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Human embryonic stem cells (hESCs) provide an important means to characterize early stages of hematopoietic development. However, the in vivo potential of hESC-derived hematopoietic cells has not been well defined. We demonstrate that hESC-derived cells are capable of long-term hematopoietic engraftment when transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Human CD45(+) and CD34(+) cells are identified in the mouse bone marrow (BM) more than 3 months after injection of hESCs that were allowed to differentiate on S17 stromal cells for 7-24 days. Secondary engraftment studies further confirm long-term repopulating cells derived from hESCs. We also evaluated two mechanisms that may inhibit engraftment: host immunity and requirement for homing to BM. Treatment with anti-ASGM1 antiserum that primarily acts by depletion of natural killer cells in transplanted mice leads to improved engraftment, likely due to low levels of HLA class I expressed on hESCs and CD34(+) cells derived from hESCs. Intra-BM injection also provided stable engraftment, with hematopoietic cells identified in both the injected and contra-lateral femur. Importantly, no teratomas are evident in animals injected with differentiated hESCs. These results demonstrate that SCID-repopulating cells, a close surrogate for hematopoietic stem cells, can be derived from hESCs. Moreover, both adaptive and innate immune effector cells may be barriers to engraftment of these cells.

Seventy to eighty percent of patients with newly-diagnosed bladder cancer will present with superficial tumors (Ta, Tis or T(1)). There is, however, a continuum between superficial and muscle-invasive cancer, with the advanced cases usually associated with less-differentiated histology and aneuploidy. Common sites of metastasis include regional lymph nodes, bone, lung, skin and liver. From the low cure rates achieved with radical cystectomy, there is strong evidence that bladder cancer, from the outset, is a systemic disease. The limitations of local treatment are well-documented: a local control rate of 30% with radiation treatment, and 50-70% with radical cystectomy; and no improvement in surgical cure was seen with the use of preoperative radiation. Over the past 30 years, since the initial reports of the effectiveness of cisplatin in the treatment of advanced bladder cancer, there has been a steady flow of chemotherapeutic agents, singly and in combination, shown to be effective in the treatment of this tumor. While response rates and CR rates have increased with the use of combination chemotherapy, this has not translated into survival in advanced disease of greater than 16 months. While the search for more effective agents and combinations continues, attention has also been given to the roles of neoadjuvant and adjuvant chemotherapy in an effort to improve the cure rate achieved with surgery alone. Although radical cystectomy, with continent diversion or neobladder construction in selected cases remains the standard of care in the United States for patients with muscle-invasive bladder cancer, several groups have explored therapeutic strategies that aim at bladder preservation. Early approaches with the goal of bladder preservation consisted of radiation treatment as monotherapy (largely abandoned) or aggressive TURBT for smaller tumors. Over the past 20 years, the Massachusetts General Hospital (MGH) and the Radiation Therapy Oncology Group (RTOG) have studied patients with muscle-invading bladder cancer utilizing tri-modality treatment: a visibly complete transurethral resection followed by radiation with concurrent radiosensitizing chemotherapy and, subsequently, adjuvant chemotherapy. Thus, chemotherapy has been used in two phases of treatment (1) as radiosensitizers, given concurrently with radiation treatment and (2) as adjuvant treatment, recognizing that survival will only be improved by the successful treatment of micrometastases. Based on preliminary information from reports of the effectiveness of gemcitabine/cisplatin in advanced disease, that combination was chosen as the adjuvant regimen in one of our earlier protocols, recently completed and reported. Our current protocol utilizes the Bellmunt regimen as our adjuvant program with the highest RR in advanced disease. This study is ongoing, with early reports of tolerance of the three-drug regimen encouraging. The treatment options for muscularis propria-invasive bladder tumors can broadly be divided into those that spare the bladder and those that involve removing it. In the United States, radical cystectomy with pelvic lymph node dissection is the standard method used to treat patients with this tumor.

Background: Many novel studies and therapies are possible with the use of human embryonic stem cells (hES cells) and their differentiated cell progeny. The hES cell derived CD34 hematopoietic stem cells can be potentially used for many gene therapy applications. Here we evaluated the capacity of hES cell derived CD34 cells to give rise to normal macrophages as a first step towards using these cells in viral infection studies and in developing novel stem cell based gene therapy strategies for AIDS., Results: Undifferentiated normal and lentiviral vector transduced hES cells were cultured on S17 mouse bone marrow stromal cell layers to derive CD34 hematopoietic progenitor cells. The differentiated CD34 cells isolated from cystic bodies were further cultured in cytokine media to derive macrophages. Phenotypic and functional analyses were carried out to compare these with that of fetal liver CD34 cell derived macrophages. As assessed by FACS analysis, the hES-CD34 cell derived macrophages displayed characteristic cell surface markers CD14, CD4, CCR5, CXCR4, and HLA-DR suggesting a normal phenotype. Tests evaluating phagocytosis, upregulation of the costimulatory molecule B7.1, and cytokine secretion in response to LPS stimulation showed that these macrophages are also functionally normal. When infected with HIV-1, the differentiated macrophages supported productive viral infection. Lentiviral vector transduced hES cells expressing the transgene GFP were evaluated similarly like above. The transgenic hES cells also gave rise to macrophages with normal phenotypic and functional characteristics indicating no vector mediated adverse effects during differentiation., Conclusion: Phenotypically normal and functionally competent macrophages could be derived from hES-CD34 cells. Since these cells are susceptible to HIV-1 infection, they provide a uniform source of macrophages for viral infection studies. Based on these results, it is also now feasible to transduce hES-CD34 cells with anti-HIV genes such as inhibitory siRNAs and test their antiviral efficacy in down stream differentiated cells such as macrophages which are among the primary cells that need to be protected against HIV-1 infection. Thus, the potential utility of hES derived CD34 hematopoietic cells for HIV-1 gene therapy can be evaluated.

The human/sheep xenograft model has proven valuable in assessing the in vivo hematopoietic activity of stem cells from a variety of fetal and postnatal human sources. CD34+/lineage- or CD34+/CD38- cells isolated from human embryonic stem cells (hESCs) differentiated on S17 feeder layer were transplanted by intraperitoneal injections into fetal sheep. Chimerism in primary transplants was established with polymerase chain reaction (PCR) and flow cytometry of bone marrow and peripheral blood samples. Whole bone marrow cells harvested from a primary recipient were transplanted into a secondary recipient. Chimerism was established as described before. This animal was stimulated with human GM-CSF, and an increase in human hematopoietic activity was noted by flow cytometry. Bone marrow aspirations cultured in methylcellulose generated colonies identified by PCR to be of human origin. We therefore conclude that hESCs are capable of generating hematopoietic cells that engraft primary recipients. These cells also fulfill the criteria for long-term engrafting hematopoietic stem cells as demonstrated by engraftment and differentiation in the secondary recipient.

Human embryonic stem (hES) cells provide an important means to evaluate specific soluble and cell-bound stimuli that regulate development of specific cell lineages. Here, we examined specific cytokines and extracellular matrix (ECM) proteins that support differentiation of hES cells to hepatocytes. Tests of several different conditions determined that addition of fibroblast growth factor (FGF)-4 and hepatocyte growth factor in completely serum-free cultures of hES cell-derived embryoid bodies subsequently allowed to attach to type I collagen-coated dishes led to maximal differentiation into cells, not only with the morphologic and phenotypic characteristics of hepatocytes but also the functional characteristics. Expression of common hepatic transcription factors including HNF-3beta, HNF-1, and GATA-4 were all significantly induced under these conditions. Hepatocyte function was demonstrated by multiple complementary criteria: production of urea and albumin, phenobarbital-induced cytochrome P450 expression, and uptake of indocyanine green. These hES cell-derived hepatocytes will serve as a resource to understand normal human hepatocyte development and for applications such as cell replacement therapies and screening of pharmacologic drugs.

