Robert P. Huben

A new approach in the diagnosis and follow-up of bladder cancer: FISH analysis of urine, bladder washings, and tumors

The aim of the present study was to ascertain whether fluorescence in situ hybridization (FISH) of urine could be a useful approach in bladder cancer. Herein, we present the cytogenetic and FISH findings in patients with and without bladder cancer. The samples examined with FISH consisted of urine, bladder washings, and tumor tissue, when available. The results obtained show that the FISH technique, particularly when used on urine, is a very useful tool in the diagnosis, early detection, and management of bladder cancer.

Assessment of Human Genitourinary Tumors and Chemosensitivity Testing In 3-Dimensional Collagen Gel Culture

A recently described collagen gel culture technique has been modified to evaluate the growth characteristics and chemosensitivity patterns of genitourinary neoplasms. Fresh human surgical explants incorporated radiolabeled DNA precursors [H3)thymidine or deoxyuridine) in 97% of 38 patient specimens (18/19 bladder, 3/3 prostate, 2/2 testis, 13/14 renal), after being maintained for three to 13 weeks/passage, with several specimens reaching their sixth passage (20 months). Control cellular DNA incorporation ranged from five to 90% (#cells labeled/#cells evaluated), with median labeling for bladder 30%, prostate 80%, testis 90%, and renal 80%. Original histopathologic classification was maintained in all cases. Tumor volume and glucose consumption were other measurable parameters. Seventy-three surgical specimen cultures were treated with chemotherapeutic agents after a minimum of four weeks in culture. Single agent exposures were 24 hours at 1X and 10X reported peak plasma concentrations. Combination agents were sequenced as in current clinical protocol for bladder tumors and fourteen day continuous fluorodeoxyuridine (FdURD) exposure for renal tumors. Sensitivity was found in 1/2 prostate and 1/10 renal tumors to Adriamycin, 8/15 bladder and 1/2 testis tumors to cisplatin, 11/28 renal tumors to FdURD and 6/16 bladder tumors to MVAC combination chemotherapy. This culture system offers the advantages of in vivo-like solid tumor growth, a high culture success rate, longevity in culture, maintenance of the primary histopathology and reproducible chemosensitivity response.

If you ‘watch and wait,’ prostate cancer may progress dramatically

PURPOSEnObservation has been proposed as an option for localized prostate cancer. However, most series reporting on watch and wait include patients treated by TUR or hormones that may affect results. We retrospectively reviewed the natural history of truly untreated prostate cancer and report the outcome for these patients.nnnMETHODS AND MATERIALSnFrom 1976 to 1992, 34 patients of median age 70 years (range 56-88) with biopsy proven localized adenocarcinoma of the prostate refused therapy. All had negative bone scan and none underwent TUR or hormone treatment. No patient was lost to follow-up (median 76 months). Failure patterns and survival were analyzed.nnnRESULTSnAt diagnosis 27 patients had palpable nodules (T2), of which 13 were well differentiated and 14 moderately differentiated. Seven had moderately differentiated T3 lesions. Mild prostatitis including nocturia, hesistancy, and urgency were reported in 16 T2 and 6 T3 patients. Within 36 months, local progression requiring therapy occurred in all T3, all T2 moderate and 5 of 13 T2 well-differentiated patients. Systemic progression occurred in 6 of 7 T3, 9 of 14 T2 (mod), and 2 of 13 T2 (well) patients. Overall 59% are alive, 26% succumbed to prostate carcinoma and 15% to other causes.nnnCONCLUSIONnObservation results in a high rate of local progression requiring intervention (77%) and excessive systemic disease development (50%) for patients with clinically palpable disease. Perhaps this strategy is viable for earlier stage lesions detected by PSA but it must be tested in a rigorous fashion before accepted.

LOCALLY RECURRENT ENDOMETRIOID ADENOCARCINOMA OF THE PROSTATE AFTER RADICAL PROSTATECTOMY

Endometrioid carcinoma of the prostate is considered a variant of classical prostatic ductal carcinoma. Endometrioid carcinoma variant often has the unique clinical presentation of gross hematuria. The propensity of this tumor to spread within the urothelium makes local failure of curative therapy commonplace. We present 2 representative cases with a review of followup surveillance procedures and treatment options for the local recurrence once identified.

