Novel GHRH antagonists suppress the growth of human malignant melanoma by restoring nuclear p27 function.

Malignant melanoma is the deadliest form of skin cancer; the treatment of advanced and recurrent forms remains a challenge. It has recently been reported that growth hormone-releasing hormone (GHRH) receptor is involved in the pathogenesis of melanoma. Therefore, we investigated the effects of our new GHRH antagonists on a human melanoma cancer cell line. Antiproliferative effects of GHRH antagonists, MIA-602, MIA-606 and MIA-690, on the human melanoma cell line, A-375, were studied in vitro using the MTS assay. The effect of MIA-690 (5 µg/day 28 d) was further evaluated in vivo in nude mice bearing xenografts of A-375. Subcellular localization of p27 was detected with Western blot and immunofluorescent staining. MIA-690 inhibited the proliferation of A-375 cells in a dose-dependent manner (33% at 10 µM, and 19.2% at 5 µM, P < 0 .05 vs. control), and suppressed the growth of xenografted tumors by 70.45% (P < 0.05). Flow cytometric analysis of cell cycle effects following the administration of MIA-690 revealed a decrease in the number of cells in G2/M phase (from 19.7% to 12.9%, P < 0.001). Additionally, Western blot and immunofluorescent studies showed that exposure of A-375 cells to MIA-690 triggered the nuclear accumulation of p27. MIA-690 inhibited tumor growth in vitro and in vivo, and increased the translocation of p27 into the nucleus thus inhibiting progression of the cell cycle. Our findings indicate that patients with malignant melanoma could benefit from treatment regimens, which combine existing chemotherapy agents and novel GHRH-antagonists.

Shrinkage of experimental benign prostatic hyperplasia and reduction of prostatic cell volume by a gastrin-releasing peptide antagonist.

Gastrin releasing-peptide (GRP) is a potent growth factor in many malignancies. Benign prostatic hyperplasia (BPH) is a progressive age-related proliferation of glandular and stromal tissues; various growth factors and inflammatory processes are involved in its pathogenesis. We have demonstrated that potent antagonists of GRP inhibit growth of experimental human tumors including prostate cancer, but their effect on models of BPH has not been studied. Here, we evaluated the effects of GRP antagonist RC-3940-II on viability and cell volume of BPH-1 human prostate epithelial cells and WPMY-1 prostate stromal cells in vitro, and in testosterone-induced BPH in Wistar rats in vivo. RC-3940-II inhibited the proliferation of BPH-1 and WPMY-1 cells in a dose-dependent manner and reduced prostatic cell volume in vitro. Shrinkage of prostates was observed after 6 wk of treatment with RC-3940-II: a 15.9% decline with 25 µg/d; and a 18.4% reduction with 50 µg/d (P < 0.05 for all). Significant reduction in levels of proliferating cell nuclear antigen, NF-κß/p50, cyclooxygenase-2, and androgen receptor was also seen. Analysis of transcript levels of genes related to growth, inflammatory processes, and signal transduction showed significant changes in the expression of more than 90 genes (P < 0.05). In conclusion, GRP antagonists reduce volume of human prostatic cells and lower prostate weight in experimental BPH through direct inhibitory effects on prostatic GRP receptors. GRP antagonists should be considered for further development as therapy for BPH.

Mechanisms of synergism between antagonists of growth hormone-releasing hormone and antagonists of luteinizing hormone-releasing hormone in shrinking experimental benign prostatic hyperplasia.

