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Surgical Oncology

The Surgical Oncology Research Laboratories on Jackson 9 have been newly renovated and now form the core of a Molecular Oncology laboratory in conjunction with the Division of Medical Oncology in the MGH Cancer Center. The laboratories are funded by the National Cancer Institute, American Cancer Society, private endowments and through biotechnology collaborations. The Surgical Oncology Research Laboratories have been extensively involved in training and education of both surgery residents and post-doctoral fellows. Residents and fellows have been successful in competition for national scholarships and research awards. Residents and fellows are encouraged to present their research at national meetings and publish their data in high impact journals. The laboratory investigators collaborate closely with investigators in the Harvard Medical School, MGH Cancer Center, Dana-Farber Cancer Institute, and the Dana-Farber/Harvard Cancer Center. Trainees learn advanced techniques, experimental design, data analysis, biostatistics, problem solving, manuscript preparation, and grant application preparation. Trainees gain broad experience in molecular biology, cell biology, gene cloning and characterization, cell culture, protein purification and assays, molecular virology, animal models, and clinical trial design. A list of former trainees and their publications is available upon request.

Principal Investigators:
James C. Cusack, M.D.
James W. Rocco, M.D., Ph.D.
Kevin Hughes, M.D.
Kenneth K. Tanabe, M.D.
James Michaelson, Ph.D.

Current research projects include the following:

Genetic Engineering, Characterization, and Animal Model Testing of Herpes Simplex Viral Mutants for Treatment of Liver Metastases:

Principal Investigator:
Kenneth K. Tanabe, M.D.
Consultant:
David Knipe, Ph.D. Harvard Medical School
Group Members:
James Donahue, M.D.
Hideki Kasuya, M.D.
Soundararajalu Chandrasekhar, Ph.D.
Hiroshi Kawasaki, M.D., Ph.D.

Viruses used for gene therapy have generally been genetically engineered to prevent viral replication and to deliver transgenes to achieve a therapeutic benefit. However, viruses engineered to remain replication-competent may be exploited for cancer gene therapy because replication within cancer cells results in oncolysis. We have examined a strategy that targets herpes simplex virus 1 (HSV-1) replication specifically to diffuse liver metastases after delivery into the portal vein. In initial proof-of-principle experiments, we used the HSV-1 mutant hrR3, which has an E. coli b-galactosidase gene inserted into the ICP6 locus. This inactivates the HSV-1 viral ribonucleotide reductase gene and renders it replication-defective. However, cellular ribonucleotide reductase can complement the absence of its viral counterpart to allow hrR3 productive replication in infected cells. We have demonstrated that cellular ribonucleotide reductase levels are extremely low in normal liver and hepatocytes and high in liver metastases. Accordingly, titers of hrR3 infectious virions recovered after infection of colon carcinoma cells are two to three log orders higher than those recovered after infection of hepatocytes. In contrast, wild-type HSV-1 replicates equally well in hepatocytes and colon carcinoma cells. hrR3 selectively replicates in liver metastases and not in normal liver, unless ribonucleotide reductase levels are experimentally raised in the normal liver. This replication is cytolytic; hrR3 destroys colon carcinoma cell lines infected in vitro with multiplicities of infection as low as one viral unit per ten tumor cells. When mice bearing diffuse liver metastases are treated with a single intra-portal administration of hrR3, the anti-tumor efficacy is striking. Histochemical staining of liver sections for b-galactosidase expression reveals significant viral replication in liver metastases and no viral replication in normal liver tissues. hrR3-mediated tumor inhibition is equivalent in immunocompetent and nude mice, suggesting that the host immune response is not the primary mechanism of tumor destruction. Infection of diffuse liver metastases with a replication-incompetent HSV-1 mutants d120 (ICP4-defective) and d27 (ICP27-defective) does not produce any measurable anti-neoplastic effects. We have also demonstrated that administration of hrR3 produces significant anti-tumor activity even in mice that have been previously vaccinated against HSV-1. These results indicate that replication-competent HSV1 mutants hold significant promise as cancer therapeutic agents.

