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Old 08-18-2008, 02:59 PM
gdpawel gdpawel is offline
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Default Assay-Directed Therapy Strategies in CLL

Bax expression correlates with cellular drug sensitivity to doxorubicin, cyclophosphamide and chlorambucil but not fludarabine, cladribine or corticosteroids in B cell chronic lymphocytic leukemia.

Bosanquet AG, Sturm I, Wieder T, Essmann F, Bosanquet MI, Head DJ, Dorken B, Daniel PT.

Bath Cancer Research, Wolfson Centre, Royal United Hospital, Bath, UK.

Summary: Bosanquet and colleagues report that (1) fludarabine resistance in the DiSC assay correlates with patient survival in CLL; (2) apoptosis-related Bax and Bcl-2 expression do not correlate with patient survival; (3) reduced Bax expression correlates with resistance to alkylators, doxorubicin, and vincristine, but not with resistance to fludarabine or glucocorticoids; (4) Bcl-2 expression does not correlate with resistance to any drug. They conclude that apoptosis-activation pathways are different in the case of fludarabine and glucocorticoids than in the cases alkylators, doxorubicin, and vincristine.

In B-CLL, non-proliferating B cells accumulate due to defective apoptosis. Cytotoxic therapies trigger apoptosis and deregulation of apoptotic pathways contributes to chemoresistance. Loss of the apoptosis-promoting Bax has been implicated in resistance to cytotoxic therapy. We therefore evaluated ex vivo drug sensitivity of CLL, producing chemoresponse data which are prognostic indicators for B-CLL, in particular in the case of purine nucleoside analogs. To analyze the underlying mechanisms of drug resistance, we compared endogenous Bax and Bcl-2 expression to ex vivo response to eight drugs, and to survival in 39 B-CLL patients. We found that reduced Bax levels correlated well with ex vivo resistance to traditional B-CLL therapies - anthracyclines, alkylating agents and vincristine (all P < 0.04). Surprisingly, no such relationship was observed for the purine nucleoside analogs or corticosteroids (all P > 0.5). Mutational analysis of p53 could not explain the loss of Bax protein expression. Levels of Bcl-2 were not associated with sensitivity to any drug. In contrast to the ex vivo data, neither Bax or Bcl-2 expression nor doxorubicin sensitivity were associated with increased survival whereas sensitivity to fludarabine correlated with better overall survival (P = 0.031). These findings suggest that the resistance to purine nucleoside analogs and corticosteroids in B-CLL is due to inactivation of pathways different from those activated by anthracyclines, vinca alkaloids and alkylating agents and may be the molecular rationale for the efficacy of purine analogs in this disease.

Source: Leukemia 2002 Jun;16(6):1035-44

[url]http://www.nature.com/bjc/journal/v76/n4/pdf/bjc1997417a.pdf

Last edited by gdpawel : 12-13-2012 at 02:16 PM. Reason: corrected url address
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Old 11-17-2010, 03:42 PM
gdpawel gdpawel is offline
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Default Chronic Lymphocytic Leukemia (CLL) as a Platform for Functional Profiling

Among the most common forms of leukemia in adults is chronic lymphocytic leukemia. This neoplasm usually arises in a subset of lymphocytes known as B-cells. However, T-cell variants also occur. The disease presents clinically as an elevation of the circulating lymphocytes. This may be associated with enlarged lymph nodes, splenomegaly or liver enlargement.

The decision to treat patients is largely based upon clinical staging systems know as the Rai or Binet classifications. Low risk patients can often be observed without treatment, while more aggressive presentations (such as those associated with anemia and low platelet counts) require intervention. More recently, molecular determinants of aggressiveness have been applied in the prognosis of this disease. These include: CD38, VH gene mutation and Zap 70. Additional findings include ATM mutations, principally in the T-cell and pro-lymphocytic variants.

For more than 40 years, the treatment of choice for this disease was oral chlorambucil. Although effective, chlorambucil resulted in the development of resistance and was associated with rather significant myelosuppression over time. The introduction of fludarabine (FAMP) and 2-CDA revolutionized the management of this disease - providing high response rates with relatively tolerable toxicities.

The introduction of 2-CDA and fludarabine in the 1980s offered an opportunity for our laboratory to examine drug interactions in CLL patients. Combining the alkylating agents (of which, chlorambucil is a member) with 2-CDA revealed synergy (supra-additvity) in 100 percent of the CLL samples we studied (Nagourney, R; et al. British Journal of Cancer, 1993). Based on this observation, we began treating patients with CLL and related lymphoid malignancies with a combination of Cytoxan and 2-CDA, resulting in dramatic and durable remissions.