Objective: To find a human cell line that could support differentiation of human embryonic stem cells (hESCs) into hematopoietic cells. To determine in detail the expression profiles of the beta-like globin genes in hESC-derived erythroid cells., Materials and Methods: FH-B-hTERT, a human fetal liver-derived cell line, and S17, a mouse bone marrow stromal cell line, were used as stromas to induce the differentiation of hESC into hematopoietic cells. The number of hematopoietic progenitors and surface antigen expression were monitored during time-course experiments using colony assays and flow cytometry. Globin expression patterns in individual erythroid colonies were determined by real-time quantitative reverse transcriptase polymerase chain reaction., Results: Comparison of coculture of hESCs with FH-B-hTERT or S17 cells revealed that the fraction of CD34(+) cells and the number of clonogenic progenitors per 250,000 cells plated were higher with FH-B-hTERT than with S17. Analysis of beta-like globin expression in individual burst-forming unit erythroid and colony-forming unit erythroid colonies revealed that erythroid cells derived from hESC cocultured for 8 to 21 days on either FH-B-hTERT or S17 produced epsilon- and gamma-globin mRNAs in similar amounts. With increasing time in coculture, the mean ratio of gamma/epsilon increased by more than 10-fold on both S17 and FH-B-hTERT stroma. Importantly, beta-globin expression was barely detectable at all time point examined., Conclusions: FH-B-hTERT can induce hESCs differentiation into hematopoietic cells more efficiently than S17. In vitro differentiation of hESCs recapitulates the epsilon-globin to gamma-globin switch but not the gamma-globin to beta-globin switch that occurs around birth. This experimental system will be useful for studying the regulation of globin gene expression during early human hematopoiesis.

Human embryonic stem cells (hESCs) provide a unique resource to analyze early stages of human hematopoiesis. However, little is known about the ability to use hESCs to evaluate lymphocyte development. In the present study, we use a two-step culture method to demonstrate efficient generation of functional NK cells from hESCs. The CD56(+)CD45(+) hESC-derived lymphocytes express inhibitory and activating receptors typical of mature NK cells, including killer cell Ig-like receptors, natural cytotoxicity receptors, and CD16. Limiting dilution analysis suggests that these cells can be produced from hESC-derived hemopoietic progenitors at a clonal frequency similar to CD34(+) cells isolated from cord blood. The hESC-derived NK cells acquire the ability to lyse human tumor cells by both direct cell-mediated cytotoxicity and Ab-dependent cellular cytotoxicity. Additionally, activated hESC-derived NK cells up-regulate cytokine production. hESC-derived lymphoid progenitors provide a novel means to characterize specific cellular and molecular mechanisms that lead to development of specific human lymphocyte populations. These cells may also provide a source for innovative cellular immune therapies.

Embryonic stem cells (ESCs) and adult stem cells both provide important resources to define the mechanisms of hematopoietic cell development. To date, studies that utilize hematopoietic stem cells (HSCs) isolated from sites such as bone marrow or umbilical cord blood have been the primary means to identify molecular and phenotypic characteristics of blood cell populations able to mediate long-term hematopoietic engraftment. Although these HSCs are very useful clinically, they are difficult to expand in culture. Now, basic research on human ESCs provides opportunities for novel investigations into the mechanisms of HSC self-renewal. Eventually, the long history of basic and clinical research with adult hematopoietic cell transplantation could translate to establish human ESCs as a suitable alternative starting cell source for clinical hematopoietic reconstitution.

Stem cell-based regenerative medicine therapies have been touted recently as a novel therapeutic approach to treat and cure a wide range of diseases. Both adult and embryonic stem (ES) cells can serve as important sources of precursor cells to derive more mature cells potentially utilized for clinical applications. Nonhuman primates have proven useful as a preclinical model, as demonstrated in studies of hematopoietic cell transplantation, gene therapy, and other areas. The derivation of nonhuman primate ES cells now provides an optimal resource to characterize and test ES cell-based therapies prior to trials with human ES cells. This review describes work to define strategies and mechanisms to derive blood and endothelial cells from nonhuman primate ES cells isolated from various species. Preclinical testing that solely relies on studies of putative therapeutic cells derived from mouse ES cells transplanted into other mice, or analyses of human ES cell-derived cells transplanted into immunodeficient or immunosuppressed rodents may not be predictive of efficacy in subsequent human trials. However, future testing using nonhuman primate ES cell-derived therapeutic cells done as an allogeneic transplant may best predict success for subsequent studies using human ES cells. Therefore, additional research on nonhuman primate ES cells, in addition to work on mouse and human ES cells, is greatly needed to facilitate clinical translation of new stem cell treatments.

Background: Effective care of each patient throughout a hospital admission involves executing a specific set of tasks to produce a favorable outcome within an appropriate time frame. The ProjectRounds methodology, which can be implemented using widely available software, incorporates the principles of project management in planning and control hospital inpatient care. It consists of four stages--clinical assessment, planning, scheduling, and tracking. OVERVIEW OF PROJECTROUNDS AND EXAMPLE: As an example, a 68-year-old-man is admitted with pneumonia. In clinical assessment, the admitting physician uses an assessment tool that prompts her to list all the patient's clinical issues, define the conditions that need to be met to discharge the patient, highlight special problems, and list any consultations, diagnostic tests, and procedures that are planned. In planning, the work breakdown structure--a tabulation of all the tasks in the "project" (the admission)--is created. In scheduling, a project schedule is generated, and in tracking, the clinical team evaluates and monitors the project's course. During interdisciplinary clinical rounds, the progress of the patient's hospital care can be tracked and quantified by employing the percent complete method. Tracking can be used as a "dashboard," providing a concise summary of the care that needs to be and has been rendered to the patient., Summary and Next Steps: Applying the tenets of project management can optimize the process of providing health care to hospital inpatients.

The Massachusetts General Hospital and the Radiation Therapy Oncology Group have been leading the charge for organ conservation in bladder cancer in North America for over two decades. In a series of six successive studies the group has refined the techniques and is now moving toward a translational future in which novel biologic agents will be combined with the best current strategies. The North American approach is characterized by its selective nature, in that it preselects patients likely to do well with a trimodality approach and then further selects according to the response to an induction course of chemotherapy and radiation. Only those who are complete responders move onto full dose. This "check point" allows salvage cystectomy to be performed on incomplete responders before they have had full-dose radiation. This preserves the urinary diversion options open to the surgeon as well as brings forward the time to a salvage procedure should it be needed.

The isolation of embryonic stem (ES) cells from human preimplantation blastocysts creates an exciting new starting point to analyze the earliest stages of human blood development. This chapter describes two methods to promote hematopoietic differentiation of human ES cells: stromal cell coculture and embryoid body formation. Better understanding of basic human hematopoiesis through the study of human ES cells will likely have future therapeutic benefits.

Purpose: To define the maximal tolerated dose, safety, and efficacy of docetaxel, carboplatin, and estramustine in patients with hormone-refractory prostate cancer (HRPC)., Methods: Patients with HRPC received docetaxel for 3 weeks, followed by a rest week. Docetaxel (20, 25, 30, 36, or 43 mg/m2) was given on days 2, 9, and 16 of a 28-day cycle. Patients also received estramustine (140 mg p.o. three times daily on days 1-5, 8-12, and 15-19) and carboplatin [area under the curve, AUC (5) or (6) on day 2]., Results: Thirty patients were treated. Five patients received carboplatin [AUC (6)] but experienced delayed thrombocytopenia. After a protocol amendment, 25 subsequent patients received carboplatin [AUC (5)]. Median age was 64 years. Median prostate-specific antigen (PSA) was 117 ng/mL. Fifty-three percent received prior ketoconazole and 10% had mitoxantrone. No dose-limiting toxicities were noted. Although maximal tolerated dose was not reached, docetaxel dose escalation was stopped at 43 mg/m2. Significant myelosuppression was not seen until the highest dose level, when seven and four patients experienced grade 3 and 4 toxicities, respectively. Among all patients, PSA declines of > or =50% occurred in 63%. At the recommended phase II dose, PSA declines of > or =50% occurred in 75% (95% confidence interval, 43-95). Four of 14 (29%) patients with measurable disease had partial responses. Median survival was 14.6 months., Conclusions: Estramustine, docetaxel, and carboplatin are well tolerated and active in HRPC. Myelosuppression is the primary toxicity. The recommended phase II dose of docetaxel is 43 mg/m2 combined with estramustine and carboplatin. PSA declines were seen at every dose level.

