Why Oncologists Don’t Like In Vitro Chemosensitivity Tests

In human experience, the level of disappointment is directly proportional to the level of expectation. When, for example, the world was apprised of the successful development of cold fusion, a breakthrough of historic proportions, the expectations could not have been greater. Cold fusion, the capacity to harness the sun’s power without the heat and radiation, was so appealing that people rushed into a field about which they understood little. Those who remember this episode during the 1990s will recall the shock and dismay of the scientists and investors who rushed to sponsor and support this venture only to be left out in the cold when the data came in.

Since the earliest introduction of chemotherapy, the ability to select active treatments before having to administer them to patients has been the holy grail of oncologic investigation. During the 1950s and 60s, chemotherapy treatments were punishing. Drugs like nitrogen mustard were administered without the benefit of modern anti-emetics and cancer patients suffered every minute. The nausea was extreme, the bone marrow suppression dramatic and the benefits – marginal at best. With the introduction of cisplatin in the pre Zofran/Kytril era, patients experienced a heretofore unimaginable level of nausea and vomiting. Each passing day medical oncologists wondered why they couldn’t use the same techniques that had proven so useful in microbiology (bacterial culture and sensitivity) to select chemotherapy.

And then it happened. In June of 1978, the New England Journal of Medicine (NEJM) published a study involving a small series of patients whose tumors responded to drugs selected by in vitro (laboratory) chemosensitivity. Eureka! Everyone, everywhere wanted to do clonogenic (human tumor stem cell) assays. Scientists traveled to Tucson to learn the methodology. Commercial laboratories were established to offer the service. It was a new era of cancer medicine. Finally, cancer patients could benefit from effective drugs and avoid ineffective ones. At least, it appeared that way in 1978.

Five years later, the NEJM published an update of more than 8,000 patients who had been studied by clonogenic assay. It seemed that with all the hype and hoopla, this teeny, tiny little detail had been overlooked: the clonogenic assay didn’t work. Like air rushing out of a punctured tire, the field collapsed on itself. No one ever wanted to hear about using human tumor cancer cells to predict response to chemotherapy – not ever!

In the midst of this, a seminal paper was published in the British Journal of Cancer in 1972 that described the phenomenon of apoptosis, a form of programmed cell death.  All at once it became evident exactly why the clonogenic assay didn’t work. By re-examining the basic tenets of cancer chemosensitivity testing, a new generation of assays were developed that used drug induced programmed cell death, not growth inhibition. Cancer didn’t grow too much, it died too little. And these tests proved it.

Immediately, the predictive validity improved. Every time the assays were put to the test, they met the challenge. From leukemia and lymphoma to lung, breast, ovarian, and even melanoma, cancer patients who received drugs found active in the test tube did better than cancer patients who received drugs that looked inactive. Eureka! A new era of cancer therapy was born. Or so it seemed.

I was one of those naive investigators who believed that because these tests worked, they would be embraced by the oncology community. I presented my first observations in the 1980s, using the test to develop a curative therapy for a rare form of leukemia. Then we used this laboratory platform to pioneer drug combinations that, today, are used all over the world. We brought the work to the national cooperative groups, conducted studies and published the observations. It didn’t matter. Because the clonogenic assay hadn’t worked, regardless of its evident deficiencies, no one wanted to talk about the field ever again.

In 1600, Giordano Bruno was burned at the stake for suggesting that the universe contained other planetary systems. In 1634, Galileo Galilei was excommunicated for promoting the heliocentric model of the solar system. Centuries later, Ignaz Semmelweis, MD, was committed to an insane asylum after he (correctly) suggested that puerperal sepsis was caused by bacterial contamination. A century later, the discoverers of helicobacter (the cause of peptic ulcer disease) were forced to suffer the slings and arrows of ignoble academic fortune until they were vindicated through the efforts of a small coterie of enlightened colleagues.

Innovations are not suffered lightly by those who prosper under established norms. To disrupt the standard of care is to invite the wrath of academia. The 2004 Technology Assessment published by Blue Cross/Blue Shield and ASCO in the Journal of Oncology and ASCO’s update seven years later, reflect little more than an established paradigm attempting to escape irrelevance.