O’Brian, Keating and other investigators at the MD Anderson then undertook this work (using fludarabine), providing for the most effective therapy for CLL in today’s literature. Unfortunately, a percentage of patients who receive this combination develop deep myelo-suppression. Therefore, the administration of this combination requires careful monitoring by the physician.

One of the most interesting aspects of the high activity observed for fludarabine was the capacity of this “anti-metabolite” to induce cell death in short term cultures of CLL cells. It was well known that CLL cells were not highly proliferative, yet the anti-metabolite class of drugs was specifically designed to stop cell proliferation at the level of DNA synthesis. We realized that 2-CDA and Fludarabine had to be killing cells, not preventing their growth.

This conundrum provided an opportunity for us to test a related anti-metabolite in this disease. We chose cytarabine (Ara-C), a drug not considered effective for CLL (e.g. low proliferative rate, no likelihood of DNA synthesis inhibition, no likelihood of cytotoxicity). To our delight, low doses or Ara-C proved highly effective in controlling even the most advanced cases of CLL as we then reported.

CLL became one of our favored models for the study of human tumor biology, enabling us to study drug responses at the molecular level. Many of the observations that we made in this hematologic malignancy granted us insights that we continue to apply in solid tumors today.

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.

[Dr. Nagourney is medical and laboratory director at Rational Therapeutics, Inc., in Long Beach, California, and an instructor of Pharmacology at the University of California, Irvine School of Medicine. He is board-certified in Internal Medicine, Medical Oncology and Hematology.]
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Last edited by gdpawel : 02-18-2011 at 11:20 AM. Reason: additional info
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Old 02-06-2011, 09:07 PM
gdpawel gdpawel is offline
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Default Why doesn't everyone receive assay-directed therapy strategies?

Robert Nagourney, M.D., PhD.

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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Last edited by gdpawel : 07-08-2012 at 12:56 AM. Reason: spelling errors
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Old 12-13-2012, 07:28 PM
gdpawel gdpawel is offline
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Default Results of CLL4 trial published

The choice of treatment according to in vitro drug response testing was developed for chronic lymphocytic leukemia (CLL) as the Differential Staining Cytotoxicity assay (DiSC). This demonstrated that fludarabine-test-resistant patients treated with fludarabine had a poorer response rate and worse median survival than fludarabine-test-sensitive patients.

[url]http://www.nature.com/leu/journal/vaop/ncurrent/full/leu2012209a.html

The TRAC assay is the same thing as the DiSC assay. There is a slight difference in the way the slides are read to make it easier for the less highly trained microscopists to score the slides. But it's the same test.

A Laboratory Oncologist tells me that the results of this study are remarkably positive. The reasonable expectations of assay-directed second line therapy of CLL in the era when both rituximab and bendamustine were being phased in but were not tested in the assay would be that survival at one year could be improved and that the assay results should correlate with survival at one year.

Both of these expectations were met. Look at the survival one year out from the time of first randomization (something that was initially missed by the study statisticians) and also look at the strong correlation between survival at one year from second randomization as a function of assay result.

This is particularly impressive given the design weaknesses of the study (i.e. oncologists were "encouraged" to "consider" assay results in treatment planning, etc.). It's not like an experienced user of the assay was basing treatment precisely on assay results. Also, CLL is not like lung cancer. Patients can live a long time following first relapse. They receive multiple lines of therapy (2nd, 3rd, 4th, etc.). But the only assay-directed therapy they got was 2nd line.

So you'd think that they'd do better for the first year, given that therapy is typically given for only 6 months to a year at most. And they did have a significantly improved survival at one year and the results correlated strongly for predicting survival at one year.

They should have then gone on to get assay-directed 3rd and 4th line therapy -- particularly as good new drugs were coming out (e.g. bendamustine), which weren't a part of the drugs tested and used for 2nd line. Also, a very important new drug was just coming into general use (rituximab) and this drug certainly had a major effect in prolonging the life of patients in the empiric therapy arm and in both arms, in later stages of follow up.

They didn't determine "time to progression," which is the endpoint used by virtually all clinical trials of new drugs these days, in many diseases. There is no question at all that time to progression would have been significantly improved and, had the assay been a new drug, a good case could have been made for FDA approval.

