patient fails a previous chemotherapy treatment, the test still can be done once a patient waits at least four weeks.

There are four endpoint measurements of cell-death that have been applied:

1. DISC assay. The delayed loss of cell membrane integrity.

2. MTT assay. The loss of mitochondrial Krebs cycle activity.

3. ATP assay. The loss of cellular ATP.

4. Caspase 3/7 assay. Directly measures key apoptosis expression markers.

The DISC assay is the only assay that involves direct visualization of the cancer cells at endpoint. This allows for accurate assessment of drug activity, discriminates tumor from non-tumor cells, and provides a permanent archival record. Originators of the MTT and ATP assays modeled assay conditions on the DISC assay. The use of complementary tests improves accuracy and provides quality control. Also, certain drugs cannot be tested reliably in all assay systems. Use of different tests with different mechanisms helps to overcome this.

These four endpoints can and do, in most cases, produce valid and reliable measurements of cell-death, which correlate very well with each other on direct comparisons of the different methods. This is not surprising any more than should the fact that auscultating heart sounds, observing spontaneous breathing, palpating a carotid pulse, measuring core body temperture, and recording an electroencelphalogram or electrocardiogram are all good and reliable methods of determing patient death.

Different investigators have favored different cell-death endpoints, depending on the laboratory and clinical situation. What is important is that each of the cell-death endpoints do give essentially the same results (except in the case of isolated drugs like taxanes and 5FU). So, it is entirely reasonable and proper to consider as a whole the clinical validation data which has been published over the last 20 years, using the above four endpoints.

Cell-death assays are not intended to be scale models of chemotherapy in the patient, anymore than the barometric pressure is a scale model of the weather. But it's always more likely to rain when the barometer is falling than when it is rising, and chemotherapy is more likely to work in the patient when it kills the patient's cancer cells in the laboratory. It is no different than any other medical test in this regard.

Not all patients will have the same response to the same chemotherapy. Special laboratories can test tumor samples from individual patients to see which chemotherapy drugs have the best likelihood of killing tumor cells and optimizing survival. The results provide medical and surgical oncologists with patient-specific tumor information that may provide additional insight when determing the appropriate course of treatment for a patient.

Assay-testing focuses on the unique characteristics of a particular cancer. The test results help the physician to determine which anti-cancer drugs are "likely" to be effective against a particular cancer. The assay test also helps the physician to determine which anti-cancer drugs are "unlikely" to affect a cancerous tumor, which can help to avoid toxic and possibly ineffective therapy.

The tests have a specifity of 0.92 and a sensitivity of 0.71, which means that a treatment regimen "not" resistant in the assays is 7-9 fold more likely to work than is a treatment regimen which "is" resistant in the assays, and evaluability rates (the ability to perform the assay on a specimen) are >95%. A preponderance of evidence would indicate that it would be worthwhile to consider the assay results in drug selection.

Literature Citation:
Functional profiling with cell culture-based assays for kinase and anti-angiogenic agents Eur J Clin Invest 37 (suppl. 1):60, 2007
Functional Profiling of Human Tumors in Primary Culture: A Platform for Drug Discovery and Therapy Selection (AACR: Apr 2008-AB-1546)

[url]http://www.cancertest.org/wp-content/uploads/2013/05/Weisenthal_Rec_Results_Cancer_Res_94_161-173_1984.pdf

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Following the description of apoptosis in the British Journal of Cancer in 1972, scientists around the world incorporated the concept of programmed cell death into their cancer research.

What is less understood is the fact that apoptosis is not synonymous with programmed cell death.

Programmed cell death is a fundamental feature of multicellular organism biology. Mutated cells incapable of performing their normal functions self-destruct in service of the multicellular organism as a whole.

While apoptosis represents an important mechanism of programmed cell death, it is only one of several cell death pathways.

Apoptotic cell death occurs with certain mutational events, DNA damage, oxidative stress and withdrawal of some growth factors particularly within the immune system.

Non-apoptotic programmed cell death includes: programmed necrosis, para-apoptosis, autophagic cell death, nutrient withdrawal, and subtypes associated with mis-folded protein response, and PARP mediated cell death.

While apoptotic cell death follows a recognized cascade of caspase mediated enzymatic events, non-apoptotic cell death occurs in the absence of caspase activation.

With the recognition of programmed cell death as a principal factor in carcinogenesis and cancer response to therapy, there has been a growing belief that the measurement of apoptosis alone will provide the insights needed in cancer biology.

This oversimplification underestimates the complexity of cell biology and suggests that cancer cells have but one mechanisms of response to injury. It has previously been shown that cancer cells that suffer lethal injury and initiate the process of apoptosis can be treated with caspase inhibitors to prevent caspase-mediated apoptosis.