Cancer chemosensitivity tests work exactly according to their well-established performance characteristics of sensitivity and specificity. They consistently provide superior response and, in many cases, time to progression and even survival. They can improve outcomes, reduce costs, accelerate research and eliminate futile care. If the academic community is so intent to put these assays to the test, then why have they repeatedly failed to support the innumerable efforts that our colleagues have made over the past two decades to fairly evaluate them in prospective randomized trials? It is time for patients to ask exactly why it is that their physicians do not use them and to demand that these physicians provide data, not hearsay, to support their arguments.

Scientifically-based Functional Profile Under Fire

Winston Churchill once said, “Democracy is the worst form of government, except for all the others that have been tried.” I am reminded of this quote by a “conversation” that recently took place on a cancer patient forum.

A patient wrote that they had requested that tissue be submitted for sensitivity analysis and their physician responded by describing this work as a scam. A scam is defined by the American Heritage Dictionary as slang for a “fraudulent business scheme.”

Continuing Churchill’s thread, we might respond, “that laboratory directed therapies are the worst form of cancer therapy, except for all the others that have been tried.”

Using functional profiling we measure the effect of drugs, radiation, growth factor withdrawal and signal transduction inhibition upon human tumors. Using our extensive database we compare the findings with the results of similar patients – by diagnosis and treatment status – to determine the most active and least toxic drug or combination for each patient.

The test isn’t perfect. Some patient’s cancer cells (about 5 – 7 percent of the time), do not survive the transport and processing, so no assay can be performed at all. Some patients are resistant to all available drugs and combinations. And finally, based on the established performance characteristics of the test, we can only double or in some circumstances triple, the likelihood of a clinical response.  This is all well documented in the peer-reviewed literature.

Despite this, it appears that in the eyes of some beholders these strikingly good results constitute a “scam.” So let us, in the spirit of fairness, and academic discourse examine their results.

First, it must be remembered that today in 2012 only a minority of cancer patients actually show objective response to available cancer therapies. Five-year survivals, the benchmark of success for advanced disease in oncology (those whose disease has spread beyond the primary site), have not changed in more than five decades.

The highly lauded clinical trial process, according to a study from the University of Florida, only provides a better outcome for a new drug over an old one, once for every seven clinical trials conducted

More disturbing, only one out of 14 clinical trials provide a survival advantage of 50 percent or greater for the successful treatment group.

According to a study from Tuft’s University, it takes 11 years and more than $1,000,000,000 dollars for a new drug to receive FDA approval.

And in a study published in the New England Journal of Medicine only 8 percent of drugs that complete Phase I (safe for human use) ever see the light of day for clinical therapy. This is the legacy of NCCN-guided, University-approved, ASCO-authorized clinical therapeutics programs to date.

As a practicing medical oncologist I am only too familiar with the failings of our modern clinical trial system. Having witnessed the good outcomes of our own patients on assay-directed protocols whose benefits derive from the intelligent use of objective laboratory data for the selection of chemotherapy drugs, I for one will NEVER return to business-as-usual oncology, regardless of what moniker the naysayers might choose to attach to this approach.

The Unfulfilled Promise of Genomic Analysis

In the March 8 issue of the New England Journal of Medicine, investigators from London, England, reported disturbing news regarding the predictive validity and clinical applicability of human tumor genomic analysis for the selection of chemotherapeutic agents.

As part of an ongoing clinical trial in patients with metastatic renal cell carcinoma (the E-PREDICT) these investigators had the opportunity to conduct biopsies upon metastatic lesions and then compare their genomic profiles with those of the primary tumors. Their findings are highly instructive, though not terribly unexpected. Using exon-capture they identified numerous mutations, insertions and deletions. Sanger sequencing was used to validate mutations. When they compared biopsy specimens taken from the kidney they found significant heterogeneity from one region to the next.

Similar degrees of heterogeneity were observed when they compared these primary lesions with the metastatic sites of spread. The investigators inferred a branched evolution where tumors evolved into clones, some spreading to distant sites, while others manifested different features within the primary tumor themselves. Interestingly, when primary sites were matched with metastases that arose from that site, there was greater consanguinity between the primary and met than between one primary site and another primary site in the same kidney. Another way of looking at this is that your grandchildren look more like you, than your neighbor.