Objective: Human embryonic stem (ES) cells can be induced to differentiate into hematopoietic lineages either by stromal cell coculture or by formation of embryoid bodies (EBs). Here, we better characterize cell-bound and secreted factors that support this hematopoietic development., Methods: Human ES cells either cocultured on the mouse bone marrow cell line S17, or allowed to form EBs, were induced to differentiate in the presence of serum, serum-free conditions, and serum-free media supplemented with defined cytokines. To better characterize the requirement for stromal cell-bound or secreted proteins, S17 conditioned media and transwell cultures were also utilized., Results: In both models, CD34(+), CD45(+), and hematopoietic colony-forming cells (CFCs) were routinely derived. While hematopoietic development was diminished without serum, here we demonstrate with the stromal cell coculture model that addition of the growth factors stem cell factor (SCF), thrombopoietin (TPO), and Flt-3 ligand (Flt3L) to serum-free media does allow isolation of hematopoietic progenitors. However, these same three growth factors added to serum-free media do not support significant hematopoiesis in the EB system. However, addition of the mesoderm-inducing factors bone morphogenic protein-4 and vascular endothelial growth factor to EBs grown in serum-free media plus SCF, TPO, and Flt-3L does improve hematopoietic development., Conclusion: These results demonstrate the utility of human ES cell to evaluate specific stimuli that regulate cell fate decisions and the survival of specific lineages. Moreover, the method used to promote differentiation of ES cells may alter the cytokines or growth factors required to isolate specific cell types.

Background: The objective of this study was to assess the biologic activity of rosiglitazone, a peroxisome proliferator-activated receptor gamma agonist that has been approved to treat type 2 diabetes, in men with recurrent prostate carcinoma using change in prostate specific antigen (PSA) doubling time (PSADT) as the primary outcome variable., Methods: Men with histologically confirmed prostate carcinoma, no recent hormone therapy, a rising serum PSA level after radical prostatectomy and/or radiation therapy, and no radiographic evidence of metastases were assigned randomly to receive either oral rosiglitazone (4 mg twice daily) or placebo. The treatment was continued until the men developed disease progression or adverse effects. A positive outcome was defined as a posttreatment PSADT > 150% the baseline PSADT and no new metastases., Results: One hundred six men were enrolled. The median treatment duration was 315 days for men in the placebo group and 338 days for men in the rosiglitazone group (P = 0.28). Forty percent of men in the in the placebo group and 38% of men in the rosiglitazone group had a posttreatment PSADT > 150% of the baseline PSADT and no new metastases (P = 1.00). In exploratory analyses, the rate of a positive outcome remained higher than expected in the placebo group, even when a positive outcome was redefined using more stringent criteria. The time to disease progression was similar between the groups., Conclusions: Rosiglitazone did not increase PSADT or prolong the time to disease progression more than placebo in men with a rising PSA level after radical prostatectomy and/or radiation therapy. The unexpected discordance between baseline and posttreatment PSADT in the placebo group reinforced the importance of randomized controlled trials in this setting., ((c) 2004 American Cancer Society.)

The purpose of the study was to evaluate response and survival in patients with metastatic urothelial cancer treated with combination gemcitabine and paclitaxel administered on a biweekly schedule at doses of 3000 mg/m2 and 150 mg/m2, respectively. Patients with adequate organ function and performance status were accrued through 7 institutions, stratified by prior therapy status, and treated as noted. Response was evaluated by 1979 bi-dimensional World Health Organization (WHO) criteria. Of 55 eligible patients, 17 had a partial and 5 had a complete response rate for an overall response rate of 40% (27-54%). One complete response and one partial response were observed in the 6 previously treated patients. Overall median survival was 11.8 months (11.9 months in the chemonaive cohort). Grade 3 or 4 myelosuppression occurred in 56%, but only 4 serious infections were observed. We conclude that because of a lower than expected complete response rate, even when corrected for prognostic groupings, this regimen is not recommended for routine use in patients with metastatic urothelial cancer. Insufficient patients with poor renal function or prior therapy were accrued to reach conclusions regarding its utility in these subgroups.

Background: Medical or surgical castration is effective in advanced prostate cancer but with profound side-effects, particularly on sexual function. Effective, less toxic therapies are needed. This study examined whether the addition of finasteride to high-dose bicalutamide enhanced disease control, as measured by additional decreases in serum prostate-specific antigen (PSA)., Patients and Methods: Forty-one patients with advanced prostate cancer received bicalutamide (150 mg/day). Finasteride (5 mg/day) was added at first PSA nadir. Serum PSA was measured every 2 weeks until disease progression. Questionnaires were administered to assess sexual function., Results: Median follow-up is 3.9 years. At the first PSA nadir, median decrease in PSA from baseline was 96.5%. Thirty of 41 patients (73%) achieved a second PSA nadir and median decrease of 98.5% from baseline. Median time to each nadir was 3.7 and 5.8 weeks, respectively. Median time to treatment failure was 21.3 months. Toxicities were minor, including gynecomastia. Seventeen of 29 (59%) and 12 of 24 (50%) men had normal sex drive at baseline and at second PSA nadir, respectively. One-third of men had spontaneous erection at both time points., Conclusion: Finasteride provides additional intracellular androgen blockade when added to bicalutamide. Duration of control is comparable to castration, with preserved sexual function in some patients.

We have used rhesus monkey embryonic stem (ES) cells to study endothelial cell development. Rhesus ES cells (R366.4 cell line) exposed to medium containing vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), and epidermal growth factor (EGF) assumed a relatively uniform endothelial cell morphology and could be propagated and expanded with a consistent phenotype and normal karyotype. When placed in Matrigel, these rhesus ES cell-derived endothelial cells (RESDECs) formed capillary-like structures characteristic of endothelial cells. Immunohistochemical and flow cytometric analysis of RESDECs showed that they take up acetylated low-density lipoprotein (LDL), express CD146, von Willebrand factor, and the integrin alpha v beta 3, and bind the lectin ulex europaeus agglutinin-1. These cells also express the VEGF receptor Flk-1 and secrete VEGF. When introduced in a Matrigel plug implanted subcutaneously in mice, RESDECs formed intact vessels and recruited new endothelial cell growth. In vivo function was demonstrated by coinjection of RESDECs with murine tumor cells subcutaneously into immunocompromised adult mice. RESDECs injected alone did not form measurable tumors. Tumor cells grew more rapidly and had increased vascularization when coinjected with the RESDECs. Immunohistochemical staining demonstrated that the RESDECs participated in forming the tumor neovasculature. RESDECs provide a novel means to examine the mechanisms of endothelial cell development, and may open up new therapeutic strategies.

