Purpose: mTOR and P70 S6 kinase (S6K) play a key role in regulating protein translation. The role of mTOR and S6K in hepatocellular carcinoma has not been investigated, but this pathway is of particular interest because an effective inhibitor, rapamycin, is available. This study was undertaken to determine the prevalence and clinicopathological correlates of mTOR pathway activation in hepatocellular carcinoma and to determine whether rapamycin inhibits the pathway in cell culture.Experimental Design: Total and phosphorylated mTOR and S6K protein expression were studied by immunohistochemistry in hepatocellular carcinomas (n ‫؍‬ 73), fibrolamellar carcinomas (n ‫؍‬ 13), and hepatic adenomas (n ‫؍‬ 15). Results were correlated with tumor growth pattern as defined by the WHO (trabecular, pseudoglandular/acinar, compact, and scirrhous), tumor size, Ki-67 proliferation index, and the modified Edmondson nuclear grade, which has a scale of 1 to 4. HepG2 and Hep3B cell lines were treated with rapamycin to see the effect on proliferation and S6K phosphorylation.Results: Increased expression of total mTOR was seen in 5% of hepatocellular carcinoma, whereas overexpression of phospho-mTOR was evident in 15% of hepatocellular carcinoma. Phospho-mTOR positivity correlated with increased expression of total S6K, which was found in 45% of cases. Total S6K overexpression was positively correlated with tumor nuclear grade, inversely with tumor size, and was unassociated with the proliferation index or WHO growth pattern. Rapamycin treatment of HepG2 and Hep3B cell lines markedly inhibited cell proliferation and reduced S6K phosphorylation in both cell lines.Conclusions: The mTOR pathway is activated in a subset of hepatocellular carcinoma. Rapamycin can inhibit proliferation of neoplastic hepatocytes in cell culture.

To identify 1H-MRS molecular biomarkers of early clinical therapeutic response in non-Hodgkin's lymphoma, an in vivo longitudinal study was performed on human non-Hodgkin's diffuse large B-cell lymphoma xenografts (WSU-DLCL2) grown in the flanks of female SCID mice. 31P-MRS measurements, which have been demonstrated to be prognostic clinical indices of response (Arias-Mendoza et al. Acad. Radiol. 2004; 11: 368-376) but which provide lower spatial resolution, were included for comparison. The animals received CHOP (cyclophosphamide, hydroxydoxorubicin, oncovin and prednisone) chemotherapy for three 1-week cycles, resulting in stable disease based on tumor volume. Localization of total choline and phosphorus metabolites in vivo was achieved with stimulated echo acquisition mode and image selected in vivo spectroscopy sequences, respectively. Significant decreases in lactate were detected by the selective multiple quantum coherence spectral editing technique after the first cycle of CHOP, whereas total choline and the phosphomonoester/nucleoside triphosphate ratio did not change until the third cycle. Ex vivo extract MRS of tumors corroborated the in vivo results. Histological staining with antibodies to Ki67 revealed a decrease in proliferation rate in CHOP-treated tumors that coincided with the decrease in lactate. This study demonstrates the utility of lactate as an early proliferation-sensitive indicator of therapeutic response in a mouse model of non-Hodgkin's lymphoma and serves as a basis for future clinical implementation of these methods.

The retinoblastoma gene product (Rb) is a tumor suppressor that affects apoptosis paradoxically. Most sporadic cancers inactivate Rb by preferentially targeting the pathway that regulates Rb phosphorylation, resulting in resistance to apoptosis; this contrasts with Rb inactivation by mutation, which is associated with high rates of apoptosis. How phosphorylated Rb protects cells from apoptosis is not well understood, but there is evidence that Rb may sequester a pro-apoptotic nuclear factor. pp32 (ANP32A) is a pro-apoptotic nuclear phosphoprotein, the expression of which is commonly increased in cancer. We report that hyperphosphorylated Rb interacts with pp32 but not with the closely related proteins pp32r1 and pp32r2. We further demonstrate that pp32-Rb interaction inhibits the apoptotic activity of pp32 and stimulates proliferation. These results suggest a mechanism whereby cancer cells gain both a proliferative and survival advantage when Rb is inactivated by hyperphosphorylation.The retinoblastoma protein (Rb) 1 is a nuclear phosphoprotein that regulates proliferation, differentiation, and apoptosis. As a tumor suppressor, Rb inhibits proliferation by repressing E2F1-mediated transcription when hypophosphorylated. Hyperphosphorylation of Rb relieves E2F1 repression and allows cell cycle progression to occur (1). The importance of Rb is underscored by the fact that Rb function is disrupted in virtually all human cancers (2). Paradoxically and inconsistent with its role as a tumor suppressor, hyperphosphorylated wild-type Rb inhibits apoptosis in both cell culