Rapid (“Warm”) Autopsy Study for Procurement of Metastatic Prostate Cancer¹

The molecular study of hormone-refractory prostate cancer is difficult because most patients with widely disseminated disease never undergo tumor resection. The general lack of primary and metastatic tumor samples limits research to either established tumor cell lines or archival material. Therefore, there is a need for high-quality tumor samples from these advanced prostate cancers. The present study describes the “warm” autopsy or tissue procurement program at the University of Michigan.

The term “warm” derives from the short interval between time of death and acquisition of tissue samples during the autopsy. The primary goal of this program was to develop a tumor donor program that would allow men with metastatic hormone-refractory prostate cancer to agree to an immediate autopsy shortly after death. This program would serve as an invaluable resource for molecular and clinical studies on advanced prostate cancer. The following report describes the mechanics and short-term results of this program. Future studies will report on specific scientific projects that have used these materials.

Patients diagnosed with hormone-refractory prostate cancer were referred to the Medical Oncology Service of the Comprehensive Cancer Center at the University of Michigan Hospitals. These patients, whose disease had progressed despite conventional therapies, were offered participation in a variety of experimental therapeutic protocols,including listing as posthumous tissue donors. The objectives and procedures for tissue donation were explained to the patient. Having agreed to participate in this Institutional Review Board-approved tumor donor program, permission for autopsy was obtained before the death,with consent provided by the patient, or by next of kin. After death,consent was obtained by monitored telephone call to the next of kin. An operator acts as the witness for this procedure and records the telephone consent.

Since September 1996, 14 immediate autopsies have been performed at the University of Michigan Hospitals under the auspices of the Specialized Program of Research Excellence in Prostate Cancer (NCI Grant CA69568). These autopsies have been referred to as “warm” or recent autopsies because of the short time interval between patient death and starting the necropsy.

When a patient enrolled in this protocol dies, the patient’s family or guardian informs the staff medical oncologist participating in the program. In most cases, the patient was either hospitalized at the time of death or under hospice care. In cases where the patient died outside of the hospital, the body was transported to the University of Michigan Hospitals morgue. Simultaneously, the oncologist assembled the autopsy and tissue procurement teams through a pyramidal notification plan. The autopsy team consisted of a staff genitourinary pathologist,genitourinary pathology fellow, pathology resident, and a pathology assistant. The tissue procurement team consisted of the medical oncologist, staff and postdoctorate researchers, laboratory assistants,and a urology resident. Most all team members were available continuously by pager to assure round-the-clock availability.

On arrival in the morgue, an external examination of the cadaver was performed before spraying the body with ethanol. The pathology team approached the internal organs through an anterior, “Y-shaped”incision, extending from the shoulders to the pubis. The skin and subcutical tissues were retracted from the thorax and abdomen, and the breastplate was removed. The internal organs, from the larynx to the pelvis, were dissected from their posterior attachments to the body,and the diaphragm was freed from its peripheral attachments. While still in continuity with the body, the rectum was cross-clamped to prevent spillage of intestinal contents into the pelvis and abdominal cavities. The dissection was then relinquished to the urology resident.

If the prostate was in place, the urology resident resected the prostate, urinary bladder, and pelvic lymph nodes in a manner similar to that of a cystoprostatectomy. If a previous prostatectomy had been performed, the urology resident removed what pelvic contents remained. This included an evaluation of the pelvic lymph nodes for disease.

