Chemically induced sarcomas of the mouse. I. Murine leukemia virus-related antigens and alloantigens on cultured fibroblasts and sarcoma cells: description of a unique antigen on BALB/c Meth A sarcoma

AB DeLeo, H Shiku, T Takahashi, M John and LJ Old

As background for a serological definition of the unique antigens of chemically induced sarcomas, we have typed a series of fibroblast and sarcoma cell lines of BALB/c and C57BL/6 origin by cytoxicity and absorption tests for murine leukemia virus (MuLV)-related cell surface antigens and known alloantigens. 7 of the 17 cultured lines expressed the range of cell surface antigens associated with MuLV (GIX, GCSA, gp70, p30), and this was invariably associated with MuLV production. In nonproducer lines of C57BL/6 (but not BALB/c) origin, a MuLV-gp70-like molecule was found on the surface of fibroblasts and sarcoma cells. The alloantigenic phenotype of these MuLV+ and MuLV- cell lines was H-2D+, H-2K+, Thy-1.2+ or -, PC.1+ or -, Lyt-1.2-, Lyt-2.2-, Ia.7-, and TL.2-. A unique antigen was defined on the BALB/c ascites sarcoma Meth A with antisera prepared in BALB/c or (BALB/c X C57BL/6)F1 mice. Tissue culture lines derived from this tumor were MuLV-, which facilitated serological study of the antigen. Absorption analysis indicated that the antigen was restricted to Meth A; it could not be detected in normal or fetal BALB/c tissue MuLV+ or MuLV- fibroblast lines, 12

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Scott Gottlieb, New York

The US Food and Drug Administration issued a new warning on the drug tamoxifen, advising doctors that it may cause an aggressive cancer of the uterus.

The labelling of tamoxifen will be changed to add a “black box” warning about the newly identified risk of uterine sarcoma, the drug agency said. Black boxes are used to draw attention to problems that are serious and potentially life threatening.

Letters advising doctors of the new warning were sent out last month, but the information was not posted on the agency’s website until Thursday.

Tamoxifen was already known to increase the risk of another, less dangerous type of uterine cancer, endometrial adenocarcinoma, which is usually detected at an early, curable stage. But the risk of the more dangerous type of cancer had not been recognised previously.

The new warning was directed only at women who have not had breast cancer but are at high risk. The warning does not tell those women to avoid tamoxifen, but it does urge them to talk to their doctors about its benefits and risks. The warning does not apply to women who have already had breast cancer and who take tamoxifen to prevent a recurrence. For those women, the FDA said, the benefits far outweigh its risks.

The warning is also aimed at women who have had a very early form of breast cancer that is still confined to the milk ducts (ductal carcinoma in situ, or DCIS). In those two groups, unlike those who have had invasive breast cancer, it has not been proved that tamoxifen prolongs life, even though it does lower the risk of breast cancer.

Uterine sarcoma is rare, estimated to occur in 0.17 women per 1000 a year who take tamoxifen. In women not taking the drug, there are far fewer casesonly 0.01 to 0.02 cases per 1000 women. Since 1978, when tamoxifen was first marketed in the United States, 159 cases of uterine sarcoma worldwide have been reported in women taking the drug.

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NEW YORK JAN 24, 2008 (Reuters Health) - The treatment and survival of adults with soft-tissue sarcoma of the extremities varies widely by race and ethnicity. Compared with whites, the disease-specific mortality rates are significantly higher in blacks and significantly lower in Asians, according to authors of a paper in the March 1st issue of Cancer.

To examine the effects of race and ethnicity on tumor characteristics and outcomes, Dr. Steve R. Martinez, of University of California at Davis Cancer Center in Sacramento, and colleagues used the Surveillance, Epidemiology, and End Results (SEER) database to identify 6406 patients with extremity soft-tissue sarcoma treated between 1988 and 2003. Included were 4636 whites, 773 blacks, 696 Hispanics, and 411 Asians.

“Hispanics tended to be diagnosed with extremity soft-tissue sarcoma at a younger age than their white, black, and Asian counterparts, which may suggest either a biologic predisposition or an environmental contributing factor for the development of these tumors,” the authors suggest.