Invasive transitional cell carcinoma (TCC) of the urinary bladder is traditionally treated with radical cystectomy. This approach results in great morbidity and lifestyle changes, and approximately half of the patients treated in this way will experience recurrent TCC despite surgery. An alternative approach using selective bladder-preservation techniques incorporates transurethral resection of bladder tumours, radiation therapy, and chemotherapy. Over the past 20 years, international experience has demonstrated that this approach is feasible, safe, and well tolerated. Furthermore, the long-term outcomes of overall survival and disease-free survival compare favourably with the outcomes from radical cystectomy. The most important predictor of response is stage, with significantly higher long-term survival in patients with T2 disease. Another important positive predictor of complete response to therapy is the ability of the urologic oncologist to remove all visible tumour through a transurethral approach prior to initiation of radiation therapy. A negative predictive factor is the presence of hydronephrosis, and age and gender do not affect disease-free survival. The majority of patients who enjoy long-term survival do so with an intact native bladder. Quality of life studies have demonstrated that the retained bladder functions well in nearly all of these patients. Selective bladder preservation will not entirely take the place of radical cystectomy, but should be offered as an important alternative to patients newly diagnosed with muscle-invasive TCC.

The ability of embryonic stem cells and adult stem cells to differentiate into specific cell types holds immense potential for therapeutic use in cell and gene therapy. Realization of this potential depends on efficient and optimized protocols for genetic manipulation of stem cells. In the study reported here, we demonstrate the use of nucleofection as a method to introduce plasmid DNA into embryonic and adult stem cells with significantly greater efficiency than electroporation or lipid-based transfection methods have. Using enhanced green fluorescent protein (eGFP) as a reporter gene, mouse embryonic stem cells were transfected both transiently and stably at a rate nearly 10-fold higher than conventional methods. The transfected cells retained their stem cell properties, including continued expression of the stem cell markers SSEA1, Oct4, and Rex1; formation of embryoid bodies; differentiation into cardiomyocytes in the presence of appropriate inducers; and, when injected into developing blastocysts, contribution to chimeras. Higher levels of transfection were also obtained with human embryonic carcinoma and human embryonic stem cells. Particularly hard-to-transfect adult stem cells, including bone marrow and multipotent adult progenitor cells, were also transfected efficiently by the method of nucleofection. Based on our results, we conclude that nucleofection is superior to currently available methods for introducing plasmid DNA into a variety of embryonic and adult stem cells. The high levels of transfection achieved by nucleofection will enable its use as a rapid screening tool to evaluate the effect of ectopically expressed transcription factors on tissue-specific differentiation of stem cells.

Human embryonic stem (ES) cells provide a unique model and an important resource to analyze early hematopoietic development. Other systems to study mammalian hematopoiesis include mouse ES cells, dissection of timed mouse embryos, or use of human postnatal hematopoietic tissue typically isolated from bone marrow or umbilical cord blood. All these models have particular strengths and weaknesses. The extensive studies on murine hematopoiesis provide a basis for work on the human developmental system. Since there are likely some important species differences, use of human ES cells now provides an optimal means to evaluate basic cellular and molecular mechanisms that regulate the beginning stages of human blood development, prior to derivation of hematopoietic stem cells (HSCs). Eventually, research on human ES cells may provide an alternative source of HSCs and other blood products for hematopoietic cell transplantation or other cellular therapies.

To examine combination cisplatin and twice-daily accelerated irradiation (RT) after aggressive transurethral resection of bladder tumor (TURBT) in an attempt to preserve the bladder and to determine the likelihood that patients who complete this regimen could then complete three cycles of methotrexate, cisplatin, vinblastine (MCV) chemotherapy. Between 1998 and 2000, 52 patients with Stage T2-T4aN0M0 disease, from 17 institutions, were entered into the trial. Forty-seven patients were deemed eligible; the planned accrual was 40. Of the 46 patients, 68% were >60 years old, 70% were men, and 96% had a Karnofsky score >/=90. The clinical T stage was T2 in 66%, T3a in 25%, and T3b in 9%. The median follow-up at the time of analysis was 26 months. The protocol required TURBT within 6 weeks of the initiation of induction therapy. Induction treatment involved 13 days of concomitant boost RT, 1.8 Gy to the pelvis in the morning followed by 1.6 Gy to the tumor 4-6 h later. For sensitization, cisplatin (20 mg/m(2)) was given on the first 3 days of each treatment week. Three to four weeks after induction, patients were evaluated cystoscopically for residual disease. Patients whose biopsies and cytologic evaluations showed no disease completed consolidation chemoirradiation. Patients with residual tumor went on to cystectomy. After either consolidation or cystectomy, patients were to complete three cycles of MCV chemotherapy. Of the 47 patients, 45% completed all phases of the protocol treatment with minor, or no, deviations. Five patients refused either the postinduction evaluation or cystectomy and 6 refused adjuvant chemotherapy. The CR rate after induction therapy was 74%. For 2 patients, residual disease after induction was limited to positive cytologic findings, and for 8 patients, biopsy of the primary site revealed persistence. Of the 8 cystectomy patients, 2 had no evidence of disease in the bladder at pathologic review of the surgery specimen. Grade 3 toxicity related to chemotherapy was observed in 11% of patients during both induction and consolidation, and in 41% during adjuvant chemotherapy. A total of 8 patients (36% of those receiving adjuvant chemotherapy) went on to develop Grade 4 neutropenia or thrombocytopenia during additional adjuvant chemotherapy. Grade 3 toxicity due to RT was seen in 4% and 0% of patients during induction and consolidation, respectively. One patient developed Grade 4 hydronephrosis during consolidation. The projected 36-month value for locoregional failure, distant metastasis, overall survival, and bladder-intact survival was 27%, 29%, 61%, and 48%, respectively. After aggressive TURBT, twice-daily accelerated RT initiated in concomitant-boost format is well tolerated and results in a rate of complete response (74%) similar to that in previous bladder-sparing trials. The projected 2-year values for locoregional control, bladder-intact survival, and overall survival were also consistent with previously reported trials of bladder-sparing treatment. With only 45% of patients completing three cycles of MCV, this form of adjuvant chemotherapy appears to be poorly tolerated by most patients.

Purpose: Transurethral resection, chemotherapy and radiation with salvage cystectomy may be used as alternatives to immediate radical cystectomy in the management of invasive bladder cancer. Concern exists about the function of the retained bladder after such therapy., Materials and Methods: Of 221 patients with clinical T2-4a bladder cancer treated at Massachusetts General Hospital from 1986 to 2000 with trimodality therapy, 71 were alive with native bladders and disease-free in 2001. These patients were asked to undergo a urodynamic study and to complete a quality of life questionnaire. A total of 69% participated in some component of this study with a median time from trimodality therapy of 6.3 years (range 1.6 to 14.9)., Results: Of 32 patients 24 had normally functioning bladders by urodynamic study. Decreased bladder compliance was seen in 7. Bladder hypersensitivity, involuntary detrusor contractions and incontinence were present in 2 women. The questionnaire showed that flow symptoms occurred in 6%, urgency in 15% and control problems in 19%. Of all women 11% wore pads. Distress from urinary symptoms was half as common as prevalence. Bowel symptoms occurred in 22% with 14% recording any level of distress. The majority of men retained sexual function. Global health related quality of life was high., Conclusions: The majority of patients treated with trimodality therapy retain good bladder function. A fifth have evidence of bowel dysfunction.

Objectives: To report the efficacy of castration after progression on finasteride and flutamide. Standard androgen deprivation strategies for prostate cancer typically lead to castrate levels of testosterone. One alternative is the use of finasteride and flutamide., Methods: A Phase II trial evaluated the combination of finasteride (5 mg/day) and flutamide (250 mg three times daily) in patients with rising prostate-specific antigen levels after local treatment for prostate cancer or with newly discovered metastatic disease. Patients were followed up for subsequent events, including castration-free, androgen-independent prostate cancer (AIPC)-free, and overall survival., Results: With a median follow-up of 88 months, 5 patients (25%) continued on finasteride and flutamide, and 12 had stopped this combination and subsequently underwent medical or surgical castration. No patients experienced a flutamide withdrawal effect. All patients experienced more than a 50% decline in prostate-specific antigen after castration (mean 89%). The median protocol treatment failure-free survival was 29.9 months, the median castration-free survival was 37 months, and the median AIPC-free survival was 48.6 months. At 5 years, the overall survival rate was 65% (95% confidence interval 47% to 90%); 29% were alive and have not required castration, and 35% were alive and free of AIPC., Conclusions: Finasteride and flutamide have a durable effect in suppressing prostate-specific antigen progression in some men with advanced prostate cancer. Furthermore, castration induces secondary responses that may be of shorter duration than if started initially, although the overall period of hormonally responsive prostate cancer is more than 4 years.

