Limitations of stereotactic biopsy in the initial management of gliomas.

Stereotactic biopsy is often performed for diagnostic purposes before treating patients whose imaging studies highly suggest glioma. Indications cited for biopsy include diagnosis and/or the "inoperability" of the tumor. This study questions the routine use of stereotactic biopsy in the initial management of gliomas. At The University of Texas M. D. Anderson Cancer Center, we retrospectively reviewed a consecutive series of 81 patients whose imaging studies suggested glioma and who underwent stereotactic biopsy followed by craniotomy/resection (within 60 days) between 1993 and 1998. All relevant clinical and imaging information was reviewed, including computerized volumetric analysis of the tumors based on pre- and postoperative MRI. Stereotactic biopsy was performed at institutions other than M. D. Anderson in 78 (96%) of 81 patients. The majority of tumors were located either in eloquent brain (36 of 81 = 44%) or near-eloquent brain (41 of 81 = 51%), and this frequently was the rationale cited for performing stereotactic biopsy. Gross total resection (>95%) was achieved in 46 (57%) of 81 patients, with a median extent of resection of 96% for this series. Diagnoses based on biopsy or resection in the same patient differed in 40 (49%) of 82 cases. This discrepancy was reduced to 30 (38%) of 80 cases when the biopsy slides were reviewed preoperatively by each of three neuropathologists at M. D. Anderson. Major neurologic complications occurred in 10 (12.3%) of 81 surgical patients and 3 (3.7%) of 81 patients undergoing biopsy. Surgical morbidity was probably higher in our series than it would be for glioma patients in general because our patients represent a highly selected subset of glioma patients whose tumors present a technical challenge to remove. Stereotactic biopsy is frequently inaccurate in providing a correct diagnosis and is associated with additional risk and cost. If stereotactic biopsy is performed, expert neuropathology consultation should be sought.

Coaxial double-lumen methylmethacrylate reconstruction in the anterior cervical and upper thoracic spine after tumor resection.

OBJECT: A unique method of anterior spinal reconstruction after decompressive surgery was used to prevent methylmethacrylate-dural contact in cancer patients who underwent corpectomy. The purpose of this study was to assess the efficacy and stability of polymethylmethacrylate (PMMA) anterior surgical constructs in conjunction with anterior cervical plate stabilization (ACPS) in these patients. METHODS: Approximately 700 patients underwent spinal surgery at The University of Texas M. D. Anderson Cancer Center over a 4-year period. The authors conducted a retrospective outcome study for 29 of these patients who underwent anterior cervical or upper thoracic tumor resections while in the supine position. These patients were all treated using the coaxial, double-lumen, PMMA technique for anterior spinal reconstruction with subsequent ACPS. No postoperative external orthoses were used. Twenty-seven patients (93%) harbored metastatic spinal lesions and two (7%) harbored primary tumors. At 1 month postsurgery, significant improvement was seen in spinal axial pain (p<0.001), radiculopathy (p<0.001), gait (p = 0.008), and Frankel grade (p = 0.002). A total of nine patients (31%) underwent combined anterior-posterior 360 degrees stabilization. Twenty-one patients (72%) experienced no complications. Complications related to instrumentation failure occurred in only two patients (7%). There were no cases in which the patients' status worsened, and there were no neurological complications or infections. The median Kaplan-Meier survival estimate for patients with spinal metastases was 9.5 months. At the end of the study, 13 patients (45%) had died and 16 (55%) were alive. Postoperative magnetic resonance images consistently demonstrated that the dura and PMMA in all patients remained separated. CONCLUSIONS: The anterior, coaxial, double-lumen, PMMA reconstruction technique provides a simple means of spinal cord protection in patients in the supine position while undergoing surgery and offers excellent results in cancer patients who have undergone cervical vertebrectomy.

Intracranial injection of human meningioma cells in athymic mice: an orthotopic model for meningioma growth.