Hispanics and blacks were less likely to receive radiation therapy than other groups, despite presentation with larger tumors. Hispanics tended to have higher rates of well-differentiated to moderately differentiated tumors, whereas blacks presented with more poorly differentiated or undifferentiated tumors.

“Hispanics, although they are subject to several of the same socioeconomic factors and exhibit several of the same poor tumor prognostic factors and comorbidities as blacks, displayed a disease-specific survival that, although not significantly superior to whites, clearly trended in that direction,” the investigators report.

There is no simple explanation for the racial and ethnic differences observed in treatment outcomes in patients with primary extremity soft-tissue sarcoma, the researchers state. Genetics, sociodemographics, and access to specialty care all likely play a role.

“The identification of disparities represents a unique opportunity to improve care by addressing the issues leading to the disparities,” Dr. Martinez told Reuters Health.

“Extremity soft tissue sarcomas are rare and can be difficult to diagnose,” he added. “If possible, patients should be referred to a nearby Cancer Center or center of excellence where patients can be treated by a multsidisciplinary team of experts.”

“We are going to narrow our focus to identify potential genetic or epigenetic explanations for the differences in survival noted in this study,” Dr. Martinez said. “In other words, are the survival differences between racial/ethnic populations due to the fact that these populations have, on a genetic and epigenetic level, very different diseases?”

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Are We at the End or Just the Beginning of Our Quest?

Sharon W. Weiss

From the Department of Pathology and Laboratory Medicine, EmoryUniversity School of Medicine, Atlanta, Georgia

Exactly 50 years ago this year the seminal paper on alveolar soft part sarcoma (ASPS) appeared in the journal Cancer.¹ Drawing on the archives of Memorial Sloan Kettering Cancer Center, Christopherson et al¹ described 12 patients, mostly young individuals, with a distinctive sarcoma composed of organoid nests of large eosinophilic cells. The loss of central cohesion within the nests created an alveolar pattern that figured prominently into the authors’ choice of names. These rare tumors with a predilection for the deep soft tissues of the extremities are characterized by an indolent, but inexorably fatal, course often spanning decades. A significant number of patients present with metastasis at the time of presentation, denoting early dissemination.² Several years after this publication, the distinctive periodic acid-Schiff (PAS) positive-diastase resistant rhomboid to polygonal crystals (and their precursor granules) which characterize this lesion, were noted.³ Consisting of membrane-bounded parallel arrays of rigid fibers with a diameter of 4 to 6 nm and a periodicity of 10 nm, these structures are a characteristic, if not pathognomonic, feature of this tumor which has fascinated pathologists for decades.⁴ While serving an important diagnostic role in separating this tumor from others which it may resemble (eg, renal cell carcinoma, paraganglioma), these crystalline structures have also tantalized us with the promise that if only we could identify their contents, we might discover the histogenesis of this tumor.

For 50 years we have been on a quest to unravel the contents of these crystals. Like many quests we have made some false starts, taken some unusual turns, and learned some important lessons in the process. The popular use of immunohistochemistry beginning in the late 1970s allowed us for the first time to turn attention to these crystals by applying a host of antibodies to these tumors with variable and sometimes conflicting results.^5-13 The most celebrated of all theories regarding the alveolar soft part sarcoma was that it represented an unusual myogenous tumor closely related to a rhabdomyosarcoma.^9,10 Numerous immunohistochemical studies, however, demonstrated muscle markers in less than half of ASPS.⁵ Actin was identified in about 20 to 30% of cases, whereas desmin, an intermediate filament associated with both smooth or skeletal muscle differentiation was expressed in nearly 40%. Since desmin may be expressed in non-myogenous tumors on an aberrant basis, the ability to identify the more specific nuclear regulatory proteins, associated with skeletal muscle differentiation (ie, myoD1 and myogenin), in tissue sections seemed a more certain and expeditious way to answer this question. However, early reports citing the presence of myoD1 in this tumor were fraught with technical and interpretive problems.⁹ It was not until the study by Wang et al,¹¹ using immunohistochemistry along with Western blot analysis, was it decisively shown that, whereas the tumors often expressed desmin, they did not express skeletal muscle nuclear regulatory proteins and, in all probability, did not represent a skeletal muscle tumor. Attempts to demonstrate catecholamines and related substances have also failed and therefore, effectively excluded the possibility that these tumors were a variant of paraganglioma.¹² Perhaps the most whimsical and short-lived of all theories was the notion that alveolar soft part sarcomas were extra-renal renin-producing tumors (so-called malignant angioreninomas) based on immunoreactivity of the granules to renin antiserum.¹³ Other studies failed to corroborate these findings,⁷ and, by way of passing, hypertension has never been reported as a paraneoplastic symptom in with ASPS. As a result of the myriad of immunohistochemical articles over the years relating to this tumor, hopefully we have learned to be cautious in accepting novel findings particularly when they relate to new antibodies that have not been extensively used or performance tested.