After the dissection of the pelvis, the rectum was transected, and the viscera were removed en bloc. The pelvic and the visceral blocks were passed to the pathology team, and primary prostate tumors and sites of metastasis were dissected. Tumor from these sites, as well as unaffected normal tissues, were sprayed with ethanol and harvested by the tissue procurement team under the direction of the staff pathologist. Although most of the tissues were well preserved because of the short interval from death to autopsy, attempts were made to harvest tumors from deep within tissues to further minimize the effects of bacterial contamination and to counter any effects that the alcohol spray may have on tissue culture. Normal and malignant tissues were collected as fresh tissue, snap frozen tissue using liquid nitrogen,and formalin-fixed tissue. A second pathology team removed the calvarium and brain and identified metastases in the skull and dura. The second team also took bone samples from the ribs and vertebral column. Examination for bone metastases was guided by the patient’s bone scan. All suspicious sites by bone scan were sampled. All bone lesions were confirmed by histology. Routinely, samples from multiple ribs, vertebral column, and skull were taken, regardless of bone scan results or presence of gross lesions. This coordinated effort allowed for rapid processing of fresh tissue samples. Histological sections stained with H&E were later prepared from the formalin-fixed,paraffin-embedded tissues to verify its malignant nature. Frozen tissue was stored at −70°C. Fresh tissue was transported to several laboratories for tissue culture experiments. Blood (20–30 ml) was collected in a purple-topped, heparinized tube for extraction of germ-line DNA. Once the tissue harvest was complete, the autopsy proceeded in accordance with the protocol established by the University of Michigan Autopsy Service. This protocol examines all organs(i.e., heart, thyroid, parathyroid, brain, and others) both grossly and microscopically. As standard with all routine autopsies, a detailed provisional and final report are issued by the Department of Pathology.

Frozen and formalin-fixed, paraffin-embedded tissue samples were kept in the Specialized Program of Research Excellence tissue core. All samples are electronically registered using a relational database(Microsoft Access). This allowed for convenient and rapid access of all samples.

Using aseptic technique, tumor tissue was harvested from several metastatic lesions. Special care was taken to avoid areas of necrosis. For example, when there were multiple metastatic tumors to the liver,the smaller metastases (2–4 cm) tended to be more viable; the larger tumors had central tumor necrosis, making them unsuitable for xenografting. The tumor issue was washed in a solution of HBSS (Life Technologies, Inc., Gaithersburg, MD) with 5.0% antibiotic/antimycotic(Life Technologies) at 4°C. Tissue was transferred to 150-mm tissue culture-treated plastic dishes (Fisher Scientific, Pittsburgh, PA) and cut into 2–5 mm² pieces using sterile #20 scalpel blades. Chunks of tumors were bathed on ice in a 1× solution of Matrigel (Becton Dickinson, Bedford, MA) and transferred to a laminar flow room housing immune-deficient animals. Nude (athymic) and SCID³ mice were briefly anesthetized by Metofane (methoxyflurane; Mallinckrodt Veterinary, Mundelein, IL) inhalation according to approved protocols. With scissors, a 5-mm incision was made over the dorso-lumbar region of each animal, and a cavity was created in the s.c. space using blunt dissection technique. Tumor pieces were inserted into the excavation using forceps, and the incision was closed with a sterile 9-mm “autoclip” staple (Fisher Scientific). Animals were monitored for a postoperative period of 9 months and sacrificed if tumor negative. Tumors were passaged into new animals every 90–100 days or when the size reached 3000 mm² using the above technique. Tumor tissue was introduced into in vitro tissue culture as described below.

During animal passage, tumor tissue was minced with scalpels and plated on tissue culture grade 150-mm plastic dishes. Cells were maintained in a humidified incubator at 37°C with 5.0% CO₂. Initially, media formulations were supplemented with growth factors presumed to augment the growth of epithelial-like cells. Specifically,a 1:1 mixture of DMEM/F-12 (Life Technologies) media supplemented with 10 μg/ml epithelial growth factor (Life Technologies), 5 μg/ml insulin (Sigma Chemical, St. Louis, MO), 10% fetal bovine serum (Life Technologies), and 1% antibiotic/antimycotic supported the viability of tumor cells introduced into cell culture. Cells are routinely passaged every 7 days, and medium was exchanged every 3–4 days. At least 1–2 million cells per 25-mm flask must be used for viable passaging.