OBJECT: Although human meningioma cells have been heterotopically implanted in nude mice, introducing these cells into intracranial locations seems more likely to reproduce normal patterns of tumor growth. To provide an orthotopic xenograft model of meningioma, the authors implanted a controlled quantity of meningioma cells at subdural and intracerebral sites in athymic mice. METHODS: Malignant (one tumor), atypical (two tumors), or benign (three tumors) meningiomas were placed into primary cell cultures. Cells (10(6)/10 microl) from these cultures and from an immortalized malignant meningioma cell line, IOMM-Lee, were injected with stereotactic guidance into the frontal white matter or subdural space of athymic mice. Survival curves were plotted for mice receiving tumor cells of each histological type and according to injection site. Other mice were killed at intervals and their heads were sectioned whole. Hematoxylin and eosin staining of these sections revealed the extent of tumor growth. CONCLUSIONS: The median length of survival for mice with malignant, atypical, or benign tumors was 19, 42, or longer than 84 days, respectively. Atypical and malignant tumors were invasive, but did not metastasize extracranially. Malignant tumors uniformly showed leptomeningeal dissemination and those implanted intracerebrally grew locally and spread noncontiguously to the ventricles, choroid plexus, convexities, and skull base. Tumors formed in only 50% of mice injected with benign meningioma cells, whereas injection of more aggressive cells was uniformly successful at tumor production. The three types of human meningiomas grown intracranially in athymic mice maintained their relative positions in the spectrum of malignancy. However, atypical meningiomas became more aggressive after xenografting and acquired malignant features, implying that there had been immune constraint in the original host. Tumor cells injected into brain parenchyma migrated to more optimal environments and grew best there. This model provides insights into the biology of meningiomas and may be useful for testing new therapies.

Chondrosarcoma of the spine: 1954 to 1997.

OBJECT: Primary chondrosarcoma of the spine is extremely rare. During the last 43 years only 21 patients with this disease were registered at The University of Texas M. D. Anderson Cancer Center. The purpose of this study was to examine the demographic characteristics, treatments, and outcomes of this set of patients. METHODS: Medical records for 21 patients were reviewed. Age, sex, race, clinical presentation, tumor histology, tumor location in the spinal column, treatments, surgical details, and response to treatment were recorded. Surgical procedures were categorized as either gross-total resection or subtotal excision of tumor. Neurological function was assessed using Frankel's functional classification. Time to recurrence and survival analyses were performed using the Kaplan-Meier method. The median age of patients was 51 years, with fairly equal gender representation. Eighteen patients underwent at least one surgical procedure for a total of 28 surgical procedures: seven radical resections and 21 subtotal excisions. Radiation therapy was used in conjunction with 10 of the 28 surgical procedures. The median Kaplan-Meier estimate of overall survival for the entire group was 6 years (range 6 months-17 years). Tumors recurred after 18 of the 28 procedures. Kaplan-Meier analysis revealed a statistically significant difference in the per-procedure disease-free interval after gross-total resection relative to subtotal excision (exact log rank 3.39; p = 0.04). The addition of radiation therapy prolonged the median disease-free interval from 16 to 44 months, although this was not statistically significant (exact log rank 2.63; p = 0.16). CONCLUSIONS: Our results suggest that gross-total resection of the chondrosarcoma provides the best chance for prolonging the disease-free interval in patients. Subtotal excision should be avoided whenever possible. Addition of radiation therapy does not appear to lengthen significantly the disease-free interval in this patient population.

Gastric cancer and metastasis to the brain.