Patients with clinical T3 or T4 prostate cancer or with elevated serum prostate-specific antigen (PSA) levels greater than 40 ng/ml are at high risk of failure with primary treatment. Chemotherapy administered at the time of diagnosis may decrease the risk of recurrence. Patients with high risk localized prostate cancer were treated with two cycles of liposomal doxorubicin (Doxil) at 50 mg/m2 every 28 days. Patients were assessed for response by digital rectal examination (DRE), PSA, and endorectal magnetic resonance imaging (MRI) (erMRI). Patients were then treated with androgen ablative therapy and prostate radiotherapy. Seven patients were treated. Three patients had T3 disease, and 4 patients had T2b disease with either PSA greater than 40 ng/ml or erMRI evidence of seminal vesicle involvement or extracapsular disease. Median PSA was 29.4 ng/ml. None of the seven patients experienced a significant response, as measured by changes in DRE, PSA, or erMRI. Toxicity was significant, with 4 of 7 patients developing skin toxicity. All seven patients responded to androgen ablative therapy and prostate irradiation. Neoadjuvant liposomal doxorubicin demonstrates no apparent activity and significant toxicity. New strategies are needed to improve outcomes in high-risk prostate cancer.

Recently cancer susceptibility syndromes have been characterized that suggest possible genetic linkages between breast cancer and prostate cancer within families. Despite these connections, male breast cancer and prostate cancer in an individual man has rarely been reported. The clinical features of 10 patients with both of these cancers are described here. One hundred sixty-one patients with male breast cancer were seen at the Dana-Farber Cancer Institute and Massachusetts General Hospital between 1977 and 2000. Of these, 10 were identified who also had prostate cancer. A retrospective review of records from these 10 patients was performed. Breast cancer preceded prostate cancer in eight of these men. The mean age of diagnosis of breast cancer was 65.7 years (range 47-72 years). Twenty percent had nodal involvement at diagnosis and two patients ultimately developed evidence of metastatic disease. The mean age of diagnosis of prostate cancer was 68.0 years (range 51-76 years) with a median prostate-specific antigen (PSA) level at diagnosis of 6 ng/ml (range 1.8-47.5 ng/ml). Seven patients had a family history of female breast cancer in a first-degree relative, while one had a family history of prostate cancer. At a median follow-up of 6.5 years from initial cancer diagnosis, one patient had died of metastatic breast cancer and another had died of metastatic prostate cancer. The clinical features and course of the breast cancers diagnosed in this series do not appear significantly different from those described for the general population of male breast cancer patients. In addition, these men do not appear to develop prostate cancer at an earlier age or more aggressive stage than the general population.

In the United States, radical cystectomy is viewed as the gold standard and, with few exceptions, is the only treatment recommended for patients with invasive bladder cancer. In many areas of cancer treatment, however, the trend in the 1990s has been toward organ conservation using combined chemotherapy and radiation with or without conservative local surgery. For patients with breast, esophageal, anal, and laryngeal cancers as well as limb sarcomas, conservative therapy often is recommended. However, invasive bladder cancer has not been viewed generally as a condition that allows for conservative management. In the past 15 years, the Radiation Therapy Oncology Group (RTOG) has completed six prospective protocols of combined-modality therapy for patients with muscle-invasive cancer who were candidates for cystectomy. Bladder preservation with intravesical surgery, chemotherapy, and radiation therapy were combined as initial treatment, with radical cystectomy recommended for incomplete responders. Five of the RTOG protocols were Phase I-II trials of concurrent chemotherapy and radiation therapy, and one protocol was a Phase III trial that tested the efficacy of adjuvant chemotherapy with methotrexate, cisplatin, and vinblastine. A total of 415 patients were entered on these trials. The 5-year overall survival rate was approximately 50%, with three-quarters of those patients achieving a cure for their bladder cancer while maintaining a functioning bladder. The current RTOG protocol and its successor are directed toward better tolerated and potentially more effective chemotherapy regimens that may result in a high protocol compliance rate and, possibly, a higher overall survival rate. The trimodality therapeutic approach used in all of these RTOG protocols was more effective compared with the radiation monotherapy offered in the 1970s and with protocols that used only chemotherapy. Trimodality therapy with selective bladder preservation is not designed to take the place of radical cystectomy; however, it may be offered as a reasonable alternative to patients with invasive bladder cancer who are not willing to undergo radical cystectomy and urinary diversion. A bladder-sparing strategy may be offered appropriately to highly selected patients with the understanding that radical cystectomy is an available option in those who fail combined radiation and chemotherapy with no diminution in survival related to the delay in cystectomy., (Copyright 2003 American Cancer Society)

Objectives: To evaluate the outcomes of patients with muscle-invasive Stage T2-4a bladder carcinoma managed by transurethral surgery and concurrent chemoradiation., Methods: A total of 190 patients were treated on institutional prospective protocols using concurrent cisplatin-containing chemotherapy and radiotherapy after rigorous transurethral resection of the bladder tumor. Patients were re-evaluated by repeated biopsy and urine cytologic analysis after 40 Gy, with the initial tumor response guiding subsequent therapy. One hundred twenty-one patients with a complete response by cytologic and histologic examination and those medically unfit for cystectomy received boost chemoradiation to 64 to 65 Gy. Those patients without a complete response were advised to undergo radical cystectomy. A total of 66 patients (35%) ultimately underwent radical cystectomy; 41 for less than a complete response and an additional 25 for recurrent invasive tumors. The median follow-up was 6.7 years for all surviving patients., Results: The 5 and 10-year actuarial overall survival rate was 54% and 36%, respectively (Stage T2, 62% and 41%; Stage T3-T4a, 47% and 31%, respectively). The 5 and 10-year disease-specific survival rate was 63% and 59% (Stage T2, 74% and 66%; Stage T3-T4a, 53% and 52%), respectively. The 5 and 10-year disease-specific survival rate for patients with an intact bladder was 46% and 45% (Stage T2, 57% and 50%; Stage T3-T4a, 35% and 34%), respectively. The pelvic failure rate was 8.4%. No patient required cystectomy because of bladder morbidity., Conclusions: The 10-year overall survival and disease-specific survival rates are comparable with the results reported for contemporary radical cystectomy for patients of similar clinical and pathologic stage. One third of patients treated on protocol with the goal of bladder sparing ultimately required a cystectomy. A trimodality approach with bladder preservation based on the initial tumor response is, therefore, safe, with most long-term survivors retaining functional bladders.

Human embryonic stem (ES) cells provide a novel opportunity to study early developmental events in a human system. We have used human ES cell lines, including clonally derived lines, to evaluate haematopoiesis. Co-culture of the human ES cells with irradiated bone marrow stromal cell lines in the presence of fetal bovine serum (FBS), but without other exogenous cytokines, leads to differentiation of the human ES cells within a matter of days. A portion of these differentiated cells express CD34, the best-defined marker for early haematopoietic cells. Haematopoietic colony-forming cells (CFCs) are demonstrated by methylcellulose assay. Myeloid, erythroid, megakaryocyte and multipotential CFCs can all be derived under these conditions. Enrichment of CD34+ cells derived from the human ES cells markedly increases the yield of CFCs, as would be expected for cells derived from adult bone marrow or umbilical cord blood. Transcription factors are also expressed in a manner consistent with haematopoietic differentiation. This system now presents the potential to evaluate specific conditions needed to induce or support events in early human blood development. Human ES cells are also a novel source of cells for transplantation therapies. The immunogenicity of ES cell-derived cells is unknown. The unique properties of ES cells afford the opportunity to explore novel mechanisms to prevent immune-mediated rejection. Potential strategies to overcome rejection will be presented, including creation of haematopoietic chimerism as a means to successfully transplant cells and tissues derived from human ES cells.