BACKGROUND: Benign prostatic hyperplasia (BPH) affects aging men. Combined therapy with antagonists of growth hormone-releasing hormone (GHRH) and of luteinizing hormone-releasing hormone (LHRH or GnRH) induces prostate shrinkage in rat models. We investigated the mechanisms of action of this combination on cell cycle traverse and expression of prostatic genes. METHODS: Effects of GHRH antagonist, JMR-132 (40 µg/day), the LHRH antagonist, cetrorelix (0.625 mg/kg), and their combination were evaluated on testosterone-induced benign prostatic hyperplasia in male Wistar rats. Influence of JMR-132, cetrorelix, and their combinations on cell viability was assessed by MTS assay in BPH-1 human prostate epithelial cells and WPMY-1 normal prostate stromal cells. Cell cycle was analyzed by laser flow cytometry. Real-time PCR arrays were performed. RESULTS: The combination of antagonists caused marked shrinkage of rat prostate (29.5%). In vitro, JMR-132 plus cetrorelix (both 5µM) produced synergistic (57.4%) inhibition of growth of BPH-1 cells, but a lesser inhibition (46%) of WPMY-1 cells. Co-treatment of with JMR-132 plus cetrorelix induced a significant increase of BPH-1 cells blocked in S-phase plus cells with lower G0 /G1 and G2 /M DNA content. Significant changes in expression of >40 gene transcripts related to growth factors, inflammatory cytokines, and signal transduction were identified. CONCLUSIONS: GHRH antagonist and LHRH antagonist combination potentiates rat prostate weight reduction and synergistically inhibits of growth of BPH-1 leading to cell cycle arrest in S-phase. These effects were lesser in normal stromal prostate cell line, WPMY-1. Our findings suggest that GHRH antagonists could be useful for BPH therapy, possibly in combination with LHRH antagonists.

GHRH antagonist when combined with cytotoxic agents induces S-phase arrest and additive growth inhibition of human colon cancer.

Treatment of colon cancer with an antagonist of growth hormone-releasing hormone (GHRH), JMR-132, results in a cell cycle arrest in S-phase of the tumor cells. Thus, we investigated the effect of JMR-132 in combination with S-phase-specific cytotoxic agents, 5-FU, irinotecan and cisplatin on the in vitro and in vivo growth of HT-29, HCT-116 and HCT-15 human colon cancer cell lines. In vitro, every compound inhibited proliferation of HCT-116 cells in a dose-dependent manner. Treatment with JMR-132 (5 µM) combined with 5-FU (1.25 µM), irinotecan (1.25 µM) or cisplatin (1.25 µM) resulted in an additive growth inhibition of HCT-116 cells in vitro as shown by MTS assay. Cell cycle analyses revealed that treatment of HCT-116 cells with JMR-132 was accompanied by a cell cycle arrest in S-phase. Combination treatment using JMR-132 plus a cytotoxic drug led to a significant increase of the sub-G 1 fraction, suggesting apoptosis. In vivo, daily treatment with GHRH antagonist JMR-132 decreased the tumor volume by 40-55% (p < 0.001) of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice. Combined treatment with JMR-132 plus chemotherapeutic agents 5-FU, irinotecan or cisplatin resulted in an additive tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts to 56-85%. Our observations indicate that JMR-132 enhances the antiproliferative effect of S-phase-specific cytotoxic drugs by causing accumulation of tumor cells in S-phase.

Combination of gastrin-releasing peptide antagonist with cytotoxic agents produces synergistic inhibition of growth of human experimental colon cancers.

We investigated the efficacy of a powerful antagonist of bombesin/gastrin-releasing peptide (BN/GRP) RC-3940-II administered as a single agent or in combination with cytotoxic agents on the growth of HT-29, HCT-116 and HCT-15 human colon cancer in vitro and in vivo. GRP-receptor mRNA and protein were found in all three cell lines tested. Exposure of HT-29 cells to 10 µM RC-3940-II led to an increase in the number of cells blocked in S phase and G 2/M and cells with lower G(0)/G(1) DNA content. Similar changes on the cell cycle traverse of HT-29 cells could also be seen at lower concentrations of RC-3940-II (1 µM) after pretreatment with 100 nM GRP (14-27), indicating a dose-dependent mechanism of action based on the blockage of BN/GRP induced proliferation of tumor cells at lower concentrations. Daily in vivo treatment with BN/GRP antagonist RC-3940-II decreased the volume of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice by 25 to 67% (p < 0.005). Combined treatment with RC-3940-II and chemotherapeutic agents 5-FU and irinotecan resulted in a synergistic tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts by 43% to 78%. In HT-29 and HCT-116 xenografts the inhibition for the combinations of RC-3940-II and irinotecan vs. single substances (p < 0.05) was significantly greater. These findings support the use of RC-3940-II as an anticancer agent and may help to design clinical trials using RC-3940-II in combinations with cytotoxic agents.

Click-iT proliferation assay with improved DNA histograms.