Conditioned medium was examined for the presence of PSA by Dr. Robert L. Vessella (University of Washington, Seattle, WA), using an automated IMx PSA immunoassay system (Abbott Laboratories, Chicago, IL),according to the manufacturer’s specifications. Confirmation of PSA expression by the xenografts was performed using formalin-fixed,paraffin-embedded pieces of xenograft tissue. Monoclonal antibodies for PSA (DAKO Corp., Carpinteria, CA) were diluted 1:20. Secondary antibodies were conjugated to biotin, and antigens were detected using the avidin/biotin complex procedure. All staining was performed using an automated stainer (Ventana E.S.; Ventana Medical Systems, Tucson,AZ).

Between September 1996 and January 1999, 14 autopsies were performed. These were full autopsies with no restrictions. The average age at time of death was 68.8 years (range, 53–79 years). The median time to death after diagnosis was 7.5 years (range, 1.8–16 years). This wide range between initial diagnosis and time of death reflects that some cases were initially diagnosed with clinically localized prostate cancer but developed widely disseminated disease after 5–10 years.

The autopsies were performed as outlined in “Materials and Methods.” The median time to autopsy was 2.8 h (range, 1–4.5 h). In most all of the cases performed after 2 h, the delay was because of transportation of the body from a home or hospice to the University Hospital. Detailed clinical information regarding the 14 patients is presented in Table 1 and summarized in Table 2. The autopsies revealed widely disseminated prostate tumor in most of the cases (Table 3). Some sites, such as the orbit(case 2), were only examined in some autopsies based on clinical suspicion. In case 2, the patient had loss of vision in one eye. A summary of autopsy results presented in Fig. 1 demonstrates the percentage distribution of prostate cancer for the 14 autopsies. The majority of cases had prostatic involvement (9 of 10). However, except in 2 cases,the tumor was seen only focally. Nine of 14 cases had liver metastases,and these were the largest tumors identified. Eight of 14 cases had some lymph node involvement. In these cases, chains of lymph nodes were involved. Periaortic and pulmonary perihilar lymph nodes were two common sites. Interestingly, pelvic lymph nodes were not involved in any of the cases. The distribution of bony metastases is presented in Fig. 2. Twelve of the 14 cases had some evidence of bony metastases. The vertebral column, dura, skull, and ribs were involved in 64% (9 of 14), 43% (6 of 14), 43% (6 of 14),and 36% (5 of 14) of the cases, respectively. Although the dura is not a true bony site, these tumors did have underlying bony involvement, as demonstrated by histological examination. Of interest, one patient with dural metastases developed a unilateral hearing loss 3 months prior to death.

One of the major goals of this project was to obtain high-quality tumor tissue to further the study of prostate cancer research. The majority of the cases yielded such tissue. Bulky tumor metastases were harvested (Fig. 3, A–C). Care was taken to remove areas of necrosis. The samples obtained yielded excellent quality material. This is evidenced by good tumor histology,immunoreactivity for PSA, and the ability to develop xenografts. At the microscopic level (Fig. 4), little autolysis was seen. The tumor samples were immunoreactive for PSA (14 of 14 cases; Fig. 4). Xenografts were successfully established from at least 8 tumors (Fig. 3D). There was a success rate of ∼5%for all of the attempted xenografts. Interestingly, this success rate was unrelated to the postmortem interval. However, it should be noted that this interval was never >4.5 h. A brief description of the xenografts and cell lines is presented in Table 4.

Autopsy series have been a valuable part of understanding the natural history of prostate cancer. The majority of these have been descriptive, reporting on the incidence and prevalence of prostate cancer (1, 2, 3, 4, 5, 6, 7, 8, 9). Some extremely valuable work has been performed by Sakr et al. (10, 11) looking at autopsies to identify premalignant and early prostate tumors in patients who died of other causes. Other groups at Johns Hopkins(Baltimore, Maryland) and Washington University (Seattle, Washington)are currently developing tissue procurement programs. The design of our program was based on these studies. Several autopsy series have concentrated on men who died of widely metastatic prostate cancer. Usually, as seen in the present study, residual tumors are seen in the prostate with metastases to the liver, lung, lymph nodes, adrenal, and pelvic soft tissues. However, metastases to the brain are rare (12), involving <5% of most autopsy series. In contrast,metastases to the bones and vertebral column are common. Tumor extension from metastatic foci in skull or meninges is a recognized but infrequently documented mechanism of brain involvement in prostate cancer. In the present study, dural metastases were seen in 40% of the cases.

