Experimental Design: We report our findings using array comparative genomic hybridization on a whole-genome BAC/PAC/cosmid array with a median clone interval of 0.97 Mb to study a series of UCC cases. TP53 status was determined by direct sequencing, and an in-house tissue microarray was constructed to identify protein expression of target genes.

Results: Array comparative genomic hybridization allowed identification of novel regions of copy number changes in addition to those already known from previous studies. A novel amplification previously unreported in UCC was identified at 1q32. A chromosome 1 tile path array was used to analyze tumors that showed gains and amplification; the mouse double minute 4 (MDM4) homologue was identified as the amplified gene. MDM4 mRNA expression correlated with copy number and tumor grade. Copy number changes of MDM4 and MDM2 occurred exclusively in tumors with wild-type p53. Overexpression of MDM4 corresponded to disruption of p53 transcriptional activity. Immunohistochemistry on an independent series by tissue microarray identified an inverse relationship between Mdm4 and Mdm2, with Mdm4 expression highest in invasive UCC.

Conclusion: The data indicate that gain/amplification and overexpression of MDM4 is a novel molecular mechanism by which a subset of UCC escapes p53-dependent growth control, thus providing new avenues for therapeutic intervention.

Experimental Design: In this study, we profiled miRNA expression in 10 early stage invasive squamous cell carcinomas (ISCC) and 10 normal cervical squamous epithelial specimens using TaqMan real-time quantitative PCR array methods. In order to evaluate the role of miR-199a, one of the most significantly overexpressed in ISCCs, we transfected cervical cancer cells (SiHa and ME-180) with anti–miR-199a oligonucleotides and assessed the cell viability.

Results: We found 70 genes (68 up-regulated, 2 down-regulated) with significantly different expression in the ISCCs compared with normal samples (P < 0.05). When we analyzed the expression of the 10 most significant miRNAs in 31 ISCCs, increased miR-127 expression was significantly associated with lymph node metastasis (P = 0.006). Transfection of anti–miR-199a oligonucleotides to cervical cancer cells suppressed cell growth in vitro, which was potentiated with the anticancer agent cisplatin.

Conclusions: Our results show that miRNA deregulation may play an important role in the malignant transformation of cervical squamous cells. In addition, they may offer new candidate targets to be exploited for both prognostic and therapeutic strategies in patients with cervical cancer.

Experimental Design: We determined the frequency of mTOR activation in endometrial carcinoma (primary tumors and cell lines) and investigated PTEN, LKB1, and TSC2 defects as underlying cause(s) of mTOR activation, and determined the ability of rapamycin to reverse these signaling defects in endometrial carcinoma cells.

Results: Activation of mTOR was a consistent feature in endometrial carcinomas and cell lines. In addition to PTEN, loss of TSC2 and LKB1 expression occurred in a significant fraction of primary tumors (13% and 21%, respectively). In tumors that retained TSC2 expression, phosphorylation of tuberin at S939 was observed with a high frequency, indicating that mTOR repression by TSC2 had been relieved via AKT phosphorylation of this tumor suppressor. In PTEN-null and LKB1-null endometrial carcinoma cell lines with functional inactivation of TSC2, phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 were able to inhibit AKT and mTOR signaling and reverse TSC2 phosphorylation. In contrast, although rapamycin inhibited mTOR signaling, it did not relieve phosphorylation of TSC2 at S939.

Conclusions: Inactivation of TSC2 via loss of expression or phosphorylation occurred frequently in endometrial carcinoma to activate mTOR signaling. High-frequency mTOR activation supports mTOR as a rational therapeutic target for endometrial carcinoma. However, whereas rapamycin and its analogues may be efficacious at inhibiting mTOR activity, these drugs do not reverse the functional inactivation of TSC2 that occurs in these tumors.

Experimental Design: A panel of 53 primary tumors (42 benign, 11 malignant) was analyzed by quantitative bisulfite pyrosequencing. Based on methylation levels in the tumor suppressor genes, p16^INK4A, CDH1, DCR2, RARB, RASSF1A, NORE1A, TP73, APC, DAPK1, p14^ARF, and PTEN, a CpG island methylator phenotype (CIMP) was defined as concerted hypermethylation in three or more genes. Mean Z scores for the hypermethylated promoters were calculated to characterize overall promoter methylation. Global DNA methylation was quantified for LINE-1 promoter sequences and by using luminescent methylation analysis.

Results: Five primary tumors (9.4%) exhibited a CIMP phenotype, four of which were malignant paragangliomas. CIMP was significantly associated with malignant behavior (P = 0.005) and younger age at presentation (P < 0.007) but did not result from BRAF V600E mutation. Global hypomethylation of LINE-1 elements was observed in tumors compared with normal adrenal samples (P < 0.02).

Conclusion: We here describe the identification of CIMP in abdominal paragangliomas and a strong association of this phenotype with malignant behavior, as well as young age at presentation. The findings raise a prospective for potential benefits of epigenetically acting drugs for a subgroup of young abdominal paraganglioma patients with adverse prognosis.

Experimental Design: We evaluated the relationships of aberrant methylation of APC, MGMT, hMLH1, P16, N33, and five MINTs to mutations in APC, KRAS, BRAF, and P53 in 208 colorectal carcinomas.

Results: We found that APC hypermethylation was age related (P = 0.04), in contrast to the other genes, and did not cluster with CpG island methylator phenotype (CIMP) markers. Hypermethylation of APC concurrently with either MGMT or hMLH1 was strongly associated with occurrence of G-to-A transitions in APC [odds ratio (OR), 26.8; P < 0.0002 from multivariable logic regression model], but C-to-T transitions had no associations. There was no relationship of hypermethylation of any gene, including MGMT, with G-to-A or C-to-T transitions in KRAS or P53, although APC hypermethylation was associated with P53 mutation (P < 0.0002). CIMP with MSI-H due to hMLH1 hypermethylation, or CIMP with loss of MGMT expression in non–MSI-H tumors, was associated with BRAF mutation (OR, 4.5; P < 0.0002). CIMP was also associated with BRAF V600E T-to-A transversion (OR, 48.5; P < 0.0002).

Conclusions: Our findings suggest that the heterogeneous epigenetic dysregulation of promoter methylation in various genes is interrelated with the occurrence of mutations, as manifested in epigenetic-genetic subgroups of tumors.

Experimental Design: We evaluated AAHs, adjacent normal lung tissue, and synchronous lung adenocarcinomas for promoter hypermethylation of genes implicated in lung tumorigenesis (p16, TIMP3, DAPK, MGMT, RARβ, RASSF1A, and hTERT).