Tracking additional mutations, these investigators found unexpected changes that involved histone methyltransferase, histone d-methyltransferase and the phosphatase and tensin homolog (PTEN). These findings were perhaps among the most interesting of the entire paper for they support the principal of phenotypic convergence, whereby similar genomic changes arise by Darwinian selection. This, despite the observed phenotypes arising from precursors with different genomic heritages. This fundamental observation suggests that cancers do not arise from genetic mutation, but instead select advantageous mutations for their survival and success.

The accompanying editorial by Dr. Dan Longo makes several points worth noting.  First he states that “DNA is not the whole story.” This should be familiar to those who follow my blogs, as I have said the same on many occasions.  In his discussion, Dr Longo then references Albert Einstein, who said “Things should be made as simple as possible, but not simpler.” Touché.

I appreciate and applaud Dr. Longo’s comments for they echo our sentiments completely. This article is only the most recent example of a growing litany of observations that call into question molecular biologist’s preternatural fixation on genomic analyses. Human biology is not simple and malignantly transformed cells more complex still. Investigators who insist upon using genomic platforms to force disorderly cells into artificially ordered sub-categories, have once again been forced to admit that these oversimplifications fail to provide the needed insights for the advancement of cancer therapeutics. Those laboratories and corporations that offer “high price” genomic analyses for the selection of chemotherapy drugs should read this and related articles carefully as these reports portend a troubling future for their current business model.

Best Chance for Colon Cancer Survival – Don’t Let It Start

Two papers in the February 23, 2012, New England Journal of Medicine reported important findings in the fight against colon cancer. The first paper (Zuber, AG et al; Colonoscopic Polypectomy and Long-Term Prevention of Colorectal Cancer Deaths) conducted by American investigators establishes the benefit of polyp removal in the prevention of death from colorectal cancer. The study conducted upon 2,602 patients who had adenomas removed reveals a 53 percent reduction in mortality from colon cancer compared with the expected death rate from the disease in this population.

To put this into perspective – virtually no intervention in the advanced disease setting provides a survival advantage. The best we can usually do once the disease is established is an improvement in time to progression. When we do observe a true survival advantage it is usually in the range of a few percentage points and never of this magnitude. How might we explain this astonishingly positive result?

One way to view this finding is to reexamine the biology of cancer. One of the leading experts in the field, Bert Vogelstein, MD, from Johns Hopkins, explained colon carcinogenesis as a pattern of gene perturbations starting at atypia, progressing to carcinoma in situ and ending with invasive, metastatic disease. According to Dr. Vogelstein, the average colon cancer found in a patient at the time of colonoscopy has been present in that person’s colon for 27 years. From there it is only a hop, skip and a jump from one-centimeter adenomatous polyp to metastatic (lethal) disease, all playing out over the last three years in the natural history of the disease. Thus, cancer truly is a disease that doesn’t grow too much, but dies too little and interrupting this process while it is still slumbering can, it would seem, lead to cures.

What I find surprising is the success of the strategy. Since it is now well established that cancer can metastasize when it has achieved the rather diminutive proportions of 0.125 cubic centimeters or less and the average polyp can only be detected at one or more cubic centimeters, it is our good fortune that so many cancers chose not to (or could not) metastasize prior to detection. Reading between the lines, those 12 patients who died of colon cancer as opposed to the expected 25.4 are presumably those with early metastasizing disease. The next frontier will be the detection of these cancers when they are teenagers and not 20-somethings. It may be that proteomic analyses will provide an avenue for earlier detection in the future.

The second article is a European study (Quintero, E et al; Colonoscopy versus Fecal Immunohistochemical Testing in Colorectal-Cancer Screening) that compared colonoscopy with fecal blood testing in a large cohort of patients. While the rates of detection for colorectal cancer were similar, the rates of detecting both advanced and early adenomas, favored colonoscopy (p < .001). This study represents an interesting adjunct to the American study described above. Specifically, if the early detection (and removal) of adenomas can confer a survival advantage then it could be argued that colonoscopy by its virtue of it’s higher detection rate of these precancerous adenomas, is the preferred “screening” modality. With over 50,000 deaths attributed to colorectal cancer in the U.S. each year, the public health benefit of colonoscopies becomes an intersecting point of discussion. Until now, fecal occult blood testing yearly or sigmoidoscopies every several years has been considered equivalent to colonoscopies every 10 years starting at age 50. Do we need to move colonoscopies to the front of the line?