The Click-iT EdU cell proliferation assay (Invitrogen) for detection of replicating cells is based on incorporation of EdU into newly synthesized DNA and its recognition by azide dyes via a copper mediated "click" reaction. In the protocol provided by Invitrogen, cells are fixed with paraformaldehyde and stained with 7-aminoactinomycin D (7-AAD) for DNA content analysis. Both of these procedures result in DNA histograms with a broad coefficient of variation. We have modified this protocol and show that after EdU incorporation, nuclei isolated by hypotonic lysis of cells can be directly labeled using the Click-iT Alexa Fluor 488 Assay kit and stained with propidium iodide. This modified procedure using isolated nuclei and propidium iodide staining results in DNA histograms with better resolution (lower coefficient of variation of the G(1) peak) and shorter processing time by eliminating the fixation and permeabilization steps.

Targeted cytotoxic somatostatin analog AN-162 inhibits growth of human colon carcinomas and increases sensitivity of doxorubicin resistant murine leukemia cells.

The effect of the targeted cytotoxic somatostatin (SST) analog AN-162, consisting of doxorubicin (DOX) conjugated to SST carrier RC-121, was investigated on the growth of human colorectal cancer (CRC) cell lines HT-29, HCT-15, and HCT-116 and a DOX-resistant mouse leukemia cell line P388/R84. mRNA for SST-receptors and high affinity binding sites for SST were detected in all CRC cell lines and in P388/R84 cells. In contrast to DOX alone, AN-162 blocked HCT-116 cells and P388/R84 cells in S/G2 phase and increased the number of apoptotic cells. In vivo, AN-162 reduced the volume of CRC xenografts more effectively than its unconjugated components. Our results suggest that AN-162 inhibits growth of experimental CRC more effectively than DOX and increases sensitivity of DOX resistant human leukemia cells.

Electronic volume, aldehyde dehydrogenase, and stem cell marker expression in cells from human peripheral blood apheresis samples.

BACKGROUND: Over-expression of aldehyde dehydrogenase and other stem cell markers is characteristic of cells with tumorigenic potential in NOD/SCID mice. Most of these studies have focused on metastatic cells in bone marrow and on solid tumors. There are no studies on correlation of marker expression with ALDH1 expression in cells from human peripheral blood apheresis (HPC-A) samples. METHODS: HPC-A samples from 44 patients were incubated with Aldefluor with or without the presence of aldehyde dehydrogenase inhibitor DEAB. Cells with high aldehyde dehydrogenase expression (ALDH1(bright)) were analyzed for stem/progenitor markers CD34, CD90, CD117, and CD133. Electronic volume measured by Coulter principal in a Quanta flow analyzer was correlated with ALDH1 and marker expression. RESULTS: In ALDH1(bright)/SSC(low) cells, 0.13% of the cells had CD34(+) expression and three distinct populations were seen. Expression of CD90 was dim and the frequency of ALDH1(bright)/SSC(low)/CD90(dim) cells amongst the nonlineage depleted samples was 0.04%. CD117(dim-bright) expression was seen in 0.17% of the samples. Three distinct populations of cells with CD133 expression were seen in ALDH1(bright)/SSC(low) nonlineage depleted cells with a frequency of 0.28%. The ALDH1(bright)/CD90(dim) cells had the smallest mean electronic volume of 264.9 microm(3) when compared with cells with CD34(bright) expression (270.2 microm(3)) and ALDH1(dim)/CD90(dim) cells (223 microm(3)). CONCLUSIONS: ALDH1(bright)/SSC(low) cells show heterogeneity in expression of the four stem cell markers studied. The CD90 cells in both the ALDH1(bright) and ALDH1(dim) populations had the smallest mean electronic volume when compared with similar cells with CD117 expression.

ALDH(+)/CD44(+)/CD24(-) expression in cells from body cavity fluids.