Results: For individual genes and the number of genes methylated, we observed a significant increase in the frequency of promoter hypermethylation in the histologic progression from normal to AAH, with low-grade or high-grade atypia, and finally to adenocarcinoma (P_trend ≤ 0.01). Multifocal AAHs from individual patients had distinct patterns of promoter hypermethylation, suggesting divergent epigenetic field defects. There were statistically significant positive associations for the presence of promoter hypermethylation of individual and multiple genes with advanced histology, with odds ratios between 4.3 and 58.5. p16 conveyed the strongest individual association for promoter hypermethylation when comparing tumor or high-grade AAH to low-grade AAH or normal tissue, with an odds ratio of 45.5 (95% confidence interval, 5.8-360.5).

Conclusion: This study shows epigenetic progression in the earliest stages of glandular neoplasia of the lung and has implications for early lung cancer detection.

Experimental Design: We searched for the candidate proteins by comparing the profiles of secreted proteins among the poorly invasive human bladder carcinoma cell line RT112 and the highly invasive cell line T24. The proteins isolated from cell culture supernatants were identified by shotgun proteomics. We found that CXCL1 is related to the tumor invasion of bladder cancer cells. We also evaluated whether the amount of the chemokine CXCL1 in the urine would be a potential marker for predicting the existence of invasive bladder tumors.

Results: Higher amount of CXCL1 was secreted from highly invasive bladder carcinoma cell lines and this chemokine modulated the invasive ability of those cells in vitro. It was revealed that CXCL1 regulated the expression of matrix metalloproteinase-13 in vitro and higher expression of CXCL1 was associated with higher pathologic stages in bladder cancer in vivo. We also showed that urinary CXCL1 levels were significantly higher in patients with invasive bladder cancer (pT1-4) than those with noninvasive pTa tumors (P = 0.0028) and normal control (P < 0.0001). Finally, it was shown that CXCL1 was an independent factor for predicting the bladder cancer with invasive phenotype.

Conclusions: Our results suggest that CXCL1 modulates the invasive abilities of bladder cancer cells and this chemokine may be a potential candidate of urinary biomarker for invasive bladder cancer and a possible therapeutic target for preventing tumor invasion.

Experimental Design: Expression levels of 156 human mature microRNAs were examined using real-time quantitative PCR (Taq Man MicroRNA Assays; Human Panel) on laser microdissected cells of 4 tongue carcinomas and paired normal tissues. Expression of mature miR-184 was further validated in 20 paired tongue SCC and the normal tissues. Potential oncogenic functions of miR-184 were evaluated in tongue SCC cell lines (Cal27, HN21B, and HN96) with miR-184 inhibitor. Plasma miR-184 levels were evaluated using real-time quantitative PCR.

Results: Using 3-fold expression difference as a cutoff level, we identified 24 up-regulated mature miRNAs including miR-184, miR-34c, miR-137, miR-372, miR-124a, miR-21, miR-124b, miR-31, miR-128a, miR-34b, miR-154, miR-197, miR-132, miR-147, miR-325, miR-181c, miR-198, miR-155, miR-30a-3p, miR-338, miR-17-5p, miR-104, miR-134, and miR-213; and 13 down-regulated mature miRNAs including miR-133a, miR-99a, miR-194, miR-133b, miR-219, miR-100, miR-125b, miR-26b, miR-138, miR-149, miR-195, miR-107, and miR-139. Overexpression of miR-184 was further validated in 20 paired tongue SCC and normal tissues (P = 0.002). Inhibition of miR-184 in tongue SCC cell lines could reduce cell proliferation rate. Down-regulation of c-Myc was observed in two cell lines in response to miR-184 inhibitor. Suppressing miR-184 could induce apoptosis in all three cell lines. Plasma miR-184 levels were significantly higher in tongue SCC patients in comparison with normal individuals, and the levels were significantly reduced after surgical removal of the primary tumors.

Conclusions: Overexpression of miR-184 might play an oncogenic role in the antiapoptotic and proliferative processes of tongue SCC. In addition, plasma miR-184 levels were associated with the presence of primary tumor. Further studies on the aberrantly expressed miRNAs in tongue SCC as well as using plasma miRNAs as novel tumor markers are warranted.

Experimental Design: CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells were detected using real-time (CK19) and nested (MGB1 and HER2) reverse transcription-PCR in the peripheral blood of 175 women with stage I to III breast cancer before the initiation of adjuvant chemotherapy. The detection of CTCs was correlated with clinical outcome. In 10 patients, immunofluorescence staining experiments were done to investigate the coexpression of cytokeratin, MGB1, and HER2 in CTCs.

Results: CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells were detected in 41.1%, 8%, and 28.6% of the 175 patients, respectively. Patients had one of the following molecular profiles: CK19mRNA+/MGB1mRNA+/HER2mRNA+ (n = 8), CK19mRNA+/MGB1mRNA+/HER2mRNA– (n = 1), CK19mRNA+/MGB1mRNA–/HER2mRNA+ (n = 42), CK19mRNA+/MGB1mRNA–/HER2mRNA– (n = 21), CK19mRNA–/MGB1mRNA+/HER2mRNA– (n = 5), and CK19mRNA–/MGB1mRNA–/HER2mRNA– (n = 98). Double-immunofluorescence experiments confirmed the following CTC phenotypes: CK+/MGB1+, CK+/MGB1–, CK–/MGB1+, CK+/HER2+, CK+/HER2–, MGB1+/HER2–, and MGB1+/HER2+. In univariate analysis, the detection of CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells was associated with shorter disease-free survival (DFS; P < 0.001, P = 0.001, and P < 0.001, respectively), whereas the detection of CK19mRNA+ and MGB1mRNA+ cells was associated with worse overall survival (P = 0.044 and 0.034, respectively). In multivariate analysis, estrogen receptor–negative tumors and the detection of CK19mRNA+ and MGB1mRNA+ cells were independently associated with worse DFS.

Conclusion: The detection of peripheral blood CK19mRNA+ and MGB1mRNA+ cells before adjuvant chemotherapy predicts poor DFS in women with early breast cancer.

Experimental Design: From our previously published list of genes whose expression correlates with both tumor grade and tumor stage progression, we selected five cell cycle–related genes to build MGI and evaluated MGI in two publicly available microarray data sets totaling 410 patients. Using two additional cohorts (n = 323), we developed a real-time reverse transcription PCR assay for MGI, validated its prognostic utility, and examined its interaction with HOXB13:IL17BR.