Despite this cautionary note, the paper in this issue by Ladanyi et al¹⁴ seems to represent the long-awaited unveiling of the identity of these granules. It is gratifying that this study also emanates from the institution that 50 years ago began the scientific dialogue about this enigmatic tumor. As is often the case with novel observations, the authors discovered the identity of the granules serendipitously in the course of characterizing a polyclonal antibody to the monocarboxylate transporter 1 (MCT1) in a variety of tissues and tumors. MCT1, one of a family of transporter proteins which catalyzes the rapid transport of monocarboxylates such as lactate across plasma membranes, is located ubiquitously in all tissues but is especially prevalent in cardiac and skeletal muscle and seems to roughly correlate with the abundance of mitochondria.¹⁵ Normally the protein is associated with the rough endoplasmic reticulum and is transported to the plasma membrane in association with its chaperone CD147 where it can be detected as surface staining. Important interactions between CD147 and MCT1 also occur in the plasma membrane. As is elegantly shown in the current article by Ladanyi et al,¹⁴ alveolar soft part sarcomas contain an abundance of MCT1 not only on the surface of the cells but also within the cytoplasm and in the region of the characteristic crystals. Immunolocalization of CD147 was observed in pattern similar to MCT1. The authors have avoided the traps of earlier studies by validating their immunohistochemical findings through Western blot analysis for the protein. Moreover, comparison of serial histochemical and immunohistochemical stains for the crystals confirmed congruence between the PAS positive crystalline material and the immunoreactivity for MCT1. Finally, using ultrastructural immunohistochemistry, MCT1 and CD147 were localized to cytoplasmic crystals, their precursor granules, and mitochondria. Thus substantial evidence is presented that the crystals of ASPS contain crystallized complexes of both proteins. Obviously, their findings do not exclude the possibility that other substances may be present in the granules as well.

To return to our earlier question, however, does the identity of the crystals help us to understand the histogenesis (or line of differentiation) of the tumor? The immediate answer to the question is no. MCT1 is a ubiquitously expressed protein involved in basic metabolic processes in a variety of cells, although its predilection for skeletal and cardiac muscle might make us pause to again reconsider a myogenic tumor. CD147 is a broadly expressed plasma membrane glycoprotein which not only serves as role as a chaperone for MCT1, but also interacts with the latter at the level of the plasma membrane suggesting a regulatory function.^16-18 Another intriguing feature of CD147 is that it is also induces the production of matrix metalloproteinases by fibroblasts¹⁹ and its overexpression by some cancer cells has been associated with increased tumorigenic potential in nude mice.¹⁹ One could, therefore, envision how a tumor with increased expression of CD147 could be associated with early invasion, vascular permeation, and metastasis, as characteristically occurs in ASPS. In fact, ASPS may be the soft tissue sarcoma in which vascular invasion is most prevalent even in early stage disease. Perhaps also, as suggested by the authors, even the characteristic alveolar pattern may be explained by matrix degradation as a result of metalloproteinase production. Although the histogenesis of the ASPS still eludes us, we may have discovered deeper biological truths about the tumor.