What is most interesting about both these reports is the low-tech nature of the study modalities – and the astonishing efficacy of their application. Colonoscopies have been conducted for decades. They are comparatively simple, do not require affymetrix chips, and yet provide demonstrable benefit that appears to exceed anything offered, to date, by the “genomic revolution.” Perhaps we should all keep an open mind about other comparatively low-tech methodologies that can provide survival advantages.

If It is Too Good to Be True . . .

The February 12, 2012, CBS 60 Minutes covered a story that has sparked a great deal of interest among cancer patients and medical professionals. The topic was an investigator named Anil Poti who, while working at Duke University developed a laboratory platform for the study of human lung cancer.

Using molecular profiling, Dr. Poti and his collaborators, reported their capacity to distinguish responding and non-responding cancer patients, providing survival curves that were nothing short of astonishing. I recall attending the original lectures given by these investigators at the American Association of Cancer Research meeting several years ago.

As an investigator in the field of drug response prediction, working in lung cancer I had a particular interest in their platform and I was extremely impressed by the outcomes they reported. At the time, I wondered how the static measurement of gene profiles could possibly characterize the nuances of human biology, to encompass the epigenetic, siRNA, pseudogene, non-coding DNA and protein kinetics that ultimately characterize the human phenotype. Nonetheless, with such compelling data I was prepared to be convinced.

That is until a relatively unheralded report in the Cancer Letter raised concerns by several biostatisticians regarding the reproducibility of Dr. Poti’s findings. And then more comments were followed by a full NIH investigation. A panel of biostatisticians was convened and a formal report provided the explanation for Dr. Poti’s excellent results.

They had been invented. The clinical outcomes were not real results. The findings had been retrofitted to match the patient responses and this was the subject of the 60 Minutes report.

What the 60 Minutes report did not address however, was the real problem. That being the inability of contemporary genetic profiling to truly define human biology. For all the reasons enumerated above, siRNA, non-coding DNA, etc., the simple measurement of gene sequences cannot accurately predict biological behavior. This is what the 60 Minutes reporters and the physicians they interviewed, never discussed. The problem at hand is not an errant investigator but an errant scientific community. Our love affair with the gene that began in 1953 (Watson and Crick) has now been confronted by a most heartbreaking example of infidelity (pun intended).

Genes do not make us what we are; they only (sometimes) permit us to become what we are, with the vagaries of transcription and translation lying between.

This leads us to the reasons I find this so critically important:

1. I cannot stress strongly enough that this is NOT what I do. Genomic analysis (their work) and functional analysis (our work) are distinctly different platforms.
2. I strenuously resist any attempt on the part of anyone to tar me or my work with this brush.
3. It is precisely because genomic analysis cannot accurately predict cancer patient outcomes, that these investigators found it necessary to invent their data.
4. Despite this, functional analyses can and do provide these types of predictive results in lung cancers and other diseases as we have reported in numerous publications.
5. Finally, while imitation is the sincerest form of flattery, this is one instance in which I would prefer to decline the compliment.

A Day at CHORI (Children’s Hospital of Oakland Research Institute)

As a hematology fellow at the Scripps Clinic in the 1980s, my friend and colleague Sheldon Hendler, MD, PHD, recommended that I read an article in Science magazine. The manuscript entitled “Cancer and Diet,” by Bruce Ames, PhD, described the mutagens and carcinogens to which we are exposed on a daily basis that are found in a normal diet. His paper then examined the defenses that we have developed as a species.

Dr. Ames has distinguished himself as a pioneer in the study of aging, degenerative disease and cancer and I have read many of his papers since then. You can imagine my delight when I received a phone call some months ago and found that my interlocutor was none other than Bruce Ames, inviting me to speak at his research institute.

On Tuesday, January 31, I traveled to Oakland to present a symposium. Dr. Ames arranged for me to meet many of his colleagues. The topics ranged from neuraminic acid residues expressed as neoantigens on dividing cancer cells, to antifungal agents as anti-cancer drugs. One discussion of particular interest surrounded sphingomyelin metabolism as an important mediator of tumor cell progression. A subject about which I knew little prior to this discussion but will certainly now examine with interest.