Results: MGI performed consistently as a strong prognostic factor and was comparable with a more complex 97-gene genomic grade index in multiple data sets. In patients treated with endocrine therapy, MGI and HOXB13:IL17BR modified each other's prognostic performance. High MGI was associated with significantly worse outcome only in combination with high HOXB13:IL17BR, and likewise, high HOXB13:IL17BR was significantly associated with poor outcome only in combination with high MGI.

Conclusions: We developed and validated a five-gene reverse transcription PCR assay for MGI suitable for analyzing routine formalin-fixed paraffin-embedded clinical samples. The combination of MGI and HOXB13:IL17BR outperforms either alone and identifies a subgroup (~30%) of early stage estrogen receptor–positive breast cancer patients with very poor outcome despite endocrine therapy.

Experimental Design: Both bone marrow and peripheral blood samples from 810 gastric cancer patients were collected at the Central Hospital, National Cancer Center (Tokyo, Japan). The samples were transferred to Kyushu University Hospital (Beppu, Japan) where they were analyzed by quantitative real-time reverse transcription-PCR for three epithelial cell markers, carcinoembryonic antigen, cytokeratin-19, and cytokeratin-7, as well as VEGFR-1.

Results: ITCs were observed in peripheral blood and bone marrow even in early stages of gastric cancer. The frequency of ITC in bone marrow was significantly associated with the stage of disease by ANOVA (P < 0.01). Gastric cancer metastasized when ITCs were observed in the presence of VEGFR-1. In the 380 patients who were ITC negative and showed low VEGFR-1 expression, synchronous (at the time of surgery) and heterochronous (recurrent) metastases were not observed.

Conclusions: ITCs circulate even in early stages of disease. Furthermore, elevated expression of VEGFR-1 facilitates the establishment of hematogenous metastases in gastric cancer. This study indicates that the simultaneous presence of ITC and VEGFR-1 expression at premetastatic sites is clinically significant for disease progression.

Experimental Design: PTOV1 expression in HG-PIN lesions from 140 patients was analyzed by immunohistochemistry in a semiquantitative manner (Histo-score). HG-PIN derived from 79 radical prostatectomies for prostate cancer and from 11 cistoprostatectomies for bladder cancer without prostate cancer were used as positive and negative controls, respectively. Fifty patients with HG-PIN without concomitant cancer at their first prostate needle biopsy were chosen as the study group. Patients were followed by a mean of 2.5 repeated prostate needle biopsies (1-5), during a mean period of 12.4 months (1-39).

Results: PTOV1 expression in HG-PIN from radical prostatectomies showed a significantly higher Histo-score (162.6) compared with specimens from cistoprostatectomies (67.0). In the study group, PTOV1 expression was significantly higher in samples with cancer in the follow-up (11 patients, 22%) compared with samples in which cancer was not detected (151.4 versus 94.6). PTOV1 expression was the only independent predictor of cancer in the multivariate analysis and the area under the curve was 0.803 (95% confidence interval, 0.728-0.878). A threshold of 100 for PTOV1 expression provided 90.9% sensitivity, 51.3% specificity, 34.5% positive predictive value, and 95.2% negative predictive value.

Conclusions: PTOV1 is overexpressed in HG-PIN associated with cancer and is a potential marker for studying the carcinogenesis and progression of prostate cancer. Prostate needle biopsy with PTOV1 expression in HG-PIN above a threshold of 100 should be repeated immediately for the likely presence of undiagnosed cancer.

Experimental Design: Twenty-two patients were studied before biopsy or between resection and starting radiotherapy. Each had a 20-minute emission scan 2 hours after i.v. injection of 7 mCi of [¹⁸F]fluoromisonidazole. Venous blood samples taken during imaging were used to create tissue to blood concentration (T/B) ratios. The volume of tumor with T/B values above 1.2 defined the hypoxic volume (HV). Maximum T/B values (T/B_max) were determined from the pixel with the highest uptake.

Results: Kaplan-Meier plots showed shorter TTP and survival in patients whose tumors contained HVs or tumor T/B_max ratios greater than the median (P ≤ 0.001). In univariate analyses, greater HV or tumor T/B_max were associated with shorter TTP or survival (P < 0.002). Multivariate analyses for survival and TTP against the covariates HV (or T/B_max), magnetic resonance imaging (MRI) T1Gd volume, age, and Karnovsky performance score reached significance only for HV (or T/B_max; P < 0.03).

Conclusions: The volume and intensity of hypoxia in glioblastoma multiforme before radiotherapy are strongly associated with poorer TTP and survival. This type of imaging could be integrated into new treatment strategies to target hypoxia more aggressively in glioblastoma multiforme and could be applied to assess the treatment outcomes.

Experimental Design: We built the first series of serial analysis of gene expression (SAGE) libraries from GBC and nonneoplastic gallbladder mucosa, composed of 21-bp long-SAGE tags. SAGE libraries were generated from three stage-matched GBC patients (representing Hispanic/Latino, Native American, and Caucasian ethnicities, respectively) and one histologically alithiasic gallbladder. Real-time quantitative PCR was done on microdissected epithelium from five matched GBC and corresponding nonneoplastic gallbladder mucosa. Immunohistochemical analysis was done on a panel of 182 archival GBC in high-throughput tissue microarray format.

Results: SAGE tags corresponding to connective tissue growth factor (CTGF) transcripts were identified as differentially overexpressed in all pairwise comparisons of GBC (P < 0.001). Real-time quantitative PCR confirmed significant overexpression of CTGF transcripts in microdissected primary GBC (P < 0.05), but not in metastatic GBC, compared with nonneoplastic gallbladder epithelium. By immunohistochemistry, 66 of 182 (36%) GBC had high CTGF antigen labeling, which was significantly associated with better survival on univariate analysis (P = 0.0069, log-rank test).

Conclusions: An unbiased analysis of the GBC transcriptome by SAGE has identified CTGF expression as a predictive biomarker of favorable prognosis in this malignancy. The SAGE libraries from GBC and nonneoplastic gallbladder mucosa are publicly available at the Cancer Genome Anatomy Project web site and should facilitate much needed research into this lethal neoplasm.

Experimental Design: Nude mice bearing LS174T liver orthotopic tumors received a single i.v. injection of fluorescently labeled A5B7 and were sacrificed at 10 minutes, 1 hour, or 24 hours postinjection. Before sacrifice, mice were injected with the perfusion marker Hoechst 33342. An anti-CD31 antibody was used to detect blood vessel distribution. Cryostat sections were processed with immunofluorescence procedures and analyzed with fluorescence microscopy and image analysis techniques. The fluorescence images were related to morphologic images of the same or adjacent tumor sections.