Of course the most intriguing question is how the recently described translocation in ASPS relates to the accumulation of crystalline deposits of MCT1 and CD147. One might logically assume that there is a relationship given the fundamental role that fusion genes and their derivative proteins play in soft tissue sarcomas in general. This translocation, resulting in der17t(X;17)(p11.2;q25), fuses the TFE3 transcription factor gene at Xp11 to a novel gene at 17q25 designated APSL.²⁰ Although the translocation in ASPS differs from that in most sarcomas in that it is unbalanced, it shares a common theme in that one partner represents a transcription factor, suggesting transcriptional deregulation as a mechanism for tumorigenesis. The most obvious and intellectually satisfying explanation of the relationship between the translocation and the crystals would be that the promotor of either MCT1 or CD147 is activated by TFE3 resulting in overproduction of the proteins. Although the MCT1 promoter is unknown, the CD147 promoter could potentially be activated by TFE3 given the sequence data. We await further information on this point. There are, of course, other explanations for the accumulation of these protein products, as suggested by the authors, such as impaired trafficking to their target destination.

Thus the paper by Ladanyi et al¹⁴ in this issue of The American Journal of Pathology has revealed (at least partially) the identity of the enigmatic crystals of ASPS and at the same time given us some plausible mechanisms for the way in which the fusion oncogene may relate to the crystals. One thing we have not learned, however, is the histogenesis (or line of differentiation) of ASPS. Implied in this question is our naive belief that all tumors must necessarily relate to some normal tissue type. In many sarcomas this is true. For example, the myxoid liposarcoma recapitulates embryonic fat and its characteristic translocation results in deregulation of normal adipocytic differentiation. Not all sarcomas may follow that paradigm and the ASPS may be the prime example. Some sarcomas may express a phenotype corresponding to no known normal tissue or a “scrambled” phenotype of several tissue types. Hopefully, we will not be disappointed, after learning the contents of the enigmatic crystals, to realize that ASPS is still a lesion of “uncertain histogenesis.” In the next half century, perhaps we should decide that it is time to rephrase the question.

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DURHAM — A quote by Winston Churchill, written at the back of a photo album made by Holly Young, encompasses the life she lived: “Never, never, never, give up.”

Young, a University of New Hampshire volleyball player, lost her three-year battle with cancer on Jan. 24. She was 22.

Tuesday night at Lundholm Gymnasium, 400 friends and family celebrated her life.

“We celebrate a life far too short, but very well lived,” said University chaplain Larry Brickner-Wood.

Young, from Dennisport, Mass., was an accomplished student-athlete at Dennis-Yarmouth Regional High School. She took her volleyball club team to a championship, set state track records, won the Boston Globe All-Scholastic Award, the Principal’s Leadership Award and was a member of the National Honor Society.

In 2004, Young came to UNH to play on the volleyball team as an outside hitter, and with her incredible vertical leap and thunderous kill she was bound to be a star. During her first semester she was diagnosed with Ewing’s Sarcoma, a rare form of bone cancer found primarily in children and adolescents.

“If you filled the (Whittemore Center) 50 times with children and adolescents and then pointed to one of them and said, ‘You have Ewing’s Sarcoma,’ that is the national statistic,” said Holly’s father, Bernard Young, of the probability of being diagnosed.

A table just inside the door to the gym was filled with mementos from Holly’s brief time at UNH, her No. 4 jersey and a pom pom, a photo album from her first semester, and a daring photo of Holly, bald from chemotherapy, elbow resting on her knee with her hand on her head, staring intensely into the camera.

“I wouldn’t have had the courage to do that,” said UNH athletic director Marty Scarano.

Outside the gym a poster with photos taken in 2005 from the same session were displayed in a frame featuring the word LIVESTRONG, written in its signature yellow. In the pictures Young was doing what she loved best, playing a sport and giving voice to a cause.

“In the middle of all that she decided to model,” said Scarano. “She was a really special kid.”

Scarano spoke of Young’s will to embrace life. He said Young taught everyone to be courageous in the face of adversity, to find humor in things that were not humorous and to fight even when the odds are stacked against them.

Young’s coach at UNH, Jill Hirschinger, recalled days when Young would finish a treatment in Boston and race back to campus to visit with her teammates and friends.

“We had team meetings where we would just share stories about Holly,” said Hirschinger.

Young’s teammate and neighbor during her first semester, Jessica Compton, spoke of Young’s inspirational spirit. Compton said Young was never one to talk about having a bad day.

“She did not confine herself to a life of pain and struggle,” she said.