BACKGROUND: Metastasis of gastric carcinoma to the brain is very uncommon. At The University of Texas M. D. Anderson Cancer Center (M. D. Anderson), less than 1% of patients with primary gastric carcinoma are found to have brain metastases. Little has been published regarding the evaluation and treatment of these patients. The purpose of this study was to review our experience with gastric cancer metastatic to the brain and to describe the efficacy of the treatment used. METHODS: Between 1957 and 1997, a total of 218,690 patients were seen for evaluation of malignant tumors at M. D. Anderson. Of these patients, 3320 (1.5%) had a diagnosis of gastric cancer; however, only 24 patients (0.7%) were found to have brain metastases on imaging studies or at autopsy. We performed a retrospective review of these 24 patients and divided them into three groups on the basis of the treatment they received. RESULTS: Group 1 included patients who received steroids alone (16 mg of dexamethasone, daily). Group 2 patients received 3000 cGy of whole-brain radiation therapy (WBRT) delivered in 10 fractions in addition to steroids. Group 3 patients were managed with surgical resection, WBRT, and steroids. There were 18 male and 6 female patients, with a median age of 53 years. The most common presenting symptoms were weakness, difficulty with balance, and headache. Of the 19 patients diagnosed antemortem, 11 patients developed neurological symptoms after the primary diagnosis of gastric carcinoma, whereas 8 patients developed neurological symptoms before the diagnosis of gastric cancer. Forty-five percent of patients had a single brain metastasis, whereas 55% had multiple lesions. All patients had systemic disease, with bone, liver, and lung involvement seen in 46%, 42%, and 29%, respectively. Nineteen of 24 patients received treatment after diagnosis of brain metastases. Four patients received steroids only (group 1), 11 patients received WBRT and steroids (group 2), and 4 patients were treated with surgery, WBRT, and steroids (group 3). Median survival was approximately 2 months for patients in groups 1 and 2, whereas group 3 patients had a median survival of slightly greater than I year. CONCLUSIONS: Our results suggest that the overall prognosis of patients with brain metastases from gastric cancer is extremely poor (median survival, 9 weeks). WBRT, as an adjuvant to steroid treatment, was not effective in improving outcome in our series. In selected patients, most of whom were relatively young and had less advanced systemic disease, surgical resection followed by WBRT was associated with relatively long survival times (median survival, 54 weeks).

Transthoracic vertebrectomy for metastatic spinal tumors.

OBJECT: Anterior approaches to the spine for the treatment of spinal tumors have gained acceptance; however, in most published reports, patients with primary, metastatic, or chest wall tumors involving cervical, thoracic, or lumbar regions of the spine are combined. The purpose of this study was to provide a clear perspective of results that can be expected in patients who undergo anterior vertebral body resection, reconstruction, and stabilization for spinal metastases that are limited to the thoracic region. METHODS: Outcome is presented for 72 patients with metastatic spinal tumors who were treated by transthoracic vertebrectomy at The University of Texas M. D. Anderson Cancer Center. The predominant primary tumors included renal cancer in 19 patients, breast cancer in 10, melanoma or sarcoma in 10, and lung cancer in nine patients. The most common presenting symptoms were back pain, which occurred in 90% of patients, and lower-extremity weakness, which occurred in 64% of patients. All patients underwent transthoracic vertebrectomy, decompression, reconstruction with methylmethacrylate, and anterior fixation with locking plate and screw constructs. Supplemental posterior instrumentation was required in seven patients with disease involving the cervicothoracic or thoracolumbar junction, which was causing severe kyphosis. After surgery, pain improved in 60 of 65 patients. This improvement was found to be statistically significant (p < 0.001) based on visual analog scales and narcotic analgesic medication use. Thirty-five of the 46 patients who presented with neurological dysfunction improved significantly (p < 0.001) following the procedure. Thirty-three patients had weakness but could ambulate preoperatively. Seventeen of these 33 regained normal strength, 15 patients continued to have weakness, and one patient was neurologically worse postoperatively. Of the 13 preoperatively nonambulatory patients, 10 could walk after surgery and three were still unable to walk but showed improved motor function. Twenty-one patients had complications ranging from minor atelectasis to pulmonary embolism. The 30-day mortality rate was 3%. The 1-year survival rate for the entire study population was 62%. CONCLUSIONS: These results suggest that transthoracic vertebrectomy and spinal stabilization can improve the quality of life considerably in cancer patients with spinal metastasis by restoring or preserving ambulation and by controlling intractable spinal pain with acceptable rates of morbidity and mortality.

Neurosurgical outcomes in a modern series of 400 craniotomies for treatment of parenchymal tumors.