Results: Fluorescently labeled antibody showed rapid, selective uptake into tumor deposits, with a strong negative correlation with tumor size at 10 minutes and 1 hour (P ≤ 0.01). By 24 hours, the correlation was no longer significant. The study showed movement of antibody across the tumor with time and a tendency to localize more uniformly by later time points (24 hours). The rate of antibody motility was similar in small and large tumor metastases, but small deposits showed more rapid antibody localization. Intratumoral vessels were positively related to tumor size (P ≤ 0.001).

Conclusion: The obtained data suggest that radioimmunotherapy can be highly efficient in an adjuvant or minimal residual disease setting.

Experimental Design: We developed a new class of recombinant antibodies called biobodies (Bb) and compared them to mAb for use in serodiagnosis. Bbs were secreted biotinylated in vivo by diploid yeast and used as affinity reagents after Ni purification. Bead-based assays for HE4 and mesothelin were developed using Bbs in combination with pAbs (Bb/pAb assays). To assess precision, reproducibility studies were done using four runs of 16 replicates at six analyte levels for each marker. Pearson correlations and receiver-operator characteristic analyses were done in 214 patient serum samples to directly compare the Bb/pAb assays to mAb assays. Diagnostic performance of the Bb/pAb assay was further assessed in an expanded set of 336 ovarian cancer cases and controls.

Results: On average across analyte levels, Bb/pAb assays yielded within-run and between-run coefficients of variations of 11.7 and 23.8, respectively, for HE4 and 14.0 and 14.5, respectively, for mesothelin. In the subset (n = 214), Pearson correlations of 0.95 for HE4 and 0.92 for mesothelin were observed between mAb and Bb/pAb assays. The area under the curves for the mAb and Bb/pAb assays were not significantly different for HE4 (0.88 and 0.84, respectively; P = 0.20) or mesothelin (0.74 and 0.72, respectively; P = 0.38).

Conclusion: Yeast-secreted Bbs can be used reliably in cost-effective yet highly sensitive bead–based assays for use in large validation studies.

Experimental Design: The Immediate Preoperative Anastrozole, Tamoxifen, or Combined with Tamoxifen (IMPACT) trial compared the preoperative use of anastrozole with tamoxifen in postmenopausal women with estrogen receptor–positive primary operable breast cancer over 12 weeks. Circulating VEGF and sVEGFR-1 were measured by ELISA in 106 patients treated with anastrozole or tamoxifen alone at baseline and after 2 and 12 weeks of treatment.

Results: The increase in serum VEGF from baseline to 12 weeks was significantly different between anastrozole and tamoxifen (anastrozole versus tamoxifen, 6% versus 38%; P = 0.047). There was a significant increase in sVEGFR-1 levels after 12 weeks of anastrozole (P = 0.037). The sVEGFR-1/VEGF ratio significantly decreased in the tamoxifen arm (P = 0.013) and the change in sVEGFR-1/VEGF ratio from baseline to 12 weeks was significantly different between anastrozole and tamoxifen (anastrozole versus tamoxifen, 24% increase versus 34% decrease; P = 0.013).

Conclusions: Treatment with anastrozole and tamoxifen resulted in differential effects on serum angiogenic markers. This may be related to the relative effectiveness of the treatments. These data provide further support for cross talk between estrogen receptor and VEGF.

Experimental Design: The hypermethylation status of the retinoid acid receptor β2 (RARβ2), RAS association domain family 1A (RASSF1A), O⁶-methylguanine-DNA methyltransferase (MGMT), and E-cadherin genes was analyzed in five salivary carcinoma cell lines and 69 human salivary gland carcinoma specimens by pyrosequencing and MSP techniques. The two datasets were compared by linear regression. Correlations between methods and with clinicopathologic characteristics were assessed by Pearson's ² test or the two-tailed Fisher exact test, as applicable, using cutoff points determined from the regression curves and empirical fitting. We also investigated the effect of demethylating agents on methylated genes in cell lines to assess their effect on the expression of these genes.

Results: Overall, regression analysis indicated high degrees of correlation of the two methods for measurement of methylation for the RARb2, RASSF1A, and MGMT genes (adjusted R² = 0.319, 0.835, and 0.178; P < 0.001, <0.001, and 0.0002, respectively) among the 69 tumors tested. However, the pyrosequencing technique yielded four more instances of methylation above background levels than MSP for RARβ2 and three more for RASSF1. Methylation of either RARβ2 and RASSF1A alone or both by pyrosequencing were correlated with tumor type (P = 0.027, 0.014, and 0.012, respectively). Methylation of RARβ2 alone and in combination with RASSF1A by pyrosequencing were also significantly correlated with tumor grade (P = 0.014 and 0.011, respectively) and 3-year survival (P = 0.002 and 0.004, respectively). The survival curves of patients who had hypermethylation at both RARβ2 and RASSF1A were significantly lower than those of patients who had hypermethylation at neither or just for the RASSF1A (P = 0.008 and 0.007, respectively). 5-Azadeoxycytidine treatment of methylated cell lines led to the reactivation of RARβ2 expression in only one of the five cell lines.

Conclusions: (a) Although the methylation status of RARb2, RASSF1A, and MGMT genes by both techniques were significantly correlated, pyrosequencing is generally more sensitive and its results correlate better with the clinical variables than those of MSP. (b) The methylation level of the RARβ2 and/or RASSF1A by pyrosequencing is significantly associated with aggressive tumor phenotypes and patients survival.

Experimental Design: PI3K, phospho-AKT (pAKT) and phospho-mTOR were assessed by immunohistochemistry on tumor specimens collected at baseline and after 6 months of treatment in 113 elderly breast cancer patients consecutively enrolled in a randomized phase II trial of primary letrozole therapy and letrozole associated with metronomic cyclophosphamide.

Results: Basal expression of the pathway was not significantly correlated with response or patient outcome. Both letrozole alone and letrozole with cyclophosphamide resulted in a significant reduction of PI3K expression (P = 0.02 and P < 0.005, respectively) and phospho-mTOR expression (P = 0.0001 and P = 0.0001, respectively). pAKT showed no change in the letrozole arm, whereas it was significantly decreased in the letrozole plus cyclophosphamide arm (P < 0.005). pAKT expression reduction was associated with a greater response rate (P = 0.05) and greater reduction in Ki67 expression (P = 0.05). Phospho-mTOR expression reduction was associated with a significantly longer disease-free survival in a multivariate analysis (P = 0.02).

Conclusions: Letrozole inhibits key molecules in the PI3K pathway that are important targets of new drugs being developed to overcome resistance. Changes in these molecules may have prognostic significance. These results should be taken into account when planning prospective trials testing up-front aromatase inhibitor with drugs targeting the PI3K/AKT/mTOR signaling pathway.