BACKGROUND: Enhanced expression of aldehyde dehydrogenase 1 (ALDH1) and phenotypic markers (CD44(+)/CD24(-)) in stem cells from breast tumors has been reported. This study was undertaken to monitor expression of these markers in cells from body cavity fluids of female patients suspected to have a malignancy. METHODS: Cells from peritoneal and pleural fluids of 100 female patients were examined by diagnostic cytology and analyzed by laser flow cytometry for enhanced ALDH1 expression. Cells from 36 body cavity fluids with ALDH1(bright) fluorescence were then analyzed for the expression of CD44 and CD24 markers. RESULTS: In samples positive for malignancy, ALDH1(bright) cells with both SSC(low) and SSC(high) were seen. In 15 body cavity fluids positive for malignancy, the percentage of ALDH1(bright) cells ranged from 0.26 to 6.34% of the total cells. The percentage of ALDH1(bright) cells with CD44(+)/CD24(-) expression in these samples ranged from 0.02 to 3.66%. ALDH1(bright) cells with CD44(+)/CD24(-) expression were also present in body cavity fluids of patients in whom diagnostic cytology could not detect any malignancy. However, the percentage of ALDH1(bright) and CD44(+)/CD24(-) cells amongst the 21 body cavity fluids with negative cytology was lower than that of samples with malignancy. CONCLUSIONS: Expression of ALDH1(bright) and the CD44(+)/CD24(-) phenotype in body cavity fluids in which diagnostic cytology could not find any malignant cells suggests that this phenotype may not be restricted to the putative breast tumor stem cells. It is possible that only subsets of cells with this phenotype are the putative breast tumor stem cells.

Flow immunocytochemistry of marker expression in cells from body cavity fluids.

Diagnostic cytology based on the examination of cells from body cavity fluids misses approximately 50% of patients with a proven malignancy. In an earlier study, we used immunohistochemical detection of epithelial membrane antigen expression with flow cytometric detection of DNA aneuploidy to reduce the number of false negatives. In the present study, we have combined DNA flow cytometry with flow cytometric detection of marker expression to analyze cells from body cavity fluids. Seventy-nine specimens of ascites and pleural fluids were analyzed by diagnostic cytology, DNA flow cytometry, and for the expression of the following markers: Ber-EP4, progesterone (PR), MUC4, and thyroid transcription factor-1 (TTF-1). DNA index of equal to or greater than 1.2 was seen in 33/79 (41.7%) of the samples. Statistical analysis of 79 samples in which data from cytology, DNA aneuploidy, and expression of at least one of the markers was available showed that by combining data from positive marker expression with that of aneuploidy, the sensitivity was increased from 58.5 to 100%. In contrast, out of the 38 samples designated as non-malignant by diagnostic cytology, nine had aneuploid DNA content and 16 of the diploid samples had a positive marker expression. Specificity was reduced from 74.7 to 31.6% due to the presence of aneuploidy and marker expression in these samples. ALDH1(pos)/CD44(pos)/CD24(neg) expression has been reported to be associated with human breast tumor stem cells. Some of our samples had cells with this phenotype. Flow cytometry offers the advantage of rapid multiparametric analysis of DNA aneuploidy and marker expression in cells from body cavity fluids based on the analysis of a large number of cells without observer bias. By further developing the use of specific markers and aneuploidy, it may be possible to refine flow cytometric analysis for rapid detection of malignant cells in body cavity fluids.

Triple-negative breast cancers express receptors for luteinizing hormone-releasing hormone (LHRH) and respond to LHRH antagonist cetrorelix with growth inhibition.

The aim of the present study was to evaluate the expression of receptors for luteinizing hormone-releasing hormone (LHRH) in human specimens of triple-negative breast cancers (TNBC). In addition, we used in vitro and in vivo models of TNBC to investigate if these receptors are suitable targets for the treatment with the LHRH antagonist cetrorelix. Receptors for LHRH were expressed in all tumor samples and in the TNBC cell lines HCC1806 and HCC1937. The proliferation of both TNBC cell lines was significantly inhibited in vitro by 1 microM cetrorelix. Injections of 3 mg cetrorelix on day 1 and 21 resulted in a significant growth inhibition of HCC1806 tumors xenografted into nude mice. Tumors of mice treated with cetrorelix expressed less mRNA for EGFR and HER3 receptors than untreated tumors. After treatment of cells with Cetrorelix a flow cytometric analysis of the cell cycle revealed a decrease in S-phase. Given the low toxicity and clinical availability of cetrorelix, this peptide antagonist should be considered for phase II studies in patients with advanced TNBC.

GHRH antagonist causes DNA damage leading to p21 mediated cell cycle arrest and apoptosis in human colon cancer cells.