We have constructed several replication-conditional HSV-1 mutants for both gene therapy and oncolysis of liver tumors. Several of these mutants are based on the design of hrR3, such that they are defective in viral ribonucleotide reductase expression which results in replication preferentially in liver tumors rather than normal liver. Other HSV-1 mutants possess engineered alterations in the g134.5 gene, which also results in HSV-1 replication preferentially in tumor rather than normal cells. We are also characterizing the anti-neoplastic efficacy of several mutants that are engineered to express therapeutic transgenes, such as the yeast cytosine deaminase gene or the rat CYP2B1 gene.

Tumor Associated Promoters for Regulation of Viral Replication and Oncolysis:

Principal Investigator:
Kenneth K. Tanabe, M.D.
Group Members:
Soundararajalu Chandrasekhar, Ph.D.
James M. Donahue, M.D.
John T. Mullen, M.D.

Although differential expression of ribonucleotide reductase between liver metastases and normal liver serves to drive differential replication of HSV-1 mutants that are defective in viral ribonucleotide reductase, we are testing the hypothesis that tumor associated promoters (e.g. CEA, muc1) can regulate immediate-viral gene expression in HSV-1 to limit viral replication (and oncolysis) specifically to tumor cells. Herpes simplex virus and adenovirus mutants whose life cycle is regulated by a tumor associated promoter have been constructed. The molecular regulation of their gene expression and replication, as well as their anti-neoplastic efficacy are currently under examination in vitro and in vivo.

Viral Oncolysis and Antiangiogenesis:

Principal Investigator:
Kenneth K. Tanabe, M.D.
Group Members:
James M. Donahue, M.D.
John T. Mullen, M.D.

Angiogenesis is required for tumor formation. Several studies have demonstrated that tumor angiogenesis is regulated by a balance between proangiogenesis and antiangiogenesis factors and that this balance varies in different organ environments. To investigate if expression of an angiogenesis inhibitor by cancer cells could alter this balance and prevent tumor formation in different organ environments, we engineered stable transfectants from RenCa mouse renal carcinoma cells and SW620 human colon carcinoma cells to constitutively secrete a mouse endostatin protein with c-myc and polyhistidine (His) tags. Conditioned medium from endostatin-transfected cells inhibit human umbilical vein endothelial cell (HUVEC) proliferation compared to conditioned medium from control cells. Following inoculation into mice, flank tumors from endostatin-transfected cells are growth-inhibited compared to flank tumors from control cells after 3 weeks. Inoculation of a cell mixture containing 25% endostatin-transfected cells and 75% control cells results in inhibition of flank tumor formation as effective as following inoculation of 100% endostatin-transfected cells. Formation of lung metastases by RenCa endostatin-transfected cells and formation of liver metastases by SW620 endostatin-transfected cells are dramatically inhibited compared to formation of metastases by control cells. These findings demonstrate that endostatin can inhibit tumor formation in different organ environments and that gene delivery of endostatin into even a minority of tumor cells may be an effective strategy to prevent progression of micrometastases to macroscopic disease. To develop oncolytic vectors with multiple mechanisms of anti-neoplastic activity, we have constructed both HSV and adenoviral vectors to express mouse endostatin. The interaction between endostatin expression and viral oncolysis is currently under examination.

Preclinical Toxicology in Non-Human Primates of Herpes Simplex Viral Vectors for Liver Tumor Therapy:

Principal Investigator:
Kenneth K. Tanabe, M.D.
Co-Investigator:
E. Antonio Chiocca, M.D., Ph.D.

Two HSV-1 mutants for treatment of liver tumors are currently undergoing preclinical toxicology testing for data required to file an IND application with the FDA in anticipation of clinical trials in patients with liver tumors in 2003. The most suitable model for these toxicologies studies in non-human primates is Aotus monkeys (owl monkeys). These are New World monkeys from Ecuador and are exquisitely sensitive to herpetic infection and encephalitis. The animals are housed in Harvard’s New England Regional Primate Research Center. Studies of toxicity, gene expression, and viral biodistribution using two different HSV vectors are currently being conducted.

Clinical Investigation in Surgical Oncology - Radiofrequency Ablation of Liver Tumors:

Principal Investigator:
Kenneth K. Tanabe, M.D.