Compton talked about Young’s beauty, her ability to stay courageous and the lessons she left.

“She taught us to care a little more about our hearts,” Compton said.

A video presentation of Young on the volleyball court was accompanied by a radio interview done shortly after she was diagnosed. Following the presentation was a slide show of pictures featuring Young and her life at UNH. Rod Stewart’s “Forever Young” played in the background.

“She never stopped smiling,” said Young’s mother, Carolyn Young. “It fills me with joy knowing such a short life touched so many.”

When it came to speaking about his daughter and her time at UNH, Bernard Young spoke not of what Young gave to everyone around her, but what they gave to her.

“You all left fingerprints on Holly,” he said.

As a tribute to Young’s memory, UNH will plant a tree on April 19 in the tree line walkway outside the field house.

UNH football coach Sean McDonnell paid tribute to the impact Young had on her community.

“She did three things for this community,” he said. “She made it better, stronger and special.”

While her time at UNH was brief, Young touched the hearts of all around her.

“She changed me as a coach, friend and person,” said Hirschinger.

As the celebration of Young’s life came to a close, the crowd smiled and clapped along as the UNH fight song poured out of the speakers.

“We only play it after a win,” said Hirschinger. “It’s what she would have wanted.”

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Muzammel Haque, David A. Davis, Victoria Wang, Isabelle Widmer, and Robert Yarchoan^*

HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-1868

Received 2 January 2003/ Accepted 19 March 2003

Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV), also known as human herpesvirus 8, is an etiologic agent of KS, primary effusion lymphoma (PEL), and multicentric Castleman’s disease. We recently demonstrated that hypoxia can induce lytic replication of KSHV in PEL cell lines. Hypoxia induces the accumulation of hypoxia-inducible factors (HIF), and we hypothesized that the KSHV genome may respond to hypoxia through functional hypoxia response elements (HREs). Here, we demonstrate the presence of at least two promoters within the KSHV genome that are activated by hypoxia or hypoxia mimics. One is in the promoter region of the gene for Rta, the main lytic switch gene, and the other is within the promoter region of ORF34, a lytic gene of unknown function. The ORF34 promoter contains three putative consensus HREs oriented in the direction of the gene. Dissection and site-directed mutagenesis studies confirmed that one of the HREs of the ORF34 promoter is functional. Under conditions of hypoxia, the ORF34 promoter was strongly upregulated by HIF-1 and HIF-2. By contrast, the promoter of the gene for Rta appeared to be preferentially upregulated by HIF-2. Reverse transcription-PCR analysis revealed that specific messages for ORF34 and ORF50 are upregulated in BCBL-1 cells exposed to hypoxia. An HIF-1 binding and competition assay demonstrated that the HRE sequence from the ORF34 promoter can compete for HIF-1 binding to an erythropoietin HRE oligonucleotide while a mutant sequence cannot. Thus, we demonstrated that a viral gene can be activated by hypoxia through activation of a functional viral HRE. To our knowledge, this is the first example of a functional HRE in a viral promoter.

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What Are the Symptoms of Uterine Sarcoma?

Finding uterine sarcoma while in its early stage makes it easier to treat. These are the symptoms that you may notice.

—      Unusual vaginal bleeding or discharge, especially after menopause. At first, the bleeding may appear watery with a small amount of blood in it. After a while, the bleeding may appear less watery with more blood in it.

—      Pain or a feeling of fullness in the pelvic area or lower abdomen

—      Fibroids that grow quickly, especially after menopause.

Many of these may be signs of other health problems. Talk with your doctor right away to find out for sure.

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CASE 1. Ewing’s Sarcoma of the Rectum

J. Vardy, A.M. Joshua, S.J. Clarke

Department of Medical Oncology, Sydney Cancer Centre, Royal Prince Alfred Hospital, Sydney, Australia

P.M. Yarrow, B.P.C. Lin

Department of Anatomical Pathology, Concord Repatriation General Hospital, Sydney, Australia

