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Chemosensitivity Testing predicts response in pancreatic cancer

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    Chemosensitivity Testing predicts response in pancreatic cancer

    Ex vivo Chemosensitivity testing predicts response towards adjuvant gemcitabine treatment in pancreatic cancer

    C W Michalski (1), M Erkan (1), D Sauliunaite (1), T Giese (2), R Stratmann (3), C Sartori (3), N A Giese (4), H Friess (1) and J Kleeff (1)

    (1) Department of General Surgery, Technische Universität München, Munich, Germany

    (2) Institute of Immunology, University of Heidelberg, Heidelberg, Germany

    (3) DCS Innov. Diagnostik-Systeme GmbH & Co KG, Hamburg, Germany

    (4) Department of General Surgery, University of Heidelberg, Heidelberg, Germany


    Efficacy of chemotherapy for pancreatic cancer may be improved by tailoring it to individual chemosensitivity profiles. Identification of nonresponders before initiation of treatment may help to avoid side effects. In this study, primary pancreatic cancer cells were isolated from 18 patients undergoing pancreaticoduodenectomy for pancreatic cancer. Eight commonly used pancreatic cancer cell lines were used as controls. Ex vivo chemosensitivity for gemcitabine, 5-fluorouracil, mitomycin-C, cisplatinum, oxaliplatinum, pa****axel and a combination of gemcitabine with oxaliplatinum or mitomycin-C was determined using a cellular ATP-based tumour chemosensitivity assay (ATP-TCA). Quantitative real-time–polymerase chain reaction was performed to determine RNA expression levels of genes implicated in chemoresistance. Chemosensitivity towards cytotoxic agents was highly variable in primary pancreatic cancer cells and pancreatic cancer cell lines. ATP-TCA results for gemcitabine correlated to the tissue expression of human equilibrative nucleoside transporter-1 (hENT1). Time to relapse in patients with gemcitabine-sensitive tumours was significantly higher than in patients with chemoresistant pancreatic cancers (P=0.01; 71 vs 269 days). Furthermore, time to relapse in gemcitabine-treated patients was related to hENT1 expression (P=0.0067). Thus, chemosensitivity testing using ATP-TCA in pancreatic cancer is feasible and correlated with time to relapse in gemcitabine-treated patients. This suggests that ATP-TCA testing could be used as a decision-making tool in the adjuvant treatment of pancreatic cancer.

    British Journal of Cancer (2008) 99, 760–767. doi:10.1038/sj.bjc.6604528

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    Gregory D. Pawelski

    3D Culture

    There are any number of variables that affect drugs, including the rate of excretion of the drugs by the kidneys and liver, protein binding and a myriad of other biological factors. In the body, these cells interact with and are supported by other living cells, both malignant and non-malignant cells. That is why cell-death functional profiling assays study cancer cells in microspheroids or microclusters.

    Three-dimensional (3D) tissue culture methods have an invaluable role in tumor biology and provides very important insights into cancer biology. As well as increasing our understanding of homeostasis, cellular differentiation and tissue organization, they provide a well defined environment for cancer research in contrast to the complex host environment of an in vivo model.

    Due to their enormous potential, 3D tumor cultures are currently being exploited by many branches of biomedical science with therapeutically orientated studies becoming the major focus of research. Recent advances in 3D culture and tissue engineering techniques have enabled the development of more complex heterologous 3D tumor models.

    Traditionally, in-vitro (in lab) cell-lines had been studied in two dimensions (2D) which had inherent limitations in applicability to real-life three dimensions (3D) in-vivo (in body) states.

    As the researchers at Johns Hopkins and Washington University at St. Louis have found out, our body is 3D, not 2D in form, undoubtedly, this novel step better replicates that of the human body. Recently, other researchers have pointed to the limitations of 2D cell line study and chemotherapy to more correctly reflect the human body.

    It has been found that newer methods of "cell-death" assays have an overall predictive accuracy of 98.2% concerning treatment response, which compares favorably with older, previously published data ranging from 75% to 92%. (Staib, al. Br J Haematol 128 (6):783-781, March 2005)

    What is the precedent for using these chemosensitivity tests?

    We have tests such as estrogen receptor, progesterone receptor, Her2/neu, BCR-ABL, C-KIT, CD-20, etc., and panels of immunohistochemical stains for subclassifying tumors. All of these tests are used to select chemotherapy in precisely the same manner as cell chemosensitivity tests are used.

    We also have the use of additional medical tests, such as serial CT, MRI, and PET scans, performed for the purpose of monitoring the size of the tumor to determine if it is shrinking or growing with chemotherapy. The purpose of this testing is to determine if chemotherapy with specific drugs should be continued or changed to different drugs. These radiographic tests are also used as an aid in making clinical decisions about the choice of chemotherapy.

    So yes, there is precedent for using chemosensitivity testing.
    Gregory D. Pawelski


      The Concept of Total Cell Kill

      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)
      Gregory D. Pawelski


        Types of Cell Death

        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.

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        Gregory D. Pawelski


          What Can We Offer Patients With Pancreatic Cancer?

          Dr. Robert Nagourney
          Medical and Laboratory Director
          Rational Therapeutics, Inc.
          Long Beach, California

          More than seven years ago, I was asked to see a patient in consultation. This vigorous 54-year-old gentleman had already undergone a Whipple procedure for the treatment of a pancreatic carcinoma. His skilled-surgeon had resected most of the tumor, but could not clear the margins. With each successive attempt, he identified additional tumor. Unable to achieve a complete surgical resection, the patient was closed, recovered and visited me for a discussion of therapeutic options.