Experimental Design: A breast cancer tissue microarray containing 102 tumors was stained with an anti-survivin antibody. Whole-slide scanning was used to capture high-resolution images. These images were analyzed using automated algorithms to quantify the staining.

Results: Increased nuclear, but not cytoplasmic, survivin was associated with a reduced overall survival (OS; P = 0.038) and disease-specific survival (P = 0.0015). A high cytoplasmic-to-nuclear ratio (CNR) of survivin was associated with improved OS (P = 0.005) and disease-specific survival (P = 0.05). Multivariate analysis revealed that the survivin CNR was an independent predictor of OS (hazard ratio, 0.09; 95% confidence interval, 0.01-0.76; P = 0.027). A survivin CNR of >5 correlated positively with estrogen receptor (P = 0.019) and progesterone receptor (P = 0.033) levels, whereas it was negatively associated with Ki-67 expression (P = 0.04), p53 status (P = 0.005), and c-myc amplification (P = 0.016).

Conclusion: Different prognostic information is supplied by nuclear and cytoplasmic survivin in breast cancer. Nuclear survivin is a poor prognostic marker in breast cancer. Moreover, CNR of survivin, as determined by image analysis, is an independent prognostic factor.

Experimental Design: Twenty patients diagnosed with serous ovarian carcinoma and eight patients treated for benign uterine disease between December 2000 and September 2003 were enrolled in this study. The microRNA expression profiles were examined using DNA microarray and Northern blot analyses.

Results: Several microRNAs were differentially expressed in serous ovarian carcinoma compared with normal ovarian tissues, including miR-21, miR-125a, miR-125b, miR-100, miR-145, miR-16, and miR-99a, which were each differentially expressed in >16 patients. In addition, the expression levels of some microRNAs were correlated with the survival in patients with serous ovarian carcinoma. Higher expression of miR-200, miR-141, miR-18a, miR-93, and miR-429, and lower expression of let-7b, and miR-199a were significantly correlated with a poor prognosis (P < 0.05).

Conclusion: Our results indicate that dysregulation of microRNAs is involved in ovarian carcinogenesis and associated with the prognosis of serous ovarian carcinoma.

Experimental Design: A phage cDNA expression library of colon cancer was built. The library was sequentially screened by a pool of 10 colon cancer sera, goat antihuman IgG, and a pool of two healthy sera to identify phage-expressed antigens recognized by tumor-associated antibodies. The clones picked out by these screening were subjected to a training set with 24 colon cancer sera and 24 healthy sera. The antigen combination, which got the most satisfactory classification, was tested by an independent set of 24 colon cancer sera with equal number of sera from normal donors. The carcinoembryonic antigen (CEA) level of these sera was detected for the additional classification analysis with or without the antigen combination.

Results: A cDNA expression library consisting of 2 x 10⁶ primary clones was prepared. After three turns of screening, 24 antigens recognized by tumor-associated antibodies were picked out for serum marker identification. The training set showed that a six-marker combination got the most satisfactory classification in a logistic regression model; leave-one-out validation achieved 91.7% sensitivity and 91.7% specificity. In a testing set with this marker panel, we correctly predicted 85% of the samples. Although according to CEA level alone, we correctly predicted 75% of the samples with 42% of cancer patients misclassified. When CEA was combined with the six markers, the sensitivity and specificity increased to 91.7% and 95.8%, respectively. The six antigen sequences in the phage display system are relatively short peptides. Only two of them showed homology to known protein sequences.

Conclusions: Autoantibodies against phage-expressed antigens derived from colon cancer tissues could be used as serum markers for the detection of colon cancer.

Experimental Design: Human microsomes were used to determine the cytochrome P450 enzyme(s) involved in the metabolism of ixabepilone. Computational docking (CYP3A4) studies were done for epothilone B and ixabepilone. A follow-up clinical study was done in patients with cancer to determine if 400 mg/d ketoconazole (inhibitor of CYP3A4) altered the pharmacokinetics, drug-target interactions, and pharmacodynamics of ixabepilone.

Results: Molecular modeling and human microsomal studies predicted ixabepilone to be a good substrate for CYP3A4. In patients, ketoconazole coadministration resulted in a maximum ixabepilone dose administration to 25 mg/m² when compared with single-agent therapy of 40 mg/m². Coadministration of ketoconazole with ixabepilone resulted in a 79% increase in AUC_0-. The relationship of microtubule bundle formation in peripheral blood mononuclear cells to plasma ixabepilone concentration was well described by the Hill equation. Microtubule bundle formation in peripheral blood mononuclear cells correlated with neutropenia.

Conclusions: Ixabepilone is a good CYP3A4 substrate in vitro; however, in humans, it is likely to be cleared by multiple mechanisms. Furthermore, our results provide evidence that there is a direct relationship between ixabepilone pharmacokinetics, neutrophil counts, and microtubule bundle formation in PBMCs. Strong inhibitors of CYP3A4 should be used cautiously in the context of ixabepilone dosing.

Experimental Design: Patients with measurable HER2⁺ metastatic breast cancer, ≤3 trastuzumab-based regimens, and left ventricular ejection fraction (LVEF) ≥55% received 8 or 6 mg/kg trastuzumab and 840 mg pertuzumab i.v. followed by 6 mg/kg trastuzumab and 420 mg pertuzumab every 3 weeks. Cardiac evaluation and tumor response were assessed every 3 and 6 weeks, respectively.

Results: Eleven patients received 64 cycles of trastuzumab plus pertuzumab. A total of 92 echocardiograms and 8 cardiac magnetic resonance imaging studies were done. With the lower limit of normal LVEF 55%, left ventricular systolic dysfunction was observed in six patients, three grade 1, two grade 2, and one grade 3 according to the National Cancer Institute Common Terminology Criteria for Adverse Events. The objective response rate was 18%. Two patients had partial responses, three had stable disease, and six had progressive disease. The median time to progression was 6 weeks. In baseline tumors from formalin-fixed paraffin-embedded primary and/or metastatic tumor biopsies, pHER2-Y1248 trended toward an increase in patients with partial response compared with those with stable disease/progressive disease (P = 0.095).

Conclusion: Trastuzumab plus pertuzumab may have clinical benefit in selected patients who have previously been treated with trastuzumab. Cardiac toxicity, although asymptomatic in most cases, was associated with this treatment. Further evaluation of efficacy of this combination is required to define the overall risks and benefits.

Experimental Design: This was a phase II, open-label, single arm study. Patients ≥15 years old with malignancies showing histologic or molecular evidence of expression/activation of imatinib-sensitive tyrosine kinases were enrolled. Patients were treated with 400 or 800 mg/d imatinib for hematologic malignancy and solid tumors, respectively. Treatment was continued until disease progression or unacceptable toxicity. The primary objective was to identify evidence of imatinib activity with tumor response as the primary end point.