OBJECTIVE: The goals were to critically review all complications resulting within 30 days after craniotomies performed for excision of intra-axial brain tumors relative to factors likely to affect complication rates and to assess the value of these data in predicting the risk of surgical morbidity, particularly for surgery in eloquent brain regions. METHODS: Neurosurgical outcomes were studied for 327 patients who underwent 400 craniotomies for removal of intra-axial parenchymal brain neoplasms in a 21-month period. Tumors removed included gliomas (206 tumors) and metastases (194 tumors) located both supratentorially (358 tumors) and infratentorially (42 tumors). RESULTS: The major complication incidence was 13%, and the operative mortality rate was 1.7%. The overall morbidity rate was 32%, but more types of complications were considered than in previous studies. The major neurological morbidity rate was 8.5%. Based on pre- versus postoperative (at 4 wk) Karnofsky Performance Scale scores, 9% of patients deteriorated neurologically, 32% improved, and 58% showed no change. The median postoperative hospital stay was 5 days. Tumors were defined as Grade I, II, or III based on their location relative to brain function, and this tumor functional grade was the most important variable affecting the incidence of any neurological deficit. Patients with tumors in eloquent (Grade III) or near-eloquent (Grade II) brain areas incurred more neurological deficits than did patients with tumors in noneloquent areas (Grade I). Neither repeat surgery for recurrent disease nor extent of surgical resection affected outcome significantly. Although most tumors in this study, including those in eloquent regions, were removed by gross total resection, this did not lead to more major neurological deficits. Regional complications (at the surgical sites) and systemic complications (medical) were more prevalent among older patients (age >60 yr) with lower preoperative Karnofsky Performance Scale scores (< or = 50) and posterior fossa masses. We showed how our data can be used to predict the total risk of surgical morbidity for a given patient, to facilitate patient counseling and surgical decision-making. CONCLUSION: The finding that gross total resections could be performed in eloquent brain regions with an acceptable level of neurological impairment suggested that the mere presence of a tumor in eloquent brain does not automatically contraindicate surgery. Our results have practical risk-predictive value, and they should aid in the construction of subsequent outcome studies, because we have identified the key areas to monitor.

Volumetric measurement of brain tumors from MR imaging.

Residual tumor volume has been considered important in predicting survival following brain surgery. The purpose of this study was to develop a procedure for quantifying pre- and postsurgical brain tumor volumes that is less subjective than the traditional qualitative grading scale still used by surgeons and radiologists to assess extent of resection (such as gross total, subtotal, and partial resection). Pre- and postsurgical magnetic resonance (MR) imaging brain scans on GE Medical System optical disks were transferred to a Macintosh personal computer using a Pioneer optical disk drive subsystem, and the MedVision 1.41 computer software program was used to analyze regions of interest (ROIs) within them for computation of the volume of tumor tissue therein. Because this procedure puts the original MRI (or CT scan) data file for a patient directly into the personal computer, it bypasses the need for scanning and digitizing MR (or CT scan) film images. Between June 1993 and May 1996, pre- and postsurgical volumetric measurements were made in more than 1,000 brain tumor resection cases and 49 radiosurgery cases. The average intra-observer error was estimated to be 1.8%. This method should facilitate the examination of the effects of various therapies on extent of brain tumor resection. The method is fast, is more precise than intraoperative visual assessment of tumor removal or qualitative comparison of pre- and postoperative scans, and it allows the computation of pre- and postsurgical (three-dimensional) volumes of even irregularly shaped tumors.

Expression and localization of urokinase-type plasminogen activator in human spinal column tumors.

We have sought to determine the production and activity of serine proteases in primary and metastatic spinal tumors and the association of these enzymes with the invasive and metastatic properties of spinal column tumors. Using immunohistochemical techniques, the cellular localization and expression of urokinase-type plasminogen activator (uPA) was assessed, whereas its activity was determined by fibrin zymography, and the amounts of enzyme were measured by an enzyme-linked immunosorbent assay (ELISA) in primary spinal column tumors (chordoma, chondrosarcoma, and giant cell tumor) and metastatic tumors of the spine arising from various malignancies (breast, lung, thyroid, and renal cell carcinomas, and melanomas). Metastatic tumors displayed higher levels of uPA activity than did primary spinal tumors (P<0.001). Immunohistochemical analysis revealed that uPA expression was highest in metastases from lung and breast carcinomas and melanomas, followed by metastatic tumors from thyroid and renal cell carcinomas. Similar results were obtained for uPA activity and enzyme level as determined by fibrin zymography and ELISA, respectively. We conclude that metastatic spinal tumors possess higher levels of uPA expression and activity than the primary spinal tumors, which tend to be less aggressive and only locally invasive malignancies. The results suggest that the plasminogen system may participate in the metastasis of tumors to the spinal column.

Expression and role of matrix metalloproteinases MMP-2 and MMP-9 in human spinal column tumors.