          We identified a two-drug combination to be used in conjunction with external beam radiation, a regimen that few — if any — investigators would have suggested. Adjusting the doses to achieve a tolerable schedule, he completed the entire course of therapy with acceptable toxicities. Contrary to his surgeon’s expectations, the patient achieved a complete and durable remission. He returned to his active lifestyle, remarried and became an advocate for the aggressive management of pancreatic cancer.

          Now, over seven years later with a rising CA 19.9, he is identified to have a focus of uptake on PET CT in the body of the pancreas. A surgical exploration to remove the tumor provided adequate tissue for functional profiling analysis. The patient was once again tested against the standard therapies used in this setting. Among the drugs we examined are the EGFR inhibitors, the taxanes, the combination of EGFR inhibitor + gemcitabine and the platinum + 5FU combination. Each one of these would be a reasonable choice. Indeed, FOLFOX, Tarceva + gemcitabine, the GTX regimen and — most recently — Taxol-gemcitabine based combinations, would all be favored choices for medical oncologists in the U.S. today. Yet, this patient was sensitive only to cisplatin + gemcitabine and none of the others.

          Following publications from a group in Scottsdale, Arizona, many oncologists are utilizing Taxol + gemcitabine. There are proponents for Tarceva + gemcitabine, and those who prefer FOLFOX. At least for this patient, none of them would’ve been right. Interestingly, after more than seven years later the patient’s profile reflects the same combination that was used initially. It is interesting to ponder, based on this finding, whether this is a new primary or a sanctuary-site recurrence with so long a disease-free interval to remain sensitive to the platinum-based combination. We now hope to provide him seven more excellent years… at the very least.

          I received a call from another patient for whom I was not the treating oncologist. Originally, she had heard about our work on a radio interview and asked her physician in Ohio to send a sample to our laboratory. The results of her assay concluded that a three-drug combination (cisplatin plus Taxol plus gemcitabine) — not commonly used in a pancreatic cancer — was her best option.

          Unbeknownst to me, after beginning therapy, the patient had a prompt and dramatic response. When the patient recently contacted me, I cursorily examined the chart prior to our discussion and noted the date of June 24. At first, I thought the patient was showing evidence of progression barely two months after our analysis. Recognizing that no test is perfect and that even our best recommendations may not work, I contacted the patient to discuss her case. It was only then that I realized that indeed the assay data was from 14 months ago and that her response had been excellent for more than a year.

          After congratulating the patient on her good outcome and discussing modifications in her therapy (predicated on some x-ray findings of early progression) I asked what her physician’s reaction to the good result had been. The response was muted. Indeed, the physician, having witnessed a rather remarkably good response, only commented that she knew the patient wouldn’t be cured. Recognizing that metastatic pancreatic cancer has an objective response rate measured in single digits and a median overall survival of 4-6 months, I was disappointed to realize that a patient who was well 14 months after diagnosis didn’t seem to impress the treating oncologist.

          We are now engaged in reviewing the patient’s diagnostic studies to determine if the functional profiling findings will provide information to further guide therapy. While I was very realistic with the patient — explaining that there is no certainty that further benefit can be obtained — there are, in fact, a number of drugs that could hold benefit for the patient. These including: Tarceva (erlotinib), Camptosar (irinotecan) and a number of novel combinations. We will be interested to see if further good results can be obtained and are gratified by the patient’s good outcome to date.
          Gregory D. Pawelski


            Anti-angiogenic activity and VEGF pathway inhibition of Tarceva

            The AngioRx Assay has identified potential responders to Avastin, Nexavar, Sutent and other anti-angiogenic drugs and assessed previously unanticipated direct and potentiating anti-angiogenic effects of targeted therapy drugs such as Tarceva and Iressa.

            Tarceva is a tyrosine kinase inhibitor. However, it also has an anti-angiogenic effect on cancer cells. There are a number of classes of drugs that target angiogenesis (VEGF). At the protein level is Avastin. At the tyrosine kinase level is Iressa, Nexavar, Sutent and Tarceva. At the intracellular metabolic pathway mTOR level is Afinitor and Torisel.

            When chemotherapy drugs work, they often cause tumors to shrink a lot, sometimes even making them disappear. But anti-angiogenesis drugs don't seem to work in the same way. In some cases they shrink tumors, but in others they just seem to stop them from growing any larger.

            Newer approaches to treatment that combine anti-angiogenesis drugs with chemotherapy, other targeted drugs, or radiation may work better than using them alone. For instance, early studies that tested the drug Avastin by itself did not find that it helped people with cancer to live longer. But later studies found that when it was combined with chemotherapy to treat certain cancers, it helped people (some subsets of patients) live longer than if they got the chemotherapy alone.

            Doctors aren't sure why this is the case. One theory is based on the fact that chemotherapy drugs may have a hard time getting to cells in the middle of tumors. Tumor blood vessels grow in a short amount of time and in an abnormal environment, so they are not as well-made and stable as normal blood vessels.

            Because of this, they tend to be leaky. This affects how well drugs can reach the inside of the tumor. The theory is that anti-angiogenesis drugs may somehow stabilize these tumor blood vessels for a short period of time, allowing the chemotherapy to reach more tumor cells and be more effective.

            Biomarker for anti-angiogenesis compounds:

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            Gregory D. Pawelski