As it is, this is now the first test proven to improve patient survival in a prospective randomized trial and this was a prospective randomized large multi-institutional trial, supported and run by the British equivalent of the U.S. NCI. It's a very high quality study, beyond reproach from an integrity point of view, even if seriously flawed from the standpoint that the deck was stacked against the assay, because the people who wrote the protocol were clinicians who were not supportive of the concept of of the assay.

Differential Staining Cytotoxicity Assay (DiSC)

[url]http://www.ncbi.nlm.nih.gov/pubmed/21516414
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Last edited by gdpawel : 01-28-2013 at 07:33 PM. Reason: corrected url address
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Old 12-18-2012, 10:28 PM
gdpawel gdpawel is offline
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Default Phar Lap and the Treatment of Leukemia

Robert A. Nagourney, M.D.

Phar Lap (1926-1932) was a thoroughbred horse bred in New Zealand. After winning the Melbourne Cup and 37 other races, his victory at the Agua Caliente racecourse in Tijuana, Mexico, established the track record in 1932.

With each victory, his detractors became more strident. He was even the target of an assassination attempt. To prevent him from winning (and thereby disrupting the betting odds) officials would add lead bricks to his saddle. On the occasion of the Melbourne cup of 1930 he carried 138 pounds of lead, yet won the race. A quote from the Sydney Morning Herald dated Wednesday, November 5, 1930, read, “The question was not which horse could win, but could Phar Lap carry the weight. Could he do what no other horse before him had done?”

It appeared that the one thing that race officialdom feared above all else, was a horse that could consistently beat the field and win the race.

The tale of Phar Lap was brought to mind after a colleague forwarded a paper published in the journal Leukemia on August 10, 2012: “The use of individualized tumor response testing in treatment selection: second randomization results from the LRF CLL4 trial and the predictive value of the test at trial entry.” (E Matutes, AG Bosanquet et al, Leukemia, Letter to the Editor.)

Published as a letter to the editor, the paper describes correlations between the TRAC (tumor response to antineoplastic compounds) assay, a short-term suspension culture cell death laboratory assay (very similar to our work) and clinical response, time to progression and overall survival in patients with chronic lymphocytic leukemia (CLL) who received chemotherapy as part of the LRF CLL4 trial conducted in England between 1999 and 2004.

The initial trial was a blinded correlation between laboratory assay results and patient response to one of three treatment regimens. An examination of the data reveals a clear and statistically significant correlation between drug sensitivity and overall survival (p = .0001). The 10-year survival of drug sensitive patients was 28 percent, while the 10-year survival for drug resistant patients was 12 percent.

Significant correlations with survival were observed for known prognostic factors like 17p and 11q deletion, as well as IGHV mutational status. Correlations were also observed between the TRAC assay results and these prognostic factors.

The report goes on to describe a second randomization that took place at the time of disease progression, either failure of first-line therapy or reoccurrence within 12 months. In this part of the study, 84 relapsed patients were allocated to standard therapy and their outcomes were compared with 84 patients allocated to treatment guided by the TRAC assay. The drugs tested in the assay-directed arm included chlorambucil, cytoxan, methylprednisolone, prednisolone, vincristine, doxorubicin, mitoxantrone, 2CDA, fludarabine and pentostatin. In vitro resistance for combinations was defined as resistance to all constituent drugs in the combination, while drug sensitivity was defined as TRAC-assay sensitivity for any of the drugs used in combination. No discussion of synergy analysis was included.

In examining this study, I cannot help but be reminded of Phar Lap. First, marshaling a study of 777 CLL patients, and conducting 544 TRAC analyses, is a phenomenal undertaking for which these authors should be commended.

Second, the observation of a significant correlation between laboratory assay results and overall survival, as well as the biological implications of this platform’s capacity to correlate with molecular markers is a demonstrable and noteworthy success, however unheralded.

Where the analogy with poor Phar Lap’s struggles, weighted down with lead, becomes most poignant is the final portion of the study wherein 84 patients received assay-directed therapy. To wit, we must remember that in 2012, drug refractory CLL remains an incurable malignancy (with the exception of a small subset of successfully transplanted patients) and that no chemotherapy-alone trial has provided a survival advantage in this group. But this only begins to explain this trial’s results.

Among the virtually insurmountable hurdles that these investigators were forced to confront was the fact that fully 52 percent of the standard treatment arm group were destined to receive fludarabine. This drug, the current gold standard for previously treated patients who fail chlorambucil (constituting 73 percent of the patients in this part of the trial), has an objective response rate of 48 – 52 percent in this population. As the drug would likely be identified as active in vitro as well, this had the impact of pitting the assay arm and the standard arm against one another, frequently using exactly the same treatment.