We have investigated a method to ablate liver tumors using radiofrequency (RF)wave energy. This approach involves placement of a needle percutaneously under image guidance into a liver tumor. The needle is then connected to a 500 kHz monopolar radiofrequency generator. RF energy is absorbed by tumor cells, which produces local ion agitation and heat. We have demonstrated that the extent of tumor hyperthermia is proportional to the amount of energy delivered and inversely proportional to liver blood flow. We have also demonstrated that RF energy deposition is limited by increased tissue impedance created by tissue vaporization and charring. We have successfully reduced impedance by cooling the tip of the electrode by perfusing it with iced saline through a closed circuit. Preclinical models have been used to test other refinements in the technique, and these refinements are then examined in patients who participate in approved clinical trials. We are presently capable of ablating liver tumors measuring up to 4 cm in diameter using this minimally invasive approach, and have treated approximately 35 patients over 2 years. Our current investigations center around investigating the anti-tumor effects of mild (42 C) hyperthermia for prolonged periods of time combined with intraarterial administration of Melphalan.

Role of NF-*B in inhibition of chemotherapy-induced apoptosis:

Principal Investigator:
James C. Cusack, Jr., M.D.
Group Members:
Rong Liu, M.D.
John Flannery, M.D.
Lijun Xia, M.D.

The genotoxic effect of conventional anticancer therapy involving many chemotherapy agents and gamma irradiation results in the induction of apoptosis in cancer cells. The ability to inhibit apoptosis appears to be a principal mechanism by which resistant cancer cells are protected from chemotherapy and radiation. Cellular mechanisms that protect cancer cells against apoptosis include lack of a functional response mechanism to apoptotic stimuli (e.g. mutated or deleted p53 tumor suppressor gene), presence of an inhibitor to apoptosis (e.g. Bcl-2, IAP) and the expression of the multidrug resistance gene (MDR). Recently, we found that the inducible activation of the nuclear transcription factor NF-kB inhibits the apoptotic response to chemotherapy and irradiation. NF-kB, a key transcription factor involved with immune and inflammatory responses as well as cell growth, is regulated primarily through interactions with an inhibitor protein known as IkB. Stimuli that activate NF-kB typically induce the recently identified IkB kinase (IKK) to phosphorylate IkB on N-terminal serines that leads to ubiquitination and subsequent degradation of the inhibitor by the proteasome. Following IkB degradation, NF-kB translocates to the nucleus where it regulates genes involved in a variety of processes, including the suppression of apoptosis.

We recently described a gene therapy approach that uses a recombinant adenovirus to transfer a modified form of IkBa. In these experiments, transfer of super-repressor IkBa resulted in significant augmentation of chemosensitivity and enhanced induction of apoptosis in a xenograft tumor model in response to chemotherapy treatment. These findings suggested that NF-kB may represent an important molecular target for the purpose of enhancing the sensitivity of certain cancer cells to apoptotic stimuli. The use of gene therapy to deliver NF-kB inhibitors is relevant to certain cancers but is limited when considering widely-disseminated metastases. An alternative strategy for the inhibition of NF-kB activation is facilitated by inhibition of proteasome function. The inhibition of proteolytic function effectively blocks degradation of cellular proteins that have undergone ubiquitination, such as IkB. In fact, proteasome inhibition is a well-established mechanism to block NF-kB in response to a variety of stimuli. A clear advantage to this therapeutic approach is the clinical availability of a systemically administered small molecule (PS-341, a potent, boronic acid dipeptide that is highly-selective for proteasome inhibition) that can potentially inhibit chemotherapy induced activation of NF-kB and enhance the apoptotic response to chemotherapy. To evaluate this therapeutic approach, we determined whether inactivation of proteasome function would inhibit inducible NF-kB activation, and result in increased levels of apoptosis in response to chemotherapy, and enhanced antitumor effects in vivo. To test our hypothesis we utilized PS-341 to evaluate the effects of proteasome inhibition on 1) chemotherapy-induced NF-kB activation in colorectal cancer cells, 2) the levels of apoptosis following treatment with chemotherapy; and 3) tumor growth in a xenograft model treated with chemotherapy. Phase I clinical trials in which we explore the ability of this approach to augment chemosensitivity in patients with refractory malignancy have been recently completed. Phase II studies are currently underway. Correlative studies will include pharmacokinetic and pharmacogenomic analysis of patient samples. Future laboratory studies will explore the role of newly developed small molecular compounds to selectively inhibit chemotherapy-induced NF-kB activation by targeting the downstream mediators of this response mechanism.