Of interest, these cells are not rescued from death. Instead, these cells committed to death, undergo a form of non-apoptotic programmed cell death more consistent with necrosis. Thus, commitment to death overrides mechanism of death.

Labs that focus on measurements of caspase activation can only measure apoptotic cell death. While apoptotic cell death is of importance in hematologic cancers and some solid tumors, it does not represent the mechanism of cell death in all tumors.

This is why cell-based functional profiling labs measure all cell death events by characterizing metabolic viability at the level of cell membrane integrity, ATP content, or mitochondrial function.

While caspase activation is of interest, comparably easy to measure and useful in many leukemias and lymphomas, it does not represent cancer cell death in all circumstances and can be an unreliable parameter in many solid tumors.

[url]http://www.youtube.com/watch?v=4bwx-Z2ANq4&feature=autoplay&list=ULV3wrVypS_Yc&index=3 &playnext=1

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By Larry Weisenthal, M.D., PhD.

The Differfential Staining Cytotoxicity (DISC) assay is the prototype for a closely related family of assays based on the concept of total cell kill or cell death occurring in the entire populations of tumor cells.

It is probably the most versatile of the cell-death endpoints in that it (1) can be applied to both solid and hematologic neoplasms, (2) can be applied to specimens in which it is not possible to obtain a pure populations of highly enriched tumor cells, and (3) can be applied to a wide variety of drugs, ranging from traditional cytotoxic agents to biological response modifiers with activity mediated through tumor-infiltrating effector cells, to "targeted" kinase inhibitors, and to anti-vascular agents, such as bevacizumab and pazopanib.

The basic principles of the assay are to culture three-dimensional (3D) fresh tumor cell clusters in anchorage-independent conditions. At the conclusion of the culture period, Fast Green dye is added to the microwells, the contents of which are then sedimented onto permanent Cytospin centrifuge slides and then counterstained with hematoxylin-eosin or Wright-Giemsa.

"Living" cells stain with cytologic stain in question and can be identified as either normal or neoplastic, based on standard morphologic criteria. "Dead" cells stain blue green. Nonviable endothelial cells appear as strikingly hyperchromatic, blue-black and often refractile objects, which may be readily distinguished from other types of dead cells.

DISC Assay Biological Response Modifiers Bibliography

1. Einhorn S, Fernberg J-O, Grand‚r D, Lewensohn R (1988) Interferon exerts a cytotoxic effect on primary human myeloma cells. Eur J Cancer Clin Oncol 24: 1505-1510

2. Lepri E, Barzi A, Menconi E, Portuesi MG, Liberati M (1991) In vitro synergistic activity of PDN-IFNà and NM + IFNà combinations on fresh bone-marrow samples from multiple myeloma patients. Hematol Oncol 9: 79-86

3. Weisenthal LM, Nagourney RA, Kern DH, Boullier B, Bosanquet AG, Dill PL, Messenger JC, Moran EM (1989) Approach to the clinical circumvention of drug resistance utilizing a non-clonogenic in vitro assay measuring the effects of drugs, radiation, and interleukin-II on largely non-dividing cells. In: Amadori D, Ravaioli A, Ridolfi R (eds) Strategies in cancer medical therapy: biological bases and clinical implications (Advances in Clinical Oncology, V.1.). Edimes, Pavia, Italy, pp 91-111

4. Weisenthal LM, Dill PL, Pearson FC (1990) Tumor and patient-specific activity of biologic response modifiers (ImuVert, tumor necrosis factor, alpha-interferon) in fresh specimens of human neoplasms detected by a sensitive and specific in vitro assay. Proc Am Assoc Cancer Res 31: 299(Abstract)

5. Weisenthal LM, Dill PL, Pearson FC (1991) Effect of prior cancer chemotherapy on human tumor-specific cytotoxicity in vitro in response to immunopotentiating biologic response modifiers. J Natl Cancer Inst 83: 37-42

6. Weisenthal LM (1991) Effect of prior chemotherapy on biologic response modifier activity. J Natl Cancer Inst 83: 790-791

7. Weisenthal LM, Dill PL (1992) In vitro effect of interleukin-2 on fresh human tumor cell cultures measured by the DiSC assay. Proc Am Assoc Cancer Res 33:Abs 3313

[url]http://www.springerlink.com/content/kv36q25802651083/#section=885954&page=1&locus=0

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Dr. Robert Nagourney
Medical and Laboratory Director
Rational Therapeutics

An article by Gina Kolata on the front page of the July 8, Sunday New York Times, “In Leukemia Treatment, Glimpses of the Future,” tells the heartwarming story of a young physician afflicted with acute lymphoblastic leukemia.

Diagnosed in medical school, the patient initially achieved a complete remission, only to suffer a recurrence that led him to undergo a bone marrow transplant. When the disease recurred a second time years later, his options were more limited.