We investigated the mechanisms of inhibitory effect of growth hormone-releasing hormone (GHRH) antagonist JMR-132 on the growth of HT29, HCT-116 and HCT-15 human colon cancer cells in vitro and in vivo. High-affinity binding sites for GHRH and mRNA for GHRH and splice variant-1 (SV1) of the GHRH receptor were found in all three cell lines tested. Proliferation of HT-29, HCT-116 and HCT-15 cells was significantly inhibited in vitro by JMR-132. Time course studies revealed that the treatment of human HCT-116 colon cancer cells with 10 muM GHRH antagonist JMR-132 causes a significant DNA damage as shown by an increase in olive tail moment (OTM) and loss of inner mitochondrial membrane potential (Delta Psi m). Western blotting demonstrated a time-dependent increase in protein levels of phospho-p53 (Ser46), Bax, cleaved caspase-9, -3, cleavage of poly(ADP-ribose)polymerase (PARP) and a decrease in Bcl-2 levels. An augmentation in cell cycle checkpoint protein p21(Waf1/Cip1) was accompanied by a cell cycle arrest in S-phase. DNA fragmentation visualized by the comet assay and the number of apoptotic cells increased time dependently as determined by flow cytometric annexinV and PI staining assays. In vivo, JMR-132 decreased the volume of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic mice up to 75% (p < 0.05) and extended tumor doubling time (p < 0.001). Our observations suggest that GHRH antagonist JMR-132 exerts its antiproliferative effect on experimental colon cancer cells through p21(Waf1/Cip1) mediated S-phase arrest along with apoptosis involving the intrinsic pathway.

Click-iT assay with improved DNA distribution histograms.

The Click-iT Assay developed and commercialized by Invitrogen is based on incorporation of a new 5-bromo-2'-deoxyuridine analog, 5-ethynyl-2'-deoxyuridine (EdU) into newly synthesized DNA and its recognition by azide dyes via a copper mediated "click" reaction. This relatively convenient and useful procedure depends on fixation of cells with paraformaldehyde and staining of the DNA with 7-aminoactinomycin-D (7-AAD). Both of these procedures result in DNA histograms with broad coefficients of variation (CV's). In this report, we have shown that after EdU incorporation, nuclei isolated by lysis can be incubated with the Click-iT Assay and stained with propidium iodide for generation of DNA histograms with low CV's. This modified procedure results in better DNA histograms by replacing 7-AAD with propidium iodide and also saves processing time by eliminating the fixation and permeabilization steps.

Hormone receptor-related gene polymorphisms and prostate cancer risk in North Indian population.

The purpose of this study was to analyse the frequency and type of mutations in the coding region of androgen receptor (AR) and to determine the role of polymorphisms in the intron 1 of ERalpha, exon 5 of ERbeta, intron 7 of progesterone, exon 7 of the aromatase (CYP19) and exon 9 of VDR genes in the risk of prostate cancer. PCR-RFLP analysis of all above the genes was on 100 prostate cancer patients and an equal number of matching controls. The study also included PCR-SSCP analyses of exons 2-8 of AR gene. The genotype containing -/- allele of ERalpha gene was statistically significant for the risk of prostate cancer pose (OR, 2.70; 95% CI, 1.08-6.70, P = 0.032) Rr genotype of ERbeta gene also have a higher risk (OR, 1.65; 95% CI, 0.52-5.23) for prostate cancer. The Cys allele of CYP19 gene was also associated with statistically significant increased risk of prostate cancer (OR; 2.28, 95% CI, 1.20-4.35, P = 0.012). tt genotype of codon 352 of VDR gene showed an OR of 0.43 for (95% CI, 0.13-1.39) and an OR for Tt genotype was 0.65 (95% CI, 0.36-1.16). Taken together, the results showed that in North Indian population, ERalpha and CYP19 genes may be playing a role in the risk of prostate cancer.

Electronic volume of CD34 positive cells from peripheral blood apheresis samples.

BACKGROUND: The authors have used a flow analyzer to measure electronic cellular volume of peripheral blood hematopoietic stem/progenitor cells obtained by granulocyte-colony stimulating factor (G-CSF) mobilization and apheresis (HPC-A) of patients with hematological malignancies. METHODS: Fifty three apheresis samples stained with CD45-fluorescein isothiocyanate (FITC) and CD34-R-phycoerythrin (PE)-labeled antibodies after erythrocyte lysis with BD FACS Lysing Solution were analyzed for electronic cell volume and two-color FITC and PE fluorescence. RESULTS: Lymphocytes, monocytes and granulocytes in the HPC-A samples had a mean electronic volume of 414, 797, and 670 microm(3), respectively corresponding to cell diameter of 9.25, 11.5, and 10.85 microm. In 53 HPC-A samples analyzed, the mean electronic volume of the CD34 positive mononuclear cells was 407 microm(3) while the CD45 positive cells had mean volume of 453 microm(3). CONCLUSIONS: CD34 positive stem/progenitor cells have a smaller volume and diameter than CD45 positive mononuclear cells in HPC-A samples. In the present study the CD34 stem/progenitor cells had a considerably larger diameter than that of stem cells previously reported in the literature. With the availability of electronic cell volume as a parameter in flow cytometric analysis, further studies can be carried out to correlate stem cell volume with specific phenotypic marker expression.