Results: One hundred eighty-six patients with 40 different malignancies were enrolled (78.5% solid tumors, 21.5% hematologic malignancies). Confirmed response occurred in 8.9% of solid tumor patients (4 complete, 9 partial) and 27.5% of hematologic malignancy patients (8 complete, 3 partial). Notable activity of imatinib was observed in only five tumor types (aggressive fibromatosis, dermatofibrosarcoma protuberans, hypereosinophilic syndrome, myeloproliferative disorders, and systemic mastocytosis). A total of 106 tumors were screened for activating mutations: five KIT mutations and no platelet-derived growth factor receptor mutations were found. One patient with systemic mastocytosis and a partial response to therapy had a novel imatinib-sensitive KIT mutation (D816T). There was no clear relationship between expression or activation of wild-type imatinib-sensitive tyrosine kinases and clinical response.

Conclusion: Clinical benefit was largely confined to diseases with known genomic mechanisms of activation of imatinib target kinases. Our results indicate an important role for molecular characterization of tumors to identify patients likely to benefit from imatinib treatment.

Experimental Design: Patients that had progressed to standard treatment were treated with pertuzumab at a fixed dose of 1,050 mg given i.v. on day 1 plus capecitabine at doses of 825-1,000-1,250 mg/m², twice daily orally on days 1 to 14 of each 21-day treatment cycle, in three sequential cohorts. The pharmacokinetics of capecitabine and pertuzumab were studied. Patients received a single dose of capecitabine in a pretreatment phase (day –7) followed by serum sampling for capecitabine and its metabolites.

Results: Nineteen patients were accrued and 18 were assessable. The combination of capecitabine and pertuzumab was well tolerated at all dose levels and no dose-limiting toxicities were observed. The most frequent adverse event was asthenia, which was grade 3 in two patients. One asymptomatic pulmonary embolism occurred. No other grade 3 or 4 adverse events or cardiac or left ventricular ejection fraction events were reported. There was no apparent change in the pharmacokinetics of capecitabine and its metabolites when combined with pertuzumab. The pharmacokinetics of pertuzumab was apparently not modified when administered with capecitabine. Disease stabilization was observed in 11 patients.

Conclusions: Pertuzumab and capecitabine were well tolerated at all dose levels. Escalation beyond the highest dose level tested was not planned, as this included the recommended doses of monotherapy for both drugs. In conclusion, this combination is ready for phase II testing.

Experimental Design: Patients with advanced solid malignancies received various doses of G3139 (continuous i.v. infusion days 1-7), carboplatin (day 4), and paclitaxel (day 4), repeated in 3-week cycles, in a standard cohort-of-three dose-escalation schema. Changes in Bcl-2/Bax transcription/expression were assessed at baseline and day 4 (prechemotherapy) in both PBMCs and paired tumor biopsies. The pharmacokinetic interactions between G3139 and carboplatin/paclitaxel were measured.

Results: Forty-two patients were evaluable for safety analysis. Primary toxicities were hematologic (myelosuppression and thrombocytopenia). Dose escalation was stopped with G3139 at 7 mg/kg/d, carboplatin at area under the curve of 6, and paclitaxel at 175 mg/m² due to significant neutropenia seen in cycle 1 and safety concerns in further escalating chemotherapy in this phase I population. With G3139 at 7 mg/kg/d, 13 patients underwent planned tumor biopsies, of which 12 matched pairs were obtained. Quantitative increases in intratumoral G3139 with decreases in intratumoral Bcl-2 gene expression were seen. This paralleled a decrease in Bcl-2 protein expression observed in PBMCs.

Conclusions: Although the maximal tolerated dose was not reached, the observed toxicities were consistent with what one would expect from carboplatin and paclitaxel alone. In addition, we show that achievable intratumoral G3139 concentrations can result in Bcl-2 down-regulation in solid tumors and PBMCs.

Experimental Design: After primary surgery and chemotherapy, high-risk epithelial ovarian cancer patients in first clinical remission received NY-ESO-1b peptide and Montanide every 3 weeks for five vaccinations. Tumor expression was evaluated by immunohistochemistry. Toxicity was monitored using National Cancer Institute Common Toxicity Criteria Scale Version 2. NY-ESO-1 specific humoral immunity (ELISA), T-cell immunity (tetramer and ELISPOT), and delayed-type hypersensitivity were assessed on weeks 0, 1, 4, 7, 10, 13, and 16.

Results: Treatment-related adverse events included grade 1 fatigue, anemia, pruritus, myalgias, and hyperthyroidism and grade 2 hypothyroidism. There were no grade 3/grade 4 adverse events. Three of four patients (75%) with NY-ESO-1–positive tumor showed T-cell immunity by tetramer (0.6-9.5%) and ELISPOT (range, 35-260 spots). Four of five patients (80%) with NY-ESO-1–negative tumor showed T-cell immunity by tetramer (1.0-12.1%) and/or ELISPOT (range, 35-400 spots). With a median follow-up of 11.3 months, six of nine patients (67%) have recurred, with a median progression-free survival of 13 months (95% confidence interval, 11.2 months–not reached). Three of nine patients remain in complete clinical remission at 25, 38, and 52 months.

Conclusion: Vaccination of high-risk HLA-A*0201–positive epithelial ovarian cancer patients with NY-ESO-1b and Montanide has minimal toxicity and induces specific T-cell immunity in patients with both NY-ESO-1–positive and NY-ESO-1–negative tumors. Additional study is warranted.

Experimental Design: One hundred fifteen patients including 72 men (median age, 63 years; range, 36-79 years) and 43 women (median age, 60 years; range, 36-73 years) received 6 cycles of l-leucovorin 100 mg/m²/day and 5-FU 370 mg/m²/day i.v. boluses (5 days every 4 weeks). Individual plasma concentrations of 5-FU and 5-FDHU were determined on day 1 of the first cycle with a validated high performance liquid chromatography method, and the main pharmacokinetic variables were determined. Follow-up of all patients was extended up to 5 years after the end of adjuvant chemotherapy, and DFS was recorded. Univariate and multivariate analyses were conducted to evaluate any correlation among 5-FU pharmacokinetics, clinical and pathologic variables, and DFS.