As a researcher at Washington University himself, this young physician had access to the most sophisticated genomic analyses in the world. His colleagues and a team of investigators put all 26 of the University’s gene sequencing machines to work around the clock to complete a whole genome sequence, in search of a driver mutation. The results identified FLT3. This mutation had previously been described in acute leukemia and is known to be a target for several available small molecule tyrosine kinase inhibitors. After arranging to procure sunitinib (Sutent, Pfizer Pharmaceuticals), the patient began treatment and had a prompt and complete remission, one that he continues to enjoy to this day.

The story is one of triumph over adversity and exemplifies genomic analysis in the identification of targets for therapy. What it also represents is a labor-intensive, costly, and largely unavailable approach to cancer management. While good outcomes in leukemia have been the subject of many reports, imatinib for CML among them, this does not obtain for most of the common, solid tumors that lack targets for these new silver bullets. Indeed, the article itself describes unsuccessful efforts on the part of Steve Jobs and Christopher Hitchens, to probe their own genomes for effective treatments. More to the point, few patients have access to 26 gene-sequencing machines capable of identifying genomic targets. A professor of bioethics from the University of Washington, Wiley Burke, raised additional ethical questions surrounding the availability of these approaches only to the most connected and wealthiest of individuals.

While brute force sequencing of human genomes are becoming more popular, the approach lacks scientific elegance. Pattern recognition yielding clues, almost by accident, relegates scientists to the role of spectator and removes them from hypothesis-driven investigation that characterized centuries of successful research.

The drug sunitinib is known for its inhibitory effect upon VEGF 1, 2 and 3, PDGFr, c-kit and FLT3. Recognizing the attributes of this drug and being well aware of C-KIT and FLT3’s role in leukemias, we regularly add sunitinib into our leukemia tissue cultures to test for cytotoxic effects in malignantly transformed cells. The insights gained enable us to simply and quickly gauge the likelihood of efficacy in patients for drugs like sunitinib.

Once again we find that expensive, difficult tests seem preferable to inexpensive, simple ones. While the technocrats at the helm of oncology research promise to drive the price of these tests down to a level of affordability, everyday we wait 1,581 Americans die of cancer. Perhaps, while we await perfect tests that might work tomorrow, we should use good tests that work today.

[url]http://www.nytimes.com/2012/07/08/health/in-gene-sequencing-treatment-for-leukemia-glimpses-of-the-future.html?_r=1&adxnnl=1&pagewanted=all&adxnnlx=1 341983089-3W4YsUQhmRPb6MrwjSNrXA

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Dr. Robert Nagourney
Medical and Laboratory Director
Rational Therapeutics

There are two types of cancer patients: those we can treat and those we can’t. As I reflect on this year and the years past during which we have applied the process of laboratory-guided treatment, I am reminded of this fact.

The EVA-PCD functional cytometric profile enables us to choose active treatments for patients, but I have sometimes wondered whether we are, in fact, choosing patients for the available drugs. While the end result may not be all that different, e.g. superior clinical outcomes over randomly administered (standard) therapies, the path to that outcome, leaves room for interesting discussion.

I first pondered this issue at the time of completion of our earliest study. That study was conducted in childhood acute lymphoblastic leukemia (ALL). Recognizing that the corticosteroids were among the most important drugs for ALL, we exposed freshly isolated lymphoblasts from ALL patients to dexamethasone (ex vivo). At the fourth day we measured the degree of cell death and separated the patients in “sensitive” and “resistant “ subgroups.

Strikingly, those children whose lymphoblasts died in the laboratory following exposure to dexamethasone (ex-vivo), virtually all survived without relapse, while those children whose lymphoblasts did not die in the laboratory following dexamethasone exposure (ex-vivo) relapsed at an alarming rate with only 25 percent still alive at the sixth year of follow up (p=0.009).

What we had succeeded in doing by Day 4 of diagnosis was something that all the known prognostic factors, like age, WBC and male vs. female could not do, namely accurately identify the responders and survivors.

Today, when I test patients in our laboratory, I consistently double or even triple the response rates over standard protocols, yet a subset of patients are not found sensitive to the available therapies. Patients who do not respond to chemotherapy are today known, in the oncologic vernacular, as “failing therapy.”

If we view these “non-responders” as a biologically distinct group (not unlike the dexamethasone-resistant ALL patients above) then our role, in the field of functional cytometric profiling, is to quickly segregate the responders (to available drugs) from the non-responders and move those “non-responders” immediately to something that will work for them.

In this light, patients no longer “fail therapies” but instead “therapies fail patients.” It is then our mandate to use the ex-vivo platforms to find (and yes, discover) novel therapies and combinations that will meet their unmet need.