Cellular volume and marker expression in human peripheral blood apheresis stem cells.

Coulter volume is far more accurate measure of cell volume than forward angle light scatter. In this report, we have used Coulter volume to determine the mean cell volume and diameter of normal human peripheral blood cells and hematopoietic progenitor cells obtained by apheresis (HPC-A) from patients with hematological malignancies. Fresh peripheral blood samples (treated with Beckman Coulter IMMUNOPrep erythrocyte lysis solution), HPC-A samples (treated with BD Biosciences FACSLysing solution), or processed by Ficoll Hypaque sedimentation method were stained with CD45-FITC and PE-labeled CD34, CD90, CD117, and CD133 antibodies and analyzed for electronic volume and two color fluorescence. The mean electronic volume and diameter of mononuclear cells from fresh peripheral blood samples prepared with IMMUNOPrep were lymphocytes (191 microm(3), 7.16 microm), monocytes (370 microm(3), 9.91 microm), and granulocytes (328 microm(3), 8.56 microm). In mononuclear cells of HPC-A samples prepared by Histopaque-1077 sedimentation, the lymphocytes had volume and diameter of 311 microm(3), 8.4 microm, monocytes were 486 microm(3), 9.76 microm, and granulocytes were 515 microm(3), 9.95 microm. In contrast, HPC-A samples prepared after lysis with FACSLysing solution had mean electronic volume and diameter of lymphocytes (414 microm(3), 9.25 microm), monocytes (797 microm(3), 11.5 microm), and granulocytes (670 microm(3), 10.85 microm). Cell volume of mononuclear cells in the HPC-A samples prepared by Histopaque-1077 sedimentation method was correlated with the expression of stem cell markers CD34, CD90, CD117, and CD133. CD90 positive cells had the smallest mean electronic volume of 299.93 microm(3) when compared with cells with positive expression of CD133 (322 microm(3)), CD117 (349 microm(3)), CD34 (407 microm(3)), and CD45 (453 microm(3)). Correlation of cell volume with stem cell marker expression may allow for the identification of small stem cells, which may not express the conventional markers used for the identification of stem cells in HPC-A samples.

Are increased tumor aneuploidy and heightened cell proliferation along with heterogeneity associated with patient outcome for carcinomas of the uterine cervix? A combined analysis of subjects treated in RTOG 9001 and a single-institution trial.

PURPOSE: To look for possible associations between measurements of DNA index (DI), S-phase fraction (SPF), and tumor heterogeneity (TH) using flow cytometry and overall survival for patients with invasive cervical carcinoma treated with definitive irradiation. METHODS AND MATERIALS: A total of 57 patients with International Federation of Obstetrics and Gynecology Stages IB(2) through IVB cervical carcinomas treated with definitive radiotherapy with or without concurrent chemotherapy were enrolled into this registry study that involved flow cytometric analysis of fresh tissue from each cervical cancer obtained by pretreatment biopsy. These specimens were evaluated for DNA aneuploidy (DI 1.5), SPF (15%), and TH (uniploid vs. multiploid). RESULTS: In these analyses 27 of the patients were treated in Radiation Therapy Oncology Group protocol 9001, and an additional 30 were offered chemoradiation at a single institution. Forty-one patients had DI 1.5. Twenty-nine patients had SPF 15%, and 2 had no determinable SPF. Forty-three patients had uniploid and 14 multiploid tumors. The 4-year estimated overall survival rate for the entire study cohort was 62% (95% confidence interval 48%-74%). With a median follow-up of 3.7 years, there were no observable associations by univariate analysis for DI, SPF, or TH concerning patient survival. CONCLUSIONS: There were no statistically significant associations among DI, SPF, or TH and patient outcome. Additional studies are indicated to identify tumor biomarkers that could predict patients at risk for disseminated disease.