Results: The area under the time/concentration curve (AUC) of 5-FU was significantly lower in 58 subjects who recurred (7.5 ± 2.9 h x mg/L) with respect to other patients (9.3 ± 4.1 h x mg/L). Furthermore, AUC values lower than 8.4 h x mg/L together with lymph node involvement and the interruption of treatment or reduction of doses were identified as risk factors at univariate analysis. The completion of 6 cycles of adjuvant treatment without dosage modifications was the only independent risk factor at multivariate analysis, despite a trend toward significance for 5-FU AUC values (cutoff value, 8.4 hxmg/L) was observed (P = 0.06).

Conclusions: Pharmacokinetics of 5-FU should be regarded as an important factor for predicting disease recurrence in colorectal cancers.

Experimental Design: Eligible patients were assigned to one of five disease-specific, parallel cohorts and given 12.5 mg deforolimus as a 30-minute infusion once daily for 5 days every 2 weeks. A Simon two-stage design was used for each cohort. Safety, pharmacokinetics, pharmacodynamics, and antitumor response were assessed.

Results: Fifty-five patients received deforolimus as follows: cohort 1 23 acute myelogenous leukemia, two myelodysplastic syndrome and one chronic myelogenous leukemia in nonlymphoid blast phase; cohort 2, one acute lymphocytic leukemia; cohort 3, nine agnogenic myeloid metaplasia; cohort 4, eight chronic lymphocytic leukemia; cohort 5, nine mantle cell lymphoma and two T-cell leukemia/lymphoma. Most patients were heavily pretreated. Of the 52 evaluable patients, partial responses were noted in five (10%), two of seven agnogenic myeloid metaplasia and three of nine mantle cell lymphoma. Hematologic improvement/stable disease was observed in 21 (40%). Common treatment-related adverse events, which were generally mild and reversible, were mouth sores, fatigue, nausea, and thrombocytopenia. Decreased levels of phosphorylated 4E-BP1 in 9 of 11 acute myelogenous leukemia/myelodysplastic syndrome patients after therapy showed mammalian target of rapamycin inhibition by deforolimus.

Conclusions: Deforolimus was well-tolerated in patients with heavily pretreated hematologic malignancies, and antitumor activity was observed. Further investigation of deforolimus alone and in combination with other therapeutic agents is warranted in patients with selected hematologic malignancies.

Experimental Design: We used the TAX-327 database to address (a) the relationship between quality of life (QoL) and pain; (b) whether minimally symptomatic patients benefit from treatment or have treatment-related decline in QoL; (c) the relationships between prostate-specific antigen (PSA) response, pain response, and QoL response; (d) the times at which these responses are first observed; and (e) whether PSA, pain, and/or QoL response predict for overall survival.

Results: At baseline, 374 of 815 men assessed for QoL had major pain; of these, 92% had substantial impairment of QoL compared with 75% without major pain (P < 0.001). Men with minimal symptoms had prolonged survival (median, 25.6 months) compared with symptomatic patients (median, 17.1 months; P = 0.009); they were more likely to have initial deterioration of QoL if treated with weekly docetaxel. PSA response and pain response, but not QoL response, were independently associated with survival in landmark analysis. Median times to PSA and pain response were 44 and 27 days, respectively; some men had initial increase in serum PSA before subsequent decline.

Conclusions: Symptoms other than pain contribute to impaired QoL in men with hormone-refractory prostate cancer. Those with minimal symptoms have prolonged survival. Both pain and PSA response are associated with survival but are not adequate to use as surrogate end points in phase 3 studies. Early increases in serum PSA (up to 12 weeks) should be ignored when determining response or progression.

Experimental Design: The effects of nitrite were evaluated on arteriole vasorelaxation, tumor cell respiration and tumor blood flow, oxygenation, and response to radiotherapy.

Results: We first showed that a small drop in pH (–0.6 pH unit) favored the production of bioactive NO from nitrite by documenting a higher cyclic guanosine 3',5'-monophosphate–dependent arteriole vasorelaxation. We then documented that an i.v. bolus injection of nitrite to tumor-bearing mice led to a transient increase in partial O₂ pressure in tumor but not in healthy tissues. Blood flow measurements failed to reveal an effect of nitrite on tumor perfusion, but we found that O₂ consumption by nitrite-exposed tumor cells was decreased at acidic pH. Finally, we showed that low dose of nitrite could sensitize tumors to radiotherapy, leading to a significant growth delay and an increase in mouse survival (versus irradiation alone).

Conclusions: This study identified low pH condition (encountered in many tumors) as an exquisite environment that favors tumor-selective production of NO in response to nitrite systemic injection. This work opens new perspectives for the use of nitrite as a safe and clinically applicable radiosensitizing modality.

Experimental Design: CS1 was analyzed by gene expression profiling and immunohistochemistry of multiple myeloma samples and numerous normal tissues. HuLuc63-mediated antimyeloma activity was tested in vitro in antibody-dependent cellular cytotoxicity (ADCC) assays and in vivo using the human OPM2 xenograft model in mice.

Results: CS1 mRNA was expressed in >90% of 532 multiple myeloma cases, regardless of cytogenetic abnormalities. Anti-CS1 antibody staining of tissues showed strong staining of myeloma cells in all plasmacytomas and bone marrow biopsies. Flow cytometric analysis of patient samples using HuLuc63 showed specific staining of CD138+ myeloma cells, natural killer (NK), NK-like T cells, and CD8+ T cells, with no binding detected on hematopoietic CD34+ stem cells. HuLuc63 exhibited significant in vitro ADCC using primary myeloma cells as targets and both allogeneic and autologous NK cells as effectors. HuLuc63 exerted significant in vivo antitumor activity, which depended on efficient Fc-CD16 interaction as well as the presence of NK cells in the mice.

Conclusions: These results suggest that HuLuc63 eliminates myeloma cells, at least in part, via NK-mediated ADCC and shows the therapeutic potential of targeting CS1 with HuLuc63 for the treatment of multiple myeloma.

Experimental Design: Human STS tissues and cell lines were used to study EGFR expression and activation. Western blot analysis was used to evaluate effects of EGFR activation on downstream signaling. Cell culture assays were used to assess the effect of EGF stimulation as well as EGFR blockade (using an EGFR tyrosine kinase inhibitor, Iressa; AstraZeneca) on STS cell growth, apoptosis, and chemosensitivity. An in vivo study (HT1080 human fibrosarcoma cell line in nude/nude mice: Iressa, doxorubicin, Iressa + doxorubicin, vehicle) was used to examine tumor growth; pEGFR, proliferating cell nuclear antigen, and terminal deoxyribonucleotide transferase–mediated nick-end labeling staining helped assess the effect of therapy in vivo on STS EGFR activation, proliferation, and apoptosis.