Treatment options for patients with metastatic renal cell carcinoma are limited. Interferon-alpha has an overall response rate of 10-15% in phase II and III clinical trials and is considered a standard option for patients. Though the anti-estrogen toremifene has shown only modest single agent activity in renal cell carcinoma, evidence for synergy of anti-estrogens with interferon-alpha exists in renal cell and other cancers. Therefore, a phase II trial was undertaken to test the combination of interferon-alpha and toremifene in advanced renal cell carcinoma. Thirteen patients with measurable metastatic or unresectable local disease were treated with interferon-alpha at a dose of 5 million units/m2 three times a week and daily oral toremifene at 300 mg daily in divided doses. Patients were treated for 12 weeks and then restaged. Clinical response was the primary endpoint of the trial. Four patients (31%) had evidence of stable disease at 12 weeks, while the remaining nine patients (69%) progressed on treatment. Toxicity was moderate, with grade 2 or 3 fatigue, nausea and anorexia each noted in 31% of patients. We conclude that the combination of interferon-alpha plus toremifene demonstrates no significant activity in advanced renal cell carcinoma.

Human embryonic stem (ES) cells are undifferentiated, pluripotent cells that can be maintained indefinitely in culture. Here we demonstrate that human ES cells differentiate to hematopoietic precursor cells when cocultured with the murine bone marrow cell line S17 or the yolk sac endothelial cell line C166. This hematopoietic differentiation requires fetal bovine serum, but no other exogenous cytokines. ES cell-derived hematopoietic precursor cells express the cell surface antigen CD34 and the hematopoietic transcription factors TAL-1, LMO-2, and GATA-2. When cultured on semisolid media with hematopoietic growth factors, these hematopoietic precursor cells form characteristic myeloid, erythroid, and megakaryocyte colonies. Selection for CD34(+) cells derived from human ES cells enriches for hematopoietic colony-forming cells, similar to CD34 selection of primary hematopoietic tissue (bone marrow, umbilical cord blood). More terminally differentiated hematopoietic cells derived from human ES cells under these conditions also express normal surface antigens: glycophorin A on erythroid cells, CD15 on myeloid cells, and CD41 on megakaryocytes. The in vitro differentiation of human ES cells provides an opportunity to better understand human hematopoiesis and could lead to a novel source of cells for transfusion and transplantation therapies.

Objectives: Although radical cystectomy remains the standard of care for invasive bladder cancer in the United States, many groups are exploring the use of trimodality therapy using transurethral resection of the bladder tumor, radiation, and chemotherapy in an attempt to spare patients the need for cystectomy. As transitional cell carcinoma often arises from a urothelial field change, there is concern that the retained bladder is at risk of subsequent superficial (Ta, T1, Tis) tumors, some of which may have lethal potential. This study reports the outcomes of those patients with superficial relapse of transitional cell carcinoma after trimodality therapy., Methods: One hundred ninety patients were treated using a series of trimodality therapy protocols between 1986 and 1998. All patients received induction chemotherapy and radiation and were selected for bladder preservation on the basis of a cytologic and histologic complete response. One hundred twenty-one patients had a complete response and formed the subjects of this study., Results: With a median follow-up of 6.7 years for patients still alive, 32 experienced a superficial relapse (26%). The median time to this failure was 2.1 years. Sixty percent of the superficial failures were carcinoma in situ (Tis) and 67% arose at the site of the original invasive tumor. The risk of superficial failure was higher among those who had Tis associated with their original muscle-invasive tumor. Twenty-seven of these 32 cases were managed conservatively with transurethral resection and intravesical therapy. The irradiated bladder tolerated this therapy well and only 3 patients required treatment breaks. The 5 and 8-year survival was comparable for those who experienced superficial failure (68% and 54%, respectively) and those who had no failure at all (n = 74, 69% and 61%, respectively). However, a substantially lower chance of being alive with the native bladder owing to the need for late salvage cystectomies (61% versus 34%) was found. Cystectomy became necessary in 31% (10 of 32) either because of additional superficial recurrence (n = 7) or progression to invasive disease (n = 3)., Conclusions: A trimodality approach to transitional cell bladder cancer mandates lifelong cystoscopic surveillance. Although most completely responding patients retain their bladders free from invasive relapse, one quarter will develop superficial disease. This may be managed in the standard fashion with transurethral resection of the bladder tumor and intravesical therapies but carries an additional risk that late cystectomy will be required.

Effective treatment options for high-risk localized prostate cancer are limited. Patients at high risk for recurrence include those with biopsy Gleason scores of 8 to 10, prostate specific antigen (PSA) levels > 20 ng/mL, and clinical stage T3 disease. Docetaxel chemotherapy is active in hormone-refractory prostate cancer, either combined with estramustine or used as a single agent. To determine if systemic therapy can improve the outcome of radical prostatectomy in men with high-risk localized prostate cancer, we are undertaking a pilot phase II clinical trial of weekly docetaxel at 36 mg/m(2) for up to 6 months, followed by surgery. Patients are monitored with weekly visits, monthly digital rectal examinations, PSA measurement, and testosterone tests, and endorectal magnetic resonance imaging done at baseline, after two cycles, and again after six cycles. To date, 15 patients have been enrolled, and 70 cycles of chemotherapy have been administered. Toxicity has been mostly grade 1 in intensity, and fatigue has been the most common grade 2 toxicity reported. The primary endpoint of the trial is measurement of pathologic complete response rate, for which data are not yet available. Recruitment to the trial is ongoing., (Copyright 2001 by W.B. Saunders Company.)

Bladder-preserving treatment for muscle-invasive disease is based on the response of the tumor to induction combined modality therapy. In the future, an organ-conserving approach will be widely offered as a safe and reasonable alternative to radical cystectomy.

The National Bladder Cancer Group, a multidisciplinary organization that was originally the therapeutic arm of the National Bladder Cancer Project, reported the results of trials using cisplatin with and without cyclophosphamide in patients with advanced bladder cancer. The objective response rate in both arms was 16% of 109 patients with no difference between the arms. Other studies included cisplatin and irradiation for patients who had comorbid conditions that prevented cystectomy (local response--cT2 11/13 patients; cT3 & cT4 2/4 patients). Seventy patients were treated on the same protocol, and 33 were alive 4 years later. Complete response versus local failure produced 57% versus 11% at 4 years. After cessation of funding, the clinicians at the Massachusetts General Hospital agreed to follow a potentially bladder-sparing multimodality protocol, which stipulated that visible tumor be resected following which the patient received methotrexate, vinblastine and cisplatin (MCV), small-volume irradiation, and more cisplatin with midcourse cystectomy if local tumor persisted. Overall survival was 45%. The Radiation Therapy Oncology Group conducted a similar study omitting MCV from one arm. The 5-year survival rate was 38% with no advantage for either arm.