Reported family history of cancer in 1,271 prostate cancer cases and 1,909 controls

Prostate cancer risk has been associated with a family history of the disease. A two- to three-fold increase in risk has been observed in several studies. Details concerning modification of this risk by age, type of familial history of prostate cancer, and possible involvement of history of cancer at other sites have been less well documented. This case-control study of 1,271 prostate cancer patients and 1,909 control subjects admitted to Roswell Park Cancer Institute in Buffalo, NY, found age-adjusted increased risk associated with reporting a history of prostate cancer in a father (RR = 2.3, 95% Cl 1.4-3.3) or brother (RR = 2.5, 95% Cl 1.6-3.9). Subjects with both a father and brother affected had a 6.5-fold (95% Cl 1.4-30.5) increased risk of prostate cancer. Greater risk were observed at younger ages of diagnosis. Risks associated with reporting a father or a brother affected were not significantly elevated for patients over age 70 at diagnosis. No significant differences in patients reporting histories of cancer other than prostate cancer were observed regardless of relationship, age at diagnosis, or type of cancer examined. These observations from a large cancer patient population may be useful when making recommendations for cost-effective prostate cancer screening and for directing investigators to the potentially most informative subjects.

Hormone Therapy of Prostatic Bone Metastases

When present at diagnosis or when developing in the course of disease, the presence of bone metastases from prostate cancer is generally considered an indication to begin endocrine therapy, as this is clearly the most effective form of treatment for this problem. Endocrine therapy can stop progression of prostate cancer in 80-85% of cases. Endocrine therapy can relieve pain, prevent pathologic fractures, and prevent neurologic complications from bone metastases from prostate cancer. Rarely, bone scans may become normal after the start of endocrine therapy, but partial improvement or stabilization of bone scans are more commonly seen. While endocrine therapy has been the first line of treatment of metastatic prostate cancer for the past 50 years, the recent development of newer forms of endocrine therapy have increased the options in the past few years. In addition to orchiectomy and estrogens, newer alternatives include inhibitors of androgen synthesis, the class of agents termed antiandrogens, and luteinizing hormone releasing-hormone (LHRH) analogues either alone or in combination. Orchiectomy causes a prompt fall in serum testosterone and is regarded by many as the standard form of endocrine therapy, but there is concern about the psychologic impact of surgery. Estrogens are being used less frequently today because of their real or potential side-effects, including cardiovascular and thromboembolic complications. The development of analogues of LHRH has resulted in another major choice for endocrine therapy, and one which is therapeutically equivalent to orchiectomy or estrogens. Since LHRH analogues may cause an early rise or flare in serum testosterone before it drops to castrate level, these agents should not be given alone to patients with severe pain or neurologic problems. The newly available antiandrogen flutamide can block the flare, and may also improve survival when used with LHRH analogues or orchiectomy, especially when disease is less advanced. Not all studies of combination therapy support this conclusion. However, the use of flutamide is increasing significantly in the United States. Both the LHRH analogues and flutamide are fairly safe, but they are very expensive. Their use, in combination, is likely to become a progressively more common form of initial endocrine therapy in the future. The growing application of prostate specific antigen (PSA) as a tumor marker for prostate cancer has made the difficulty in interpreting changes in bone scans a much less critical problem in determining response to endocrine or other forms of therapy for advanced prostate cancer.

Growth of human renal cortical tissue on collagen gel.