Clinical Investigation: Identification of women at high risk of Hereditary Breast & Ovarian Cancer in Bermuda

Principal Investigator:
Kevin S. Hughes, M.D.
Co-Investigator:
Constance A. Roche, MSN, RN, CS

The Island of Bermuda provides a unique opportunity for studying hereditary breast and ovarian cancer. The island has a population of 60,000 with only 1% immigration and emigration per year. The island is served by a single hospital and has a single tumor registry. Over the course of the last three years, we have collected family histories of patients with breast or ovarian cancer diagnosed since 1980. We are currently in the process of analyzing this data to determine the incidence of hereditary breast and ovarian cancer on the island and to develop ways of identifying and notifying those families who are high risk.

Clinical Investigation: Breast Cancer in African Americans

Principal Investigator:
Kevin S. Hughes, M.D.
Co-Investigator:
Constance A. Roche, MSN, RN, CS

While breast cancer is a less common disease in African Americans in the United States than in Caucasians, the mortality rate is higher in African Americans and cancer tends to occur at a younger age. It has been known for some time that breast cancer in African Americans presents at a higher stage. This has been felt to be due to a lack of access to screening and medical care. Overall, we wish to examine the efficacy of screening in this population and the characteristics of breast cancer. There is the possibility that in addition to underutilization of screening, the breast cancer in African Americans is a more aggressive cancer that may not be as amenable to early detection as Caucasian breast cancer. In order to study this, we will be looking at the records of patients who have had breast cancer and compare African American and Caucasians in terms of stage at diagnosis, biologic characteristics, and mammographic features such as calcifications, breast density, etc. The ultimate goal will be to determine whether there are differences in the efficacy of screening between these two populations, to determine if there are biologic differences between the cancers in these populations, and to develop means to identify these cancers at an earlier stage and potentially increase the cure rate for these cancers.

Clinical investigation: Efficacy of mammography in BRCA-1 and BRCA-2 carriers

Principal Investigator:
Kevin S. Hughes, M.D.
Co-Investigator:
Constance A. Roche, MSN, RN, CS

Mutations in the BRCA-1 and BRCA-2 gene place a woman at high risk for hereditary breast or ovarian cancer, and women who develop breast cancer tend to develop it at a very young age. While we are currently recommending mammography beginning at age 25 in this population, it is not clear that mammography will be effective in identifying cancer at an early treatable stage. The purpose of this study is to determine the characteristics of breast cancer and of breast tissue relative to mammographic identification of cancer in the BRCA-1 and BRCA-2 population. Women who are known BRCA-1 or BRCA-2 carriers or who are at high risk of carrying a mutation, will be identified, and all mammograms from these patients will be collected and digitized. We plan to collect a mammography bank on these patients. We will then use this bank to identify mammographic characteristics of breast cancers in this population, specifically looking at rate of growth, calcifications, DCIS, density relative to surrounding tissue, etc. In addition, we will study the density of the breast tissue by age and look for correlations between mammographic density and risk. We will also look at how mammographic density varies with increasing age, use of tamoxifen, use of hormone replacement therapy, oophorectomy, etc.

Clinical investigation: Identification of women at high risk of breast cancer

Principal Investigator:
Kevin S. Hughes, M.D.
Co-Investigator:
Constance A. Roche, MSN, RN, CS

Women at high risk of hereditary breast and ovarian cancer will benefit from specific management strategies implemented at a young age. Currently we are at a disadvantage in that many women with hereditary breast and ovarian cancer risk are unaware of their status. The purpose of this research will be to develop methods of identifying high risk women either through a web-based family history questionnaire or a scannable hand-completed questionnaire. A prototype of the web site is currently being developed, and the scannable questionnaire is currently being used in mammography. The next steps will be to identify an algorithm to cull out the high risk women from this data, to develop means of contacting these women to notify them of their risk and to develop methods to see these women and care for them once they have been identified.

The Prediction of Cancer Survival

Principal Investigator:
James Michaelson, PhD
Collaborators:
Kenneth K. Tanabe, M.D.
Barbara Smith, M.D.
Melvin Silverstein, M.D.
Kevin Hughes, M.D.