A previously healthy 53-year-old male house painter of Greek origin presented to his local doctor after a single episode of bright rectal bleeding. Additional questioning determined that he had experienced a sensation of heaviness in the rectum for several months. Sigmoidoscopy revealed a fungating, ulcerated posterior wall rectal mass. Treatment necessitated a low anterior resection with formation of a temporary ileostomy. The tumor extended through the full thickness of the rectal wall and to within 7 mm of the resection margin. Histopathology showed an undifferentiated small round blue cell tumor with high mitotic rate and extensive areas of necrosis . Immunoperoxidase stains were positive for MIC-2  and vimentin. There was focal keratin positivity with CAM 5.2. Stains for keratin AE1/AE3, S-100, chromogranin, desmin, and smooth muscle actin were negative. Neuron-specific enolase staining was nonspecific. The tumor cells contained abundant glycogen demonstrated by the periodic acid Schiff stain before and after diastase treatment. No rosette formation was seen. The histologic appearance and immunoperoxidase profile supported a diagnosis of extraosseous Ewing’s sarcoma (EOE). Additional staging using blood results; computed tomography of chest, abdomen, and pelvis; and a whole-body bone scan showed no evidence of disseminated malignancy.

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Some people use statistics to try to figure out their chance of getting cancer or being cured of it. However, statistics only show what happens to large groups of people. Because no 2 people are alike, you cannot use statistics to predict what might happen to you.

These US statistics are from the American Cancer Society’s Cancer Facts & Figure 2006.

—      This year about 41,200 women will be told they have uterine cancer. Between 2% and 4%   of these cases will be uterine sarcoma. So it’s estimated that between 824 and 1,648 women will be told they have uterine sarcoma this year.

—      Uterine cancer is the fourth most common cancer in women. Breast, lung, and colon cancer are the 3 most common cancers in women.

—      About 7,350 women will die of cancer of the uterus this year.That figure is for all types of uterine cancer combined, not just uterine sarcoma.

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Neil H. Segal, Paul Pavlidis, William S. Noble, Cristina R. Antonescu, Agnes Viale, Umadevi V. Wesley, Klaus Busam, Humilidad Gallardo, Dianne DeSantis, Murray F. Brennan, Carlos Cordon-Cardo, Jedd D. Wolchok, Alan N. Houghton

From the Memorial Sloan-Kettering Cancer Center and Columbia Genome Center, Columbia University, New York, NY.

Address reprint requests to N.H. Segal, MD, PhD, c/o Alan N. Houghton MD, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021

Purpose: To develop a genome-based classification scheme for clear-cell sarcoma (CCS), also known as melanoma of soft parts (MSP), which would have implications for diagnosis and treatment. This tumor displays characteristic features of soft tissue sarcoma (STS), including deep soft tissue primary location and a characteristic translocation, t(12;22)(q13;q12), involving EWS and ATF1 genes. CCS/MSP also has typical melanoma features, including immunoreactivity for S100 and HMB45, pigmentation, MITF-M expression, and a propensity for regional lymph node metastases.

Materials and Methods: RNA samples from 21 cell lines and 60 pathologically confirmed cases of STS, melanoma, and CCS/MSP were examined using the U95A GeneChip (Affymetrix, Santa Clara, CA). Hierarchical cluster analysis, principal component analysis, and support vector machine (SVM) analysis exploited genomic correlations within the data to classify CCS/MSP.

Results: Unsupervised analyses demonstrated a clear distinction between STS and melanoma and, furthermore, showed that CCS/MSP cluster with the melanomas as a distinct group. A supervised SVM learning approach further validated this finding and provided a user-independent approach to diagnosis. Genes of interest that discriminate CCS/MSP included those encoding melanocyte differentiation antigens, MITF, SOX10, ERBB3, and FGFR1.

Conclusion: Gene expression profiles support the classification of CCS/MSP as a distinct genomic subtype of melanoma. Analysis of these gene profiles using the SVM may be an important diagnostic tool. Genomic analysis identified potential targets for the development of therapeutic strategies in the treatment of this disease.

Supported in part by the Etta Weinheim Memorial Fund, New York, NY (J.D.W.); National Institutes of Health, Bethesda, MD, grant no. CA-47179 (M.F.B. and A.N.H.); Swim Across America, New York, NY; the Kennedy Family Fund, Naples, FL; and National Science Foundation, Arlington, VA, grant no. IIS-0093302 (W.S.N.).

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