SummaryA model system for 3-dimensional “native-state” culture of tissues on collagen gels (Proc. Natl. Acad. Sci. USA 86:2013–2017; 1989) has been applied in this study to histologically normal human renal cortical tissue from 11 patients undergoing nephrectomy for renal cell carcinoma elsewhere in the kidney. Microbial contamination occurred in 12/90 cultures, the rest (78) were studied by visual inspection, histology, immunohistochemical analysis for pankeratin (epithelial cell origin), vimentin (mesenchymal cell origin), andp-glycoprotein (associated with proximal tubules), transmission electron microscopy (EM), incorporation of tritiated thymidine (3HTdR). In the first 10 days, explants showed3HTdR-labeled cells in tubule structures. The surrounding gel was invaded by cells forming tubule structures, sometimes with basement membrane. Some of these cells showed labeling by3HTdR and immunostaining positive for pankeratin andp-glycoprotein. EM showed well-polarized epithelial cells in tubule structures with tight junctions, interdigitating lateral processes, and microvilli characteristic of proximal and distal convoluted tubules.3HTdR-labeled cells in tubule structures were observed even 2 mo. after Passage 1, 6 mo. after the initial explantation. Tubule growth was most active and fibroblast proliferation was negligible from 2 to 4 wk postexplantation. The proliferation of tubulelike cells and formation of tubulelike structures in this system represents an opportunity to study human renal cortical tissue in vitro, under conditions more closely resembling in vivo circumstances than are present in other in vitro systems suitable for long-term study. This model has potential use for in vitro toxicology studies and studies of renal physiology.

In Vitro Study of the Interaction of Doxorubicin, Thiotepa, and Mitomycin-C, Agents Used for Intravesical Chemotherapy of Superficial Bladder Cancer

Several cytotoxic agents have been identified as effective in the treatment of superficial transitional cell carcinoma of the bladder, including doxorubicin, thiotepa and mitomycin-C. An in vitro study was conducted to assess the interactions of these three drugs against a well differentiated human bladder tumor cell line, RT-4, to identify and evaluate synergistic combinations among these agents. Cytotoxicity was evaluated by a colorimetric assay based on the capacity of viable cells to metabolize a tetrazolium dye, MTT, to produce a colored formazan product. The analyses of drug interactions were done by the isobolographic method (construction of isoeffect plots). The combination of doxorubicin and thiotepa was found to be the most synergistic, followed by the combination of doxorubicin and mitomycin-C. The combination of mitomycin C and thiotepa demonstrated an unpredictable effect. These findings suggest the combination of doxorubicin and thiotepa has potential advantage for chemotherapy of superficial bladder tumors.

Effect of Perioperative Chemoimmunotherapy with Cyclophosphamide and Autologous Tumor Vaccine in Murine MBT-2 Bladder Cancer

The in vitro cytotoxic activity of splenocytes from C3H/He mice implanted subcutaneously with 10(6) syngeneic MBT-2 tumor cells on day 0 was significantly enhanced after cyclophosphamide (100 mg./kg., intraperitoneally) given 2 days before tumor resection on day 17, with or without active specific immunization with BCG plus autologous irradiated tumor cells (vaccine) 1 week after tumor resection. Furthermore, a significantly lower tumor incidence was seen in mice challenged with 10(5), but not 10(6), tumor cells per mouse 24 hours after tumor resection on day 17 and treated with cyclophosphamide on day 15 and postoperatively with vaccine than was found in nontreated tumor resected mice. Phenotypic analysis of cells from spleen showed that cyclophosphamide pretreatment and postoperative vaccine, either singly or in combination, induced a significant increase of both CD44+ memory T cells and CD11b+ myeloid/macrophage cells. Thus, in addition to a specific antitumor immune response, a nonspecific cytolytic mechanism may also play a role in the observed antitumor effect.

Glucose consumption end point in primary histoculture indicates recovery of human tumors from drug treatment