It has long been appreciated that both tumor size and the presence of cancer in the regional lymph nodes are indicators of invasive breast cancer outcome, although it has not been obvious as to how to integrate these two qualities into an overall assessment of prognosis. The unifying concept, which makes these calculations possible, was our finding that the lethal distant spread of cancer cells, which render patients incurable by local treatment, occurs with a definable probability per cell. This lead to a simple expression relating tumor size to survival:
(1)
where F is the fraction of patients surviving, e is the exponential constant, and D is the tumor diameter, and Z and Q are coefficients whose values can be determined from survival data. We have found that this expression accurately predicts the relationship between tumor size and population-wide survival for both breast cancer and melanoma.

Eq. (1) offers the possibility of making improved population-wide estimates of cancer survival from data on tumor size, but among individuals, additional factors may need to be taken into account, especially the presence of cancer in the local lymph nodes. To address this question, we examined Kaplan-Meier survival estimates (15?years), by tumor size and nodal status, for 1352 women with invasive breast cancer seen at the Van Nuys Breast Center before 1991. To isolate the individual contributions to survival of tumor size and nodal status, the data were sorted by both tumor size and survival. These survival values revealed that for women with equivalent nodal status, tumor size was associated with increased lethality, and that for women with tumors of equivalent size, lethality increased with the number of positive nodes. Notably, it was not node positivity, per se, but the number of positive nodes that appeared to indicate extra risk of death. Women that had only a single positive node, or had either one or two positive nodes, had very similar survivals to node negative women with tumors of the same size. However, as the number of positive nodes increased, there was a graded increase in lethality with each unitary increase in the number of positive nodes, such that there was approximately an extra 3.5% chance of death for each positive lymph node. The lethal contribution from the primary site was found to be well fit to eq. (1), with the values of Q and Z estimatable from survival data of women with node negative cancer. The presence of each positive node was found to contribute an extra 3.5% lethality. The overall lethality was found to be the sum of the two components, and this provided the potential to make improved estimates of survival for individual patients.

Future studies will concern the determination of how accurately survival estimates can be made among various populations of patients, estimation of the role of local metastasis in the prediction of melanoma survival, and the extension of these methods to the estimation of lung and colon cancer survival.

Analysis of Cancer Screening

Principal Investigator:
James Michaelson, PhD
Collaborators:
Kenneth K. Tanabe, M.D.
Barbara Smith, M.D.
Michelle Gadd, M.D.
Melvin Silverstein, M.D.
John Wyatt PhD
Daniel Kopans, M.D.
Griffin Webber
Dhruv Puri
Richard Moore

Randomized controlled trials have shown that mammographic screening reduces breast cancers death, but it remains to be established how to use screening to its maximal life-sparing effect, particularly as regards the optimal screening interval. Over the last several years, my colleagues and I have assembled data on the rates of breast cancer growth and spread as well as on the limits of mammographic detectability. We have also developed of a computer simulation model of breast cancer growth and spread, which can calculate such things as the relationship between the screening interval and the fraction of women likely to die from breast cancer. The results of these studies have indicated that great reductions in breast cancer death should be achievable by prompt compliance with the annual screening recommendation, and that even greater numbers of lives might be saved by screening more frequently than once a year. However, from the analysis of the patterns of utilization among women who use screening, it is clear that full utilization of screening in very incomplete, and this provides a barrier to the realization of the full life-sparing potential of screening mammography

Ongoing research concerns the collection of data for improved estimates of breast cancer growth, spread, and operational detectability, as well as data on the operational aspects of breast cancer screening. Particular interest concerns what determines whether a woman will return (or fail to return) for screening, as well as development of tools, such as computer driven automated telephony reminder systems, which can improve the utilization of screening.

Laboratory of Head and Neck Molecular Oncology:

Our laboratory is focused on understanding p16’s biological role during the progression of normal human squamous epithelium to invasive cell carcinoma:

Identification and characterization of a novel p16-dependent checkpoint that is independent of p16’s ability to elicit cell cycle arrest

Principal Investigator:
James W. Rocco M.D., Ph.D.
Group Members:
Takafumi Katayama, M.D.
Jennifer Shin, M.D.