Matrix metalloproteinases (MMPs) have been implicated in the process of tumor invasion and metastasis formation. Thus, we determined the expression of MMPs in various primary and metastatic spinal tumors in order to assess the role of these enzymes in spinal invasion. MMP expression was examined by immunohistochemical localization, and quantitative evaluation of MMP protein content was determined by enzyme-linked immunosorbant assay (ELISA) and Western blotting. MMP enzyme activity was determined by gelatin zymography. Lung carcinomas and melanomas metastatic to the spine were shown to have higher levels of MMP-9 activity than those of breast, thyroid, renal metastases and primary spinal tumors. Immunohistochemical analysis revealed similar difference in expression of MMP-9 in tissue samples. When the tissue samples were subjected to gelatin zymography for examination of MMP-2 and MMP-9 activity and to ELISA and Western blotting for quantitative estimation of protein content, the most striking results were obtained for lung carcinomas and melanomas relative to the other tumors. Lung carcinomas and melanomas metastatic to the spine had considerably higher levels of MMP-9 activity than those of primary spinal tumor or breast, thyroid, and renal carcinoma metastases. Within the metastatic tumor category, neoplasms that are known to be associated with the shortest overall survival rates and most aggressive behavior, such as lung carcinomas and melanomas, had the highest levels of MMP-2 and MMP-9 activity compared to those less aggressive metastatic tumors such as breast, renal cell, and thyroid carcinomas. Our results suggest that MMPs may contribute to the metastases to the spinal column, and overexpression of these enzymes may correlate with enhanced invasive properties of both primary and metastatic spinal tumors.

Total sacrectomy and Galveston L-rod reconstruction for malignant neoplasms. Technical note.

Although radical resection is the best treatment for malignant sacral tumors, total sacrectomy for such tumors has been performed in only a few instances. Total sacral resection requires reconstruction of the pelvic ring plus establishment of a bilateral union between the lumbar spine and iliac bone. This technique is illustrated in two patients harboring large, painful, sacral giant-cell tumors that were unresponsive to prior treatment. These patients were treated with complete en bloc resection of the sacrum and complex iliolumbar reconstruction/stabilization and fusion. Surgery was performed in two stages, the first consisting of a midline celiotomy, dissection of visceral/neural structures, and ligation of internal iliac vessels, followed by an anterior L5-S1 discectomy. The second stage consisted of mobilization of an inferiorly based myocutaneous rectus abdominis pedicle flap for wound closure, followed by an L-5 laminectomy, bilateral L-5 foraminotomy, ligation of the thecal sac, division of sacral nerve roots, and transection of the ilia lateral to the tumor and sacroiliac joints. Placement of the instrumentation required segmental fixation of the lumbar spine from L-3 down by means of pedicle screws and the establishment of a bilateral liaison between the lumbar spine and the ilia by using the Galveston L-rod technique. The pelvic ring was then reestablished by means of a threaded rod connecting left and right ilia. Both autologous (posterior iliac crest) and allograft bone were used for fusion, and a tibial allograft strut was placed between the remaining ilia. The patients were immobilized for 8 weeks postoperatively and underwent progressive rehabilitation. At the 1-year follow-up review, one patient could walk unassisted, and the other ambulated independently using a cane. Both patients controlled bowel function satisfactorily with laxatives and diet and could maintain continence but required self-catheterization for bladder emptying. The authors conclude that in selected patients, total sacrectomy represents an acceptable surgical procedure that can offer not only effective local pain control, but also a potential cure, while preserving satisfactory ambulatory capacity and neurological function.

Intraoperative monitoring of spinal cord function using motor evoked potentials via transcutaneous epidural electrode during anterior cervical spinal surgery.