While this does not mean that the assay arm could not succeed, it does have an enormous impact upon the sample size calculations used to determine the number of patients required to achieve significance. No pharmaceutical company would ever allow a registration trial to be conducted against an “unknown” control arm, particularly one using the same therapy as the study arm – not ever! Despite these burdens, the assay-directed arm had a superior one-year survival, while virtually all other trends favored the group who received assay-selected therapy. The results of this study are worthy of recognition and further support the clinical relevance, predictive validity and importance of functional analyses. Yet, this interesting study in CLL is unceremoniously relegated to the status of a Letter to the Editor in Leukemia. Perhaps, like Phar Lap, no one really wants to upset the odds.
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Old 08-06-2014, 01:11 AM
gdpawel gdpawel is offline
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Default With an EVA-PCD Assay, It Can Be That Simple

Robert A. Nagourney, M.D.

Shortly after I left the university and joined a medical oncology group, one of the junior members of the practice asked if I would cover for him during his summer vacation. Among the patients he signed over to me was a gentleman in his 60s with what he described as “end-stage” chronic lymphocytic leukemia (CLL). As the patient had already received the standard therapies, second line regimens and experimental drugs available at the time, the physician had run out of options. My charge was to keep him comfortable. I asked if it would be all right for me to study his cells in my lab and the doctor agreed.

I met the patient the next day. He was a very pleasant tall, slender black man lying in bed. He had lost a great deal of weight making the already enlarged lymph nodes in his neck appear that much more prominent. As I was engaged in the study of CLL as my principal tumor model, I asked if I might examine his circulating CLL cells as part of our IRB-approved protocol. He graciously obliged and I obtained a few ccs of blood. We were deeply ensconced in tumor biology analyses and his cells were used to explore membrane potentials, DNA degradation and glutathione metabolism as correlates with drug response profiles by EVA-PCD analysis. A large number of those studies have since been published.

What struck me about the patient’s EVA-PCD profile was the exquisite sensitivity to corticosteroids. Corticosteroids in the form of prednisone, Medrol, Solu-Medrol and Decadron are the mainstays of therapy for lymphoid malignancies like CLL. Everyone receives them. Indeed this patient had received them repeatedly including his first line chlorambucil plus prednisone, his second-line CHOP and his third line ESHAP. It was only after he had failed all of these increasingly intensive regimens that he finally moved on to an experimental agent, homoharringtonine, a drug that finally received FDA approval in 2012, after almost 40 years of clinical development. Unfortunately for him homoharringtonine did not work and it seemed we were well beyond conventional therapies, or were we?

I pondered the corticosteroid sensitivity finding and decided to start the patient on oral prednisone. It would be another two weeks before his physician returned and there really weren’t many options. The patient responded overnight. The lymph nodes melted away. The spleen diminished. He began to eat and gained weight. Within a few days he felt well enough to go home. I discharged the patient and remember writing his prednisone prescription, 40 mg by mouth each morning.

A week later, my colleague returned from his retreat in the Adirondacks. He inquired about his patients and surmised that this gentleman, no longer in the hospital, had died. I explained that he had been discharged.

“Discharged . . . how?” he asked. I described the findings of our EVA-PCD study, the sensitivity to steroids and the patient’s miraculous clinical response to this, the simplest of all possible treatments. The physician then turned to me and said “Prednisone . . . hmmm . . . I could have done that.”

I am reminded of this story almost daily. It is emblematic of our work and of those who choose not to use it. Good outcomes in cancer do not occur by chance. They also do not require blockbuster new drugs or brilliant doctors. They require individualized attention to the needs of each patient.

A recurring theme, exemplified by this patient among others, is that cancer cells can only defend themselves in a limited number of ways. Once a selection pressure, in a Darwinian sense, is removed (e.g. corticosteroids were not used during the homoharringtonine treatments) the surviving cells, sensitive to steroids, re-emerge to be identified and captured in our laboratory platform.

It is remarkable how often heavily pretreated patients with ovarian cancer are found sensitive to Taxol after they had received it years earlier, but not since; or breast cancer patients who fail every new agent only to prove responsive to CMF, the earliest of all of the breast cancer drug combinations developed in the 1970s. Our job as oncologists is to find those chinks in armor of cancer cells and exploit them. The EVA-PCD platform, in the eyes of some, may not be groundbreaking . . . it just happens to work!
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