Since its discovery as an inhibitor of cyclin dependent kinase 4 and 6, the tumor suppressor p16 has continued to gain widespread importance in cancer. The high frequency of deletions of p16 in tumor cell lines first suggested an important role for p16 in carcinogenesis. This initial genetic evidence was subsequently strengthened by numerous studies documenting p16 inactivation in kindred with familial melanoma. Moreover, a high frequency of p16 gene alterations were found in primary tumors, while recent studies have identified p16 promoter methylation as a major mechanism of tumor suppressor gene silencing. Additional insight into p16’s role in cancer has come from the genetic analysis of precancerous lesions and various tissue culture models. Micro-satellite analysis of human aerodigestive tumors suggests that p16 loss occurs early and often in the progression to malignancy and likely results in the clonal expansion of premalignant cells.

We believe that p16 has a biological role independent of it’s ability to regulate G1/S cell cycle progression. It is well known that primary epithelial cells will undergo apoptotic cell death (Anoikis) when they lose contact with the extra-cellular matrix, unlike their malignant counterparts. Based on this hypothesis, we have observed that the reintroduction of p16 into squamous cell carcinoma cell lines deprived of contact with the extra-cellular matrix will induce apoptosis. The identification of p16 as a regulator of anoikis (induction of apoptosis after loss of anchorage), is an important first step in linking the observations from molecular genetic analysis (p16 loss occurs early and often) with data from biochemical studies (p16 as a regulator of the G1/S restriction point). Consequently, during malignant progression, early p16 loss allows epithelial cells to become resistant to anoikis. This results in an important growth advantage and allows for the clonal expansion of premalignant cells. This model has particular relevance to cancers of the aerodigestive tract (i.e., lung, esophagus, and head and neck squamous cell cancer), where premalignant fields of clonal cells have been identified and are potentially condemned to a fate of progression to cancer.

Identification of p16 upstream regulators:

Principal Investigator:
James W. Rocco M.D., Ph.D.
Group Members:
William Michaud, Ph.D.

It is now believed that loss of p16 is an early and often critical event in tumor progression. Consequently, p16 is a major tumor suppressor gene whose frequent loss occurs early in many human cancers, and by even the strictest of measures, p16 fulfills all the criteria necessary to be labeled a bona fide tumor suppressive gene. Despite these advances, some confusion still surrounds p16’s exact tumor suppressor role in cancer. At the genetic level, this lack of clarity can be attributed to the disparities between human molecular genetic data and the cancer-prone phenotype of mouse knockout models at the INK4a/ARF locus. From a functional viewpoint, there is a true lack of understanding of the critical signals that regulate p16 function.

Apart from cell cycle control and potentially Anoikis, p16 has also been implicated in another fundamental cellular process, senescence. Primary epithelial cells grown in culture will eventually stop dividing and develop a flattened morphology referred to as senescence. The senescent phenotype is not well understood, although characteristic features are present. One identifying feature is the accumulation of elevated levels of p16 protein coinciding with permanent cell cycle arrest. In addition, cells that have escaped replicative senescence often have lost p16 function.

To identify potential upstream mediators of the p16 transcriptional response, we have developed a senescent model based on the expression of oncogenic ras from an inducible promoter in human oral keratinocytes immortalized with retroviral TERT. In this system, ras expression elicits a strong p16 transcriptional response that is easily assayed using a p16-promoter-luciferase construct. We are now assaying a number of ras mutant constructs to identify the specific pathways involved. Parallel studies based on ras expressed from a replication-defective adenovirus in primary oral keratinocytes are also in progress.

Adenoviral Targeting of Dysregulated E2F Activity with p14ARF

Principal Investigator:
James W. Rocco M.D., Ph.D.
Group Members:
Michael Rho, M.D.
Thongchai Bhongmakapat, M.D.

The alternate protein from the INK4a/ARF locus on chromosome 9, p14ARF is a tumor suppressor gene that regulates the activity of the tumor suppressor p53. We have noted p53-independent effects of p14ARF on adenovirus replication during the construction of a replication defective adenovirus in HEK-293 cells lacking demonstrable p53 function. We have since developed a p14ARF inducible cell line that clearly demonstrates a p53-independent role for p14ARF in regulating viral replication. Based on this observation we are attempting to develop an adenovirus vector that will only replicate in cells that have lost the ability to appropriately suppress E2F transcriptional activity. Since inappropriate and unregulated cell proliferation is a hallmark of all cancer, it is possible to develop a virus that will only replicate in tumor cells with high levels of dysregulated E2F activity.

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