Results: EGFR was expressed and activated in STS cell lines and tumors, probably due to ligand binding rather than EGFR mutation. Stimulation caused activation of AKT and mitogen-activated protein kinase pathways. EGFR blockade inhibited these effects and also caused increased apoptosis, a p53-independent G₀-G₁ cell cycle arrest, and decreased cyclin D1 expression. In vivo, Iressa + doxorubicin had markedly synergistic anti-STS effects.

Conclusion: EGFR blockade combined with conventional chemotherapy results in anti-human STS activity in vitro and in vivo, suggesting the possibility that combining these synergistic treatments will improve anti-STS therapy.

Experimental Design: Using the photosensitizer HPPH (2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide), a wide range of PDT doses that included clinically relevant photosensitizer concentrations was evaluated. Magnetic resonance imaging and oxygen tension measurements were done along with the Evans blue exclusion assay to assess vascular response, oxygenation status, and tumor necrosis.

Results: In contrast to high-incident laser power (150 mW), low-power regimens (7 mW) yielded effective tumor destruction. This was largely independent of PDT dose (drug-light product), with up to 30-fold differences in photosensitizer dose and 15-fold differences in drug-light product. For all drug-light products, the duration of light treatment positively influenced tumor response. Regimens using treatment times of 120 to 240 min showed marked reduction in signal intensity in T2-weighted magnetic resonance images at both low (0.1 mg/kg) and high (3 mg/kg) drug doses compared with short-duration (6-11 min) regimens. Significantly greater reductions in pO₂ were observed with extended exposures, which persisted after completion of treatment.

Conclusions: These results confirm the benefit of prolonged light exposure, identify vascular response as a major contributor, and suggest that duration of light treatment (time) may be an important new treatment variable.

Experimental Design: The effects of a combination of 9cRA plus trastuzumab on the inhibition of cell growth in HLF human HCC cells which express constitutive activation of HER2 protein were examined.

Results: The combination of 9cRA plus trastuzumab synergistically inhibited the growth of HLF cells without affecting the growth of Hc normal human hepatocytes. Combined treatment with these agents acted synergistically to induce apoptosis in HLF cells. The treatment of HLF cells with trastuzumab alone inhibited the phosphorylation of HER2, RXR, ERK, Akt, and Stat3 proteins and these effects were enhanced when the cells were cotreated with 9cRA. Reporter assays indicated that the combination of 9cRA plus trastuzumab markedly increased both the retinoic acid responsive element and retinoid X responsive element promoter activities in HLF cells.

Conclusion: 9cRA and trastuzumab cooperatively inhibit the activation of HER2 and its downstream signaling pathways, subsequently inhibiting the phosphorylation of RXR and the growth of HCC cells. This combination might therefore be effective for the chemoprevention and chemotherapy of HCC.

Experimental Design: We established two pairs of parental and radioresistant (R) esophageal carcinoma cell lines (Seg-1, Seg-1R and TE-2, TE-2R) by fractionated irradiation. Stem cell markers were measured by Western blotting and flow cytometry. Serial sorting was used to enrich stem-like side population cells. Telomerase activity, transgene expression, antitumor activity, apoptosis induction, and viral replication were determined in vitro and/or in vivo.

Results: Expression of the stem cell markers β-catenin, Oct3/4, and β₁ integrin in Seg-1R cells was 29.4%, 27.5%, and 97.3%, respectively, compared with 4.8%, 14.9%, and 45.3% in Seg-1 cells (P < 0.05). SP levels in Seg-1R and TE-2R cells were 14.6% and 2.7%, respectively, compared with 3.4% and 0.3% in Seg-1 and TE-2 cells. Serial sorting of Seg-1R SP cells showed enrichment of the SP cells. Telomerase activities in Seg-1R, Seg-1R SP, and TE-2R cells were significantly higher than in Seg-1, Seg-1R non-SP, and TE-2 cells, respectively (P < 0.05). Seg-1R and TE-2R cells were more sensitive to Ad/TRAIL-E1 than parental cells. Increased Coxsackie-adenovirus receptor and elevated transgene expressions were found in the radioresistant cells. Ad/TRAIL-E1 resulted in significant tumor growth suppression and longer survival in Seg-1R–bearing mice (P < 0.05) with no significant toxicity.

Conclusion: Radioresistant cells established by fractionated irradiation display CSC-like cell properties. Ad/TRAIL-E1 preferentially targets radioresistant CSC-like cells.

Experimental Design: GS-9219 was selected through screening in proliferation assays and through pharmacokinetic screening. The activation pathway of GS-9219 was characterized in lymphocytes, and its cytotoxic activity was evaluated against a panel of hematopoietic and nonhematopoietic cell types. To test whether the prodrug moieties present in GS-9219 confer an advantage over PMEG in vivo, the pharmacokinetics, pharmacodynamics (lymph node germinal center depletion), and toxicity of equimolar doses of GS-9219 and PMEG were evaluated after i.v. administration to normal beagle dogs. Finally, proof of concept of the antitumor efficacy of GS-9219 was evaluated in five pet dogs with spontaneous, advanced-stage non–Hodgkin's lymphoma (NHL) following a single i.v. administration of GS-9219 as monotherapy.

Results: In lymphocytes, GS-9219 is converted to its active metabolite, PMEG diphosphate, via enzymatic hydrolysis, deamination, and phosphorylation. GS-9219 has substantial antiproliferative activity against activated lymphocytes and hematopoietic tumor cell lines. In contrast, resting lymphocytes and solid tumor lines were less sensitive to GS-9219. GS-9219, but not PMEG, depleted the germinal centers in lymphoid tissues of normal beagle dogs at doses that were tolerated. In addition, GS-9219 displayed significant in vivo efficacy in five dogs with spontaneous NHL after a single administration, with either no or low-grade adverse events.

Conclusion: GS-9219 may have utility for the treatment of NHL.

Experimental Design: We assessed antitumor effects on RENCA tumors and the degree of donor chimerism after several doses of irradiation followed by allogeneic SCT and compared the results with those of cyclophosphamide-based cell therapy.

Results: Allogeneic SCT following sublethal irradiation (6 Gy) induced almost complete donor chimerism, whereas cyclophosphamide-based cell therapy produced low levels of donor chimerism. Nonetheless, GVT activity was much more potent in cyclophosphamide-based cell therapy than irradiation-conditioned SCT. Furthermore, cyclophosphamide-conditioned SCT induced more potent immune reconstitution with less severe graft-versus-host disease than irradiation-conditioned SCT.

Conclusions: Our results indicate that a high level of chimerism is not essential for the in vivo antitumor effect of nonmyeloablative allogeneic cell therapy against solid tumor and that the recovery of peripheral lymphocytes after the initial immunosuppression might be a critical event for the elicitation of in vivo antitumor effects of that treatment modality.