One limitation of any autopsy series is the potential to underestimate the extent of disease. For example, in this present study, metastatic disease to the bone was not seen in 2 of 14 cases. Were these cases really negative? Because the medical oncologists taking care of the patients are involved with the autopsy program, relevant clinical data are known at the time of autopsy. This includes the results of the patient’s bone scan. This information helps guide the team to concentrate on all suspicious areas. In the dissection of the pelvis,an experienced urology resident performed a careful dissection looking for any evidence of pelvic lymph nodes. Therefore, the negative findings for pelvic lymph nodes suggest that local disease was not present, as opposed to a lack of sampling in this area. However, the issue of sampling error cannot be excluded.

Most of the metastatic tumors seen in the current study were growing in sheets of poorly differentiated cells. In the cases where primary tumor was identified, the tumor demonstrated either treatment effect or was poorly differentiated. In all cases, a PSA immunostain was performed to confirm prostatic origin. In cases where the tumor is widely metastatic, it is common to find poorly differentiated tumor. Brawn and Speights (13) looked at 100 autopsies of men with widely metastatic prostate cancer; 70% of tumors were poorly differentiated,and 18% were only focally poorly differentiated. This is in contrast to men with metastatic disease to lymph nodes only; in such cases, 41%were differentiated, and 43% predominantly differentiated. Metastatic tumor identified as an incidental finding in pelvic lymph nodes of men undergoing radical prostatectomy for clinically localized disease demonstrated histologically differentiated disease. In most cases, the prostatic cancer maintained a glandular appearance. Therefore, the type of metastatic prostate cancer identified in the present study has a distinct histological appearance from the early metastases seen in pelvic lymph nodes found incidentally at time of prostatectomy for clinically localized prostate cancer. Yet these tumors still expressed PSA.

The present study provided ample frozen tissue for research purposes. The quality of this material has been excellent, as demonstrated by the ability to grow xenografts. Eight xenografts were developed and have now been serially passaged in mice. Of these eight xenografts, five were developed from bone, two from connective tissue,and one from soft tissue metastatic sites. H&E stain showed them to be consistent with prostatic adenocarcinoma. Histological sections from each xenograft stained positive for PSA. These tumors are a valuable resource to study essentially pure prostate tumor. In addition, because the original tumor (usually metastatic tumor) is available, comparisons can be made with regards to the histology, protein expression, DNA, and RNA.

Xenografts have been extremely helpful in identifying new candidate tumor suppressor genes such as PTEN/MMAC1(14, 15). Novel putative genes involved in prostate cancer have also been identified using xenografts. We are currently isolating and characterizing cell lines from each xenograft, one of which has shown strong evidence of being metastatic to bone in SCID mice. These cell lines may be a valuable addition to existing models of metastatic prostate cancer by providing both in vivo and in vitro model systems developed from the same tissue source. The autopsy study has been particularly useful because of the abundant amount of tumor tissue for potential implantation into SCID or athymic mice. Other samples, such as radical prostatectomy specimens, usually contain only a small amount of tumor; in our institution, cases with grossly recognizable tumor are uncommon.

One great obstacle in developing and maintaining a rapid autopsy program are the resources required to support such an activity. Centers with support from the Specialized Program of Research Excellence or O’Brian programs have been able to maintain vigorous programs. The personnel also need to be available on an on-call basis for the proper functioning of this program. Perhaps most vital to the success of this program is the participation of a clinical oncologist(s), who can recruit potential donors.

The future goal of this program is to continue to recruit men with advanced prostate cancer. By expanding this program, this should make the distribution of these valuable tumor samples and xenografts available to other researchers.

This work is dedicated to the men and their families who through their generous tissue donations have facilitated the study of advanced prostate cancer.