Stem cells are unique cell populations with the ability to undergo both self-renewal and differentiation. A wide variety of adult mammalian tissues harbors stem cells, yet "adult" stem cells may be capable of developing into only a limited number of cell types. In contrast, embryonic stem (ES) cells, derived from blastocyst-stage early mammalian embryos, have the ability to form any fully differentiated cell of the body. Human ES cells have a normal karyotype, maintain high telomerase activity, and exhibit remarkable long-term proliferative potential, providing the possibility for unlimited expansion in culture. Furthermore, they can differentiate into derivatives of all three embryonic germ layers when transferred to an in vivo environment. Data are now emerging that demonstrate human ES cells can initiate lineage-specific differentiation programs of many tissue and cell types in vitro. Based on this property, it is likely that human ES cells will provide a useful differentiation culture system to study the mechanisms underlying many facets of human development. Because they have the dual ability to proliferate indefinitely and differentiate into multiple tissue types, human ES cells could potentially provide an unlimited supply of tissue for human transplantation. Though human ES cell-based transplantation therapy holds great promise to successfully treat a variety of diseases (e.g., Parkinson's disease, diabetes, and heart failure) many barriers remain in the way of successful clinical trials.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear hormone receptor that plays a key role in the differentiation of adipocytes. Activation of this receptor in liposarcomas and breast and colon cancer cells also induces cell growth inhibition and differentiation. In the present study, we show that PPARgamma is expressed in human prostate adenocarcinomas and cell lines derived from these tumors. Activation of this receptor with specific ligands exerts an inhibitory effect on the growth of prostate cancer cell lines. Further, we show that prostate cancer and cell lines do not have intragenic mutations in the PPARgamma gene, although 40% of the informative tumors have hemizygous deletions of this gene. Based on our preclinical data, we conducted a phase II clinical study in patients with advanced prostate cancer using troglitazone, a PPARgamma ligand used for the treatment of type 2 diabetes. Forty-one men with histologically confirmed prostate cancer and no symptomatic metastatic disease were treated orally with troglitazone. An unexpectedly high incidence of prolonged stabilization of prostate-specific antigen was seen in patients treated with troglitazone. In addition, one patient had a dramatic decrease in serum prostate-specific antigen to nearly undetectable levels. These data suggest that PPARgamma may serve as a biological modifier in human prostate cancer and its therapeutic potential in this disease should be further investigated.

In the USA radical surgery remains the golden standard for invasive bladder cancer. Yet in most other areas of surgical oncology the trend of the 1990s has been towards organ conservation with chemoradiation with or without limited local surgery. Patients with breast, oesophageal, anal, lung and larynx cancer are routinely offered conservative therapies as valid options in the management of their diseases but bladder stands apart from the crowd. Evidence is presented here to show that this need not be the case. Four older randomized trials failed to show a survival advantage when immediate cystectomy was compared with radiation followed by salvage cystectomy, if required. Five and 8-year survival rates for clinically staged patients treated by transurethral resection and chemoradiation (trimodality therapy) in several modern, large and mature series show survival rates comparable to those reported in contemporary radical cystectomy series. Eighty per cent of those alive 5 years after chemoradiation still retain their native bladder. Although superficial relapse occurs in 20% of cases, it remains responsive to BCG (Bacilles bilie de Calmette-Guerin) in the manner of de novo superficial disease. Quality-of-life studies show that the retained bladder functions well. At the Massachusetts General Hospital and in the multicentre prospective trials, less than 1% of patients needed cystectomy for bladder morbidity. It is of note that continent diversions may be performed as salvage after contemporary radiation therapy. Trimodality therapy is a novel and contemporary approach that owes little to the radiation treatment offered in the 1970s. While it will never entirely take the place of radical cystectomy, it should be offered as a reasonable alternative to patients with a new diagnosis of bladder cancer. This multidisciplinary approach will allow uro-oncology to keep in step with the oncological vanguard.

Purpose: To assess the safety, tolerance, and efficacy of transurethral surgery plus concomitant cisplatin, 5-fluorouracil (5-FU), and radiation therapy in conjunction with selective bladder preservation in patients with muscle-invading bladder cancer. Patients and Methods. Thirty-four eligible patients with clinical stage T2-T4a, Nx M0 bladder cancer without hydronephrosis were entered into a protocol aimed at selective bladder preservation. Treatment began with as complete a transurethral resection as possible followed by induction chemoradiation. This consisted of cisplatin 15 mg/m(2) i.v. and 5-fluorouracil (5-FU) 400 mg/m(2) i.v. in the mornings on d 1, 2, 3, 15, 16, and 17. On d 1, 3, 15, and 17, radiation was given immediately following the chemotherapy using twice-a-day 3 Gy per fraction cores to the pelvis for a total radiation dose of 24 Gy. Response was evaluated by cystoscopy, cytology, and rebiopsy four weeks later. Patients with a complete response received consolidation therapy with the same drugs and doses on d 1, 2, 3, 15, 16, and 17 combined with twice-daily radiation therapy to the bladder and bladder tumor volume of 2.5 Gy per fraction for a total consolidation dose of 20 Gy and a total induction plus consolidation dose to the bladder and bladder tumor of 44 Gy. Patients who did not achieve a complete response were advised to undergo prompt cystectomy, as were those with a subsequent invasive recurrence. The median follow up is 29 months., Results: Of the 34 eligible patients, 26 had a visibly complete transurethral resection. One patient did not complete induction treatment due to acute hematologic toxicity. After induction treatment, 22 (67%) of the 33 patients had no tumor detectable on urine cytology or rebiopsy. Of the 11 patients who still had detectable tumor, six underwent radical cystectomy and five underwent consolidation chemoradiation (one because of refusal to have the recommended cystectomy and four because the treating institutions erroneously assigned them to receive consolidation chemoradiation rather than cystectomy). No patient has required a cystectomy for radiation toxicity. Six patients have died of bladder cancer. The actuarial overall survival at three years is 83%. The probability of surviving with an intact bladder is 66% at three years. A total of seven patients (21%) developed grade 3 or grade 4 hematologic toxicity in conjunction with this treatment., Conclusion: This aggressive protocol comprising local surgery plus concurrent 5-FU, cisplatin, and high-dose hypofractionated radiation has been associated with moderately severe hematologic toxicity. Longer follow-up will be necessary to assess efficacy. Both the 67% complete response rate to induction therapy and the 66% three-year survival with an intact bladder are encouraging.

Purpose: We update the results of tri-modality treatment for patients with muscle invading bladder tumors with selection for bladder preservation based on tumor response to induction therapy., Material and Methods: We reviewed the literature on modern tri-modality bladder preserving approaches using transurethral resection, radiation and concurrent chemotherapy followed by either bladder conservation with careful surveillance for complete responding patients or prompt cystectomy in those whose tumors persist after induction therapy., Results: The published experiences from 3 centers and 2 prospective trials done by the Radiation Therapy Oncology Group were evaluated for 5-year overall survival of patients selected for bladder preservation or prompt cystectomy (49 to 63%) and for those with a conserved bladder (38 to 43%). The overall 5-year survival rates were comparable to other series of immediate cystectomy based approaches in patients of similar age and presenting with tumors of similar clinical stage. Of patients treated with the bladder preserving approach 20 to 30% cured of muscle invading cancer will subsequently have a new superficial tumor. The superficial tumors have responded well to intravesical drug therapy. Modern bladder preserving treatments usually result in a well functioning bladder without incontinence or significant hematuria. However, concurrent systemic chemotherapy and radiation have the potential for acute morbidity. Presently the ideal candidate for bladder preservation has primary clinical stage T2 tumor, no associated ureteral obstruction, visibly complete transurethral resection and complete response after induction chemoradiation based on endoscopic evaluation including re-biopsy and cytology., Conclusions: It is recommended that tri-modality treatment be administered by dedicated multimodality teams. In this country this approach to treatment is available at many of the institutions participating in the Radiation Therapy Oncology Group study. This treatment may be considered a reasonable alternative in patients who are deemed medically unfit for cystectomy and for those who are seeking an alternative to radical cystectomy.