Because false-positive results are not infrequent when monitoring somatosensory evoked potentials during surgery, monitoring of motor evoked potentials (MEPs) has been proposed and successfully used during the removal of spinal cord tumors. However, this often requires direct visual placement of an epidural electrode after a laminectomy. We evaluated the use of MEPs, recorded via a transcutaneously placed epidural electrode, to monitor motor pathway functional integrity during surgery on the anterior cervical spine. Sixteen patients underwent anterior cervical vertebral decompression and fusion for cervical myelopathy and/or radiculopathy. Before surgery, an epidural monitoring electrode was placed transcutaneously at the midthoracic level and was used to record MEPs after transcranial cortical electrical stimulation. Electrode placement was successful in all patients but one, and satisfactory baseline spinal MEPs were obtained except for one patient who had cerebral palsy with significant motor dysfunction. Patients showed no significant changes in spinal MEPs during surgery, and all had baseline or better motor function postoperatively. None had complications from epidural electrode placement or electrical stimulation. We conclude that motor pathways can be monitored safely during anterior cervical spinal surgery using spinal MEPs recorded via a transcutaneously placed epidural electrode, that MEP preservation during surgery correlates with good postoperative motor function, and that cerebral palsy patients may possess too few functional motor fibers to allow MEP recording.

Purification of the adenomatous polyposis coli (APC) gene product.

Mutation of the adenomatous polyposis coli (APC) gene is responsible for familial adenomatous polyposis and is an etiologic factor for digestive tract malignancies. Although the APC gene product (APC) is believed to play a role in growth suppression of colonocytes, the underlying mechanism is not clear. However, recent evidence does suggest that APC is a microtubule-associated protein (MAP), and like other MAPs, it can be phosphorylated, as we have shown. To facilitate studies of APC function, we purified the APC protein. To purify the full-length APC protein, HCT116 human colon cancer cells were lysed and the particulate fraction from the lysate was extracted with ammonium sulfate followed by Sepharose 4B and DEAE-Sephacel column fractionation and then by sucrose zonal density gradient centrifugation. The final purified APC fraction was determined to be about 1000-fold enriched in APC. The availability of purified APC will be valuable in investigating possible growth-suppressing mechanisms of APC including specific sites of APC phosphorylation and APC's interaction with other cellular proteins.

Retinoblastoma gene product-associated proteins in human colon cancer cell lines.

The expression of cellular proteins that associate with the human retinoblastoma (RB1) gene protein was examined in three colorectal carcinoma cell lines (SW480, SW403, DiFi) by immunoprecipitation analysis, using the mouse monoclonal antibody (RB-MAb-1) directed against the RB1 gene product (RB). The potential of RB-MAb-1 to detect RB-associated proteins by immunoprecipitation analysis was confirmed using three control cell lines (WI-38, J82, T24) known to express RB and/or RB-associated proteins. In all colon cancer cell lines tested, multiple RB-associated protein (RAP) bands were found with molecular weights of 30-50 kDa. Because involvement of the RB1 gene in colorectal tumorigenesis appears to differ from its role in other cancer types (wherein RB1 is lost or inactivated), it will be important to characterize the role of RAPs in RB growth regulatory mechanisms of colonic epithelial cells.

RB1 protein in normal and malignant human colorectal tissue and colon cancer cell lines.

The pattern of human retinoblastoma (RB1) gene protein expression was directly examined in normal and malignant human colorectal tissues and in seven colorectal carcinoma cell lines by immunohistochemistry using the mouse monoclonal antibody (RB-MAb-1) directed against the retinoblastoma protein (RB). This is the first demonstration of RB immunostaining in adult human colonic epithelium and colorectal carcinomas. Specificity using RB-MAb-1 was confirmed by western blot analysis, which showed bands of 110-116 kDa corresponding to the sizes of unphosphorylated and phosphorylated RB. RB staining of normal adult colonic epithelium was confined to the nucleus and was most intense in the transitional zone of the crypt, whereas lumenal cells (fully differentiated) were RB negative. Primary colorectal carcinomas and all the colon cancer cell lines stained positively for nuclear RB, but the expression was heterogeneous with varying fractions of RB negative cells present. Because we and others have previously shown that loss or inactivation of the RB1 gene is infrequent in colorectal carcinomas, reduced RB expression in such cells is probably due to a cellular regulatory mechanism. For example, RB negative cells may be those in early-G1 phase (known to have reduced RB levels) or growth-arrested cells that have differentiated. The ability to directly detect RB in primary colorectal carcinomas will permit assessment of whether heterogeneous expression of the RB1 gene product has prognostic significance for survival of patients with this cancer.

Transforming growth factor-alpha production and autoinduction in a colorectal carcinoma cell line (DiFi) with an amplified epidermal growth factor receptor gene.