It is my hope that I might forge collaborations with some of these investigators. But, there is little that could have prepared me for the pleasure I experienced when sitting across the table from Dr. Ames, while sipping a freshly brewed espresso (deftly prepared by Dr. Ames himself), while we discussed Bruce’s six decades of extraordinary discoveries. Everywhere I looked was an award or a textbook that he had authored. Despite his many accomplishments he was humble, engaging and very witty.

My symposium that afternoon introduced the attendees to human tumor primary culture studies as predictors of response to cancer therapy. I then moved through the accumulated data supporting the clinical outcomes and finally examined our developmental work, finishing with our published collaboration with investigators at NYU and Cornell on the study of a novel class of Wnt inhibitors. Lively discussion ensued.

Among the attendees was Bengt Mannervik, who asked several good questions. I note his presence for he is one of the leading experts in the field of glutathione metabolism and a scientist who I had met several times before. As one of the fathers of glutathione s-transferase chemistry, Bengt’s work had influenced my earlier studies. It was an unexpected honor to have him in the audience, as a visiting professor on sabbatical from Uppsala.

As I have noted before, the reception from the scientists in these fora improves as they examine the data on its own merit, unaffected by the clinical dogma and politicking that contaminates so much discourse in medical oncology today. There was no agenda, just scientific interest and open discussion. It was a refreshing departure and a welcome opportunity to interact with open-minded investigators.

In the audience was Dr. Ames’ wife, Giovanna, a former professor of biochemistry at Berkeley, and a scientist whose work included the earliest discovery of the ABC transporters, now recognized as the basis for the human p-glycoprotein drug resistance mechanisms. At the end of the lecture, Giovanna Ames, impressed by the data, raised her hand and asked, “If what you need is a small portion of each patient’s tumor to conduct these studies, what do we have to do to be sure that every doctor sends you a piece of tumor?” While I’m not sure I that have the answer to her question, I am very sure that I like the way she thinks.

What Exactly are the Targets of Targeted Therapy?

The term “targeted therapy” has entered common parlance. Like personalized medicine, targeted therapy is a generic description of drugs and combinations that inhibit specific cancer-related pathways. I am impressed by how quickly esoteric phenomena like the downstream signal in the insulin factor pathway have entered the lexicon of medical oncologists. With the advent of temsirolimus and everolimus, both rapamycin derivatives that target mTOR, we now have at our disposal agents that are every bit a part of the therapy repertoire. Unlike erlotinib that targets a specific tyrosine kinase, mTOR is a complex and multifaceted target.

There are actually two separate forms of mTOR, TORC1 and TORC2, and they sit at a critical point in cellular determination. Stimulated by the insulin growth pathway, cells must decide whether they will grow in size or divide. The mTOR proteins participate in this process by regulating protein synthesis and glucose uptake among other functions. In turn, the mTOR pathway is regulated by numerous other factors like AMP kinase and AKT. The current crop of mTOR inhibitors all target TORC1.

New classes of compounds are being developed that inhibit both TORC1 and TORC2. More interesting are the compounds that influence upstream signaling, including phosphoinositol kinase (PI3K) and AKT. What we are coming to learn, however, is that these are not targets but collections of targets. Indeed, the PI3K inhibitors themselves have influence on one, two or all of the distinct classes of phosphoinositol kinases.

Most of the studies to date have used compounds that affect all the classes equally (pan-inhibitors). Pharmaceutical companies are now developing highly selective inhibitors of this fundamental pathway. In addition, duel inhibitors that target both PI3K and mTOR are in clinical trials. What we are coming to realize is the complexity of these pathways. What may prove more vexing still is their redundancy. One well-established by-product of successful inhibition of mTOR (principally TORC1) is the upstream activity of AKT via a feedback loop. This has the undesirable affect of redoubling mTOR stimulation through the very pharmacological manipulation that was designed to inhibit it. Again, an unintended consequence of a well laid plan.

To unravel the complexities and redundancies of these processes, we have utilized the primary culture platform. It enables us to examine the end result of signal inhibition and dissect disease specific profiles. Using this approach we can partner with collaborators to define the specific operative pathways in each disease entity.

Biological complexity is the hallmark of life. We ignore it at our peril.

The TEDx Experience

On Saturday July 16, I had the opportunity to present at the TEDxSoCal conference held here in Long Beach. The overall theme for this event was “thriving,” and appropriately, I presented in the afternoon session called, “well-being.” My lecture was entitled “The Future of Cancer Research Lies Behind Us.”