Dear Editor: It is well established that individuals with the same histologic type of cancer do not respond uniformly tO currently used anticancer agents. Investigators have developed a wide array of in vitro assays utilizing both animal and human cancer models in an effort to understand these biological differences (1,5-7). A further aim of these test systems is to predict the response of individual patients and study the determinants of drug action at the tissue and cellular level to develop improved therapeutic agents and strategies (7). Hoffman and coworkers have developed a drug-response assay using collagen-sponge-gels to histocuhure tumors which allows the maintenance of tissue architecture and function in vitro (14). Histopathological analysis, DNA precursor uptake (labeling index) and ability to reduce tetrazolium dyes have been studied as growth parameters of human tumors in the histoculture assay (2,3,7,8,11,13). However, these end points, although useful, are destructive techniques requiring termination of the culture at the time of analysis. The measurement of glucose consumption, however, is nondestructive, allowing serial determinations over extended periods in culture. In this report, we describe the use of the glucose-consumption end point in the histocuhure drug-response assay and the new insights it reveals into the ability of tumors to recover from drug treatment. We have previously demonstrated a high success rate of growing human urological tumors in histocuhure on collagen sponge gel (1,11) and have chosen this tumor type in histocuhure to test the end point of glucose consumption. Tumor tissue identified at the time of radical nephrectomy was transported under sterile conditions to the laboratory within 1 hour of excision. Tumor specimens were reduced to 2 × 2 mm pieces with 5 pieces implanted directly onto rehydrated collagen sponge gels (1 × 1 cm 2) (Health Design Indust., Rochester, NY). Each gel occupied one well of a six-well plate (Falcon, Lincoln Park, N J). 2.5 ml of medium which was 90% Eagles minimal essential medium (MEM) (Gibco, Grand Island, NY) and 10% fetal bovine serum (Gibco) were added. Gentamycin (Gibco) was present at a final concentration of 50/.tg/ml, and cefotaxime (Hoechst, Somerville, N J) was present at a final concentration of 1 #g/ml. l~he final volume of medium was sufficient to reach the upper gel surface without immersing it (2,8,13). Covered cultured plates were maintained in a humidified 5%-CO2 incubator at 37 ° C. Cultures underwent sterile-media changes every 72 hours. Fifty ttl of culture medium were taken every 24 hours for determination of glucose content in triplicate using the (HK20) assay kit from Sigma (St. Louis, MO). Measurements are made by monitoring the change in optical density at 340 nm due to reduction of NAD through glucose consumption by hexokinase. The glucose content of the medium was plotted as a semilog plot versus time after medium renewal using Sigmaplot (Jandel Scientific, Corte Madera, CA). A simple exponential model of glucose consumption was then fitted to the data with the program Systat (Systat, Inc., Evanston, IL). The half-life of glucose was calculated from the slope parameter of this model using the equation t 1/2 = 0.693/s, where s =slope of the best-fit linear-regression line of the natural log of the glucose concentration plotted versus time. Glucose content of the medium was measured daily for 3 days. The log values over 3 days were plotted vs. time and the slope of best fit line was taken as the glucose consumption rate during the 3-day period (one period) (Fig. 1 A-C). 5-fluoro-2-deoxyuridine (FdUrd) (Sigma) was added in final concentrations of 0.6 /~M and 6.0 #M. Continuous exposure to FdUrd for 12 days was chosen to reflect a current clinical protocol for long-term infusion in patients with metastatic renal carcinoma (9). The concentrations of drug chosen represent values that can be achieved clinically (12). Analysis of glucose consumption demonstrated that FdUrd produced a transient suppression of glucose consumption with recovery to pretreatment baseline by Day 77 post-treatment (Fig. 1 A-C). Although growth in this experiment was evident even by gross examination, this growth was slow (over a period of 6 months), and did not appear to change the glucose consumption rate in the controls over the 80 days monitored in these experiments. Individual specimens were fixed in 10% buffered formalin (pH 7.4) for 24 hours and embedded in paraffin. Sections five microns thick were stained with Harris hematoxylin and eosin. The primary specimen, control and drug-treated specimens were examined by a pathologist (K. T.). Histopathological changes (necrosis, cellular disorganization, degenerative nuclear changes) and intact tumor cells were observed in all drug treated specimens. Such damage was most pronounced in specimens treated at the highest FdUrd dose (6.0 #M), yet even these cultures recovered. The collagen-sponge-gel-supported histocuhure system allowed the study of renal cell carcinoma in vitro for several months, while maintaining tissue architecture present in the original tumor. The histoculture system appears, therefore, to be useful for monitoring the long-term growth of primary renal cell carcinoma and is a suitable system for quantitation of drug effects by determination of glucose consumption rates which is nondestructive and may be used over long time periods. Since the specimen is studied in vitro in the tissue form, use of the system may provide new information on chemotherapeutic effects at the tissue level. We have made no attempt to determine whether anaerobic or aerobic metabohsm of glucose predominates in these tissues. Regardless of the pathway of glucose utilization (4,10), however, glucose consumption rates appear to be quite stable for a given culture,