The DiFi colorectal carcinoma cell line, derived from a patient with familial adenomatous polyposis, was examined for gene expression and production of the autocrine growth factor transforming growth factor alpha (TGF-alpha) and for epidermal growth factor receptor (EGFR) gene expression and gene copy number. DiFi cells expressed TGF-alpha transcripts as identified on Northern (RNA) blots. Addition of TGF-alpha (10 ng/ml) or EGF (10 ng/ml) to DiFi cell cultures (lacking EGF or serum) up-regulated DiFi cell basal TGF-alpha mRNA levels, suggesting that autoinduction of TGF-alpha occurs in these cells. DiFi cell cultures in log phase growth secreted measurable amounts of TGF-alpha (347 pg/10(6) cells/24 h) into their culture medium, as determined by radioimmunoassay. DiFi cells showed strong overexpression of the EGFR gene on Northern blots relative to three other colon cancer cell lines examined. Immunoperoxidase staining showed enhanced EGFR expression in a cell subpopulation among the original (uncultured) ascitic fluid cells from which the DiFi cell line was established. This cell subpopulation was observed to expand dramatically between passages 1 and 25. Immune complex kinase assay of DiFi cells showed that EGFR were functional as determined by their ability to autophosphorylate. The EGFR gene in these cells was not found to be rearranged or genetically altered using Southern blot analysis. Dot blot analysis of DiFi cell DNA revealed EGFR gene amplification in the range of 60-80 copies/cell, which is approximately twice the copy number seen in A-431 epidermoid carcinoma cells. To our knowledge DiFi cells represent the first example of EGFR gene amplification in a colorectal adenocarcinoma. Because DiFi colorectal cancer cells uniquely show production and auto-induction of TGF-alpha in addition to amplification and overexpression of the EGFR gene, these cells represent a valuable tool for studying the role(s) of the EGFR in the regulation of tumor cell growth.

Characterization of the DiFi rectal carcinoma cell line derived from a familial adenomatous polyposis patient.

The DiFi human colorectal cancer cell line was recently established from a familial adenomatous polyposis patient with extracolonic features characteristic of the Gardner syndrome. These cells have now been propagated for 150 passages in standard culture media and vessels without feeder layers or collagen coatings. They retain features of colonic epithelial cells such as surface microvilli, secretory vesicles, and desmosomes. Cytosol of DiFi cells contains a high level (502 U/mg protein) of the mucin CA 19-9. In addition, DiFi cells produce carcinoembryonic antigen, and induce tumors in athymic mice. Cytoskeleton analysis of DiFi cells by fluorescence microscopy showed a pronounced disorganization of actin cable structure. The isozyme genetic signature of DiFi cells is unique (0.01 probability of finding the same genetic signature in a different cell line), differs from that of HeLa cells, and has expressional features seen in other colorectal cell lines. The DiFi cell karyotype is tetraploid, contains many marker chromosomes, and shows numerous episomal particles. Two copies of chromosome 18 were absent, and only a single normal chromosome 17 was found. This parallels detection of allelic losses from DiFi cell DNA at loci on chromosomes 17p and 18 using molecular (cDNA) probes. DiFi cells clearly express transcripts for the c-myc proto-oncogene, the c-myb proto-oncogene, and the p53 tumor suppressor gene. Transforming growth factor beta inhibits DiFi cell growth in soft agar and suppresses c-myc expression in these cells. The value of this cell line in the study of genetic alterations in colorectal cancer is discussed.

Growth effects of tamoxifen on Lovo colon carcinoma cells and cultured cells from normal colonic mucosa.

The growth effects of tamoxifen (TAM) were studied using normal and malignant colonic epithelial cells. Addition of TAM (0.8 to 10 microM) to cultured normal human colon epithelial cells and Lovo colon adenocarcinoma cells produced a dose response decrease in growth of 42 to 76%. Histamine (1 to 10 microM) did not affect cell growth and did not negate the TAM effect. Calmodulin (2 micrograms/ml) totally blocked growth inhibitory effects of a calmodulin antagonist, trifluoperazine (10 microM), but did not block the TAM inhibitory effect. These data suggest that antiestrogen binding sites (AEBS) may play an important role in the growth-inhibitory effects of TAM on colon cells. Competition with estrogen and antagonism with histamine or calmodulin do not appear to be significant in this regard.