I chose this topic in light of the growing recognition that genomic analyses are not providing the therapeutic insights that our patients so desperately need. As I have written before in this blog, the Duke University lung cancer gene program, which has received much attention recently, is emblematic of the hubris associated with contemporary genomic analytic platforms.

I reviewed the contemporary experience in clinical trials, examined the potential pitfalls of gene-based analysis, and described the brilliant work conducted by biochemists and cell biologists, like Hans Krebs and Otto Warburg, who published their seminal observations decades before the discovery of the double helix structure of DNA.

I described insights gained using our ex-vivo analytic platform, that lead to treatments used today around the world, all of which were initially discovered using cell-based studies. More interesting still will be the opportunity to use these platforms to explore the next generation of cancer therapies – those treatments that influence the cell at its most fundamental level – its metabolism.

Many attendees stopped me after my lecture to thank and congratulate me for my presentation. Fearing that my topic might have been too esoteric, I was delighted by the reception and more convinced than ever that there are many enlightened individuals who thirst for new approaches to cancer treatment. It is these people who will forge the next generation of therapy.

So What Happened to the PARP Inhibitors in Breast Cancer Anyway? ASCO 2011

Many of you may recall that we described our studies with the small molecules BSI201 (iniparib) and AZD2281 (olaparib) (Nagourney, et al. ASCO 2011). Based upon the exciting Phase II data reported by Dr. Joyce O’Shaughnessy, first at the ASCO meeting, then in the NEJM, describing the remarkable efficacy of BSI201 (iniparib) combined with carboplatin and gemcitabine in triple negative breast cancer (TNBC), we initiated a study of both iniparib and olaparib in human breast cancer specimens. Our results were reported at the American Society of Clinical Oncology meeting.

Despite the enthusiasm that surrounded Dr. O’Shaughnessy’s initial observations, the confirmatory clinical trial using iniparib combined with carboplatin and gemcitabine, then compared with carboplatin and gemcitabine did not achieve statistical significance. That is, the trial was negative and the combo of inabirib with carboplatin plus gemcitabine was not proven superior.

So, what happened? Quite a few things.

It turned out that BSI201, a member of the benzamine chemical family, at physiological concentrations achievable in humans is not a PARP inhibitor. This, in retrospect, should have been obvious because a full-dose PARP inhibitor, plus a potent combination of carboplatin plus gemcitabine would not likely be tolerable if PARP inhibition were achieved.

Second, the patients receiving the drug are probably not a homogeneous population. That is, some TNBC patients may be similar to the BRCA patients, while others may not have the DNA repair deficiencies associated with PARP inhibitor response.

Finally, it was our group that originally reported the carboplatin plus gemcitabine combination in breast cancer, as a split-dose doublet in 2008 (Nagourney, Clin Breast Cancer Research, 2008). We observed, in that original clinical trial, that even a lower starting dose of gemcitabine (i.e. 800mg/ml2 vs. the O’Shaughnessy 1000 mg/m2) resulted in significant toxicity and in our concluding comments in that paper, we suggested 600mg/ml2. At 1000 mg/m2, Dr. O’Shaughnessy’s trial nearly doubled our recommended dose in this patient population.

While our abstract did not receive the fanfare of the clinical trial, it was, in fact, remarkably prescient. We, like other investigators, entered into our original studies of these molecules believing iniparib to be a PARP inhibitor. To our surprise, and, in retrospect, to our credit, a direct comparison of olaparib (AZD2281) to inapaprib (BSI201) revealed no correlation. We described this in our abstract, “Of interest, BSI201 & AZD2281 activity did not correlate in parallel analyses (R = 0.07, P > 0.5).”  Thus, our human tumor primary culture analysis scooped the ASCO investigators. Unfortunately, it appears they weren’t listening.

So, what have we learned? First, we’ve learned that iniparib is not a true PARP inhibitor.

Second, we learned that the combination of platins plus gemcitabine in breast cancer is synergistic, highly active and can be toxic (particularly at the doses chosen for this trial).

Finally, we learned that TNBC, indeed all breast cancers, even more to the point, all cancers in general, are heterogeneous. That is precisely why the use of human tumor primary culture analyses are so instructive and should be incorporated into clinical trials for these and other targeted agents.

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.