Purpose: To assess the efficacy of neoadjuvant methotrexate, cisplatin, and vinblastine (MCV) chemotherapy in patients with muscle-invading bladder cancer treated with selective bladder preservation., Patients and Methods: One hundred twenty-three eligible patients with tumor, node, metastasis system clinical stage T2 to T4aNXMO bladder cancer were randomized to receive (arm 1, n=61 ) two cycles of MCV before 39.6-Gy pelvic irradiation with concurrent cisplatin 100 mg/m2 for two courses 3 weeks apart. Patients assigned to arm 2 (n=62) did not receive MCV before concurrent cisplatin and radiation therapy. Tumor response was scored as a clinical complete response (CR) when the cystoscopic tumor-site biopsy and urine cytology results were negative. The CR patients were treated with an additional 25.2 Gy to a total of 64.8 Gy and one additional dose of cisplatin. Those with less than a CR underwent cystectomy. The median follow-up of all patients who survived is 60 months., Results: Seventy-four percent of the patients completed the protocol with, at most, minor deviations; 67% on arm 1 and 81% on arm 2. The actuarial 5-year overall survival rate was 49%; 48% in arm 1 and 49% in arm 2. Thirty-five percent of the patients had evidence of distant metastases at 5 years; 33% in arm 1 and 39% in arm 2. The 5-year survival rate with a functioning bladder was 38%, 36% in arm 1 and 40% in arm 2. None of these differences are statistically significant., Conclusion: Two cycles of MCV neoadjuvant chemotherapy were not shown to increase the rate of CR over that achieved with our standard induction therapy or to increase freedom from metastatic disease. There was no impact on 5-year overall survival.

Purpose: We describe a protocol designed to evaluate the use of twice daily radiation used together with cisplatin and 5 fluorouracil (5-FU) in the treatment of operable transitional cell carcinoma of the bladder with potential bladder preservation., Materials and Methods: A total of 18 consecutive patients with T2-T4a bladder tumors underwent as complete a transurethral resection as possible, which was visibly complete in 14 cases. They then received twice daily radiation and infusion cisplatin and 5-FU during an induction phase. No therapy was given for 3 weeks, following which patients were reevaluated cystoscopically. Cases of clinical complete response by biopsy and cytology were consolidated with further chemotherapy/radiation using the same chemotherapeutic agents and radiation schedule. Patients who had incomplete responses were advised to undergo an immediate radical cystectomy. Of the 18 patients 15 subsequently received 3 cycles of adjuvant chemotherapy, consisting of methotrexate, cisplatin and vinblastine. Median followup for the entire group is 32 months., Results: Of the 18 patients 14 had no detectable tumor after induction therapy. Of the 4 patients with persistent tumor 2 underwent radical cystectomy and 2 refused cystectomy, 1 of whom was treated with partial cystectomy and the other with consolidation chemotherapy/radiation. The actuarial overall survival at 3 years was 83%. The chance of a patient being alive at 3 years with a native bladder was 78%. No patient required cystectomy for hematuria or bladder shrinkage. Three patients in whom superficial tumors developed were treated successfully with bacillus Calmette-Guerin. Small bowel obstruction in 1 case was corrected surgically., Conclusions: This pilot study demonstrates a high rate of response to this combined chemotherapy/radiation regimen in conjunction with a visibly complete transurethral resection. Reevaluation after a short induction phase allows for the early selection of patients with persistent disease for radical cystectomy.

Objective: To analyze the effects of 5-fluorouracil (5-FU) chemotherapy combined with preoperative irradiation and the role of intraoperative electron beam irradiation (IOERT) on the outcome of patients with primary locally advanced rectal or rectosigmoid cancer., Methods: From 1978 to 1996, 145 patients with locally advanced rectal cancer underwent moderate- to high-dose preoperative irradiation followed by surgical resection. Ninety-three patients received 5-FU as a bolus for 3 days during the first and last weeks of radiation therapy (84 patients) or as a continuous infusion throughout irradiation (9 patients). At surgery, IOERT was administered to the surgical bed of 73 patients with persistent tumor adherence or residual disease in the pelvis., Results: No differences in sphincter preservation, pathologic downstaging, or resectability rates were observed by 5-FU use. However, there were statistically significant improvements in 5-year actuarial local control and disease-specific survival in patients receiving 5-FU during irradiation compared with patients undergoing irradiation without 5-FU. For the 73 patients selected to receive IOERT, local control and disease-specific survival correlated with resection extent. For the 45 patients undergoing complete resection and IOERT, the 5-year actuarial local control and disease-specific survival were 89% and 63%, respectively. These figures were 65% and 32%, respectively, for the 28 patients undergoing IOERT for residual disease. The overall 5-year actuarial complication rate was 11%., Conclusions: Treatment strategies using 5-FU during irradiation and IOERT for patients with locally advanced rectal cancer are beneficial and well tolerated.

Purpose: Combined modality therapy has become the standard oncologic approach to achieve organ preservation in many malignancies., Methods and Materials: Although radical cystectomy has been considered as standard treatment for invasive bladder carcinoma in the United States, good results have been recently reported from several centers using multimodality treatment, particularly in patients with clinical T2 and T3a disease who do not have a ureter obstructed by tumor., Results: The components of the combined treatment are usually transurethral resection of the bladder tumor (TURBT) followed by concurrent chemotherapy and radiation therapy. Following an induction course of therapy a histologic response is evaluated by cystoscopy and rebiopsy. Clinical "complete responders" (tumor site rebiopsy negative and urine cytology with no tumor cells present) continue with a consolidation course of concurrent chemotherapy and radiation. Those patients not achieving a clinical complete response are recommended to have an immediate cystectomy. Individually the local monotherapies of radiation, TURBT, or multidrug chemotherapy each achieve a local control rate of the primary tumor of from 20 to 40%. When these are combined, clinical complete response rates of from 65 to 80% can be achieved. Seventy-five to 85% of the clinical complete responders will remain with bladders free of recurrence of an invasive tumor., Conclusions: Bladder conservation trials using combined modality treatment approaches with selection for organ conservation by response of the tumor to initial treatment report overall 5-year survival rates of approximately 50%, and a 40-45% 5-year survival rate with the bladder intact. These modern multimodality bladder conservation approaches offer survival rates similar to radical cystectomy for patients of similar clinical stage and age. Bladder-conserving therapy should be offered to patients with invasive bladder carcinoma as a realistic alternative to radical cystectomy by experienced multimodality teams of urologic oncologists.

Purpose: To analyze the impact of patient and treatment parameters in concurrent chemoradiation treatment for anal carcinoma., Methods and Materials: Retrospective review of 50 MO anal cancer patients treated from 1984-1994. Most patients received concurrent 5-FU, mitomycin, and radiation. Local control and disease-free/overall survival were determined and analyzed according to patient and treatment parameters., Results: With 43 month median follow-up, projected overall survival is 66% at 5 and 8 years. Disease-free survival is 67% at 5 years and 59% at 8 years. Local control is 70% at 5 and 8 years. Doses of > or =54 Gy are associated with improved 5-year survival (84 vs. 47%, p = 0.02), disease-free survival (74 v. 56%, p = 0.09), and local control (77 vs. 61%, p = 0.04). Although local control, disease-free survival, and overall survival were improved in patients whose overall treatment time was <40 days, this was not statistically significant. Outcome in the four patients with pretreatment hemoglobin (Hgb) <10 appeared worse with 3-year overall survival 50 vs. 68% (p = 0.07), disease-free survival 0 vs. 67% (p = 0.11), and local control 0 vs. 74% (p = 0.05). Projected 5-year overall survival, relapse-free survival, and local control in 4 HIV(+) patients is 0, 75, and 75%. Multivariate analysis reveals that dose (p = 0.02) and Hgb (p = 0.05) independently affect local control, dose (p = 0.02) affects disease-free survival, and dose (p = 0.01), Hgb (p = 0.03), T-stage (p = 0.03), and HIV-status (0.07) independently influence overall survival., Conclusion: Radiation doses of > or =54 Gy are associated with significantly improved survival and local control in anal cancer patients treated with chemoradiation. Overall treatment times of less than 40 days are associated with a trend towards improved outcome, but this is not significant. Pretreatment hemoglobin <10 is associated with worse treatment outcome. Survival of HIV (+) patient is poor, but the majority of such patients in this series died of intercurrent disease with their anal carcinomas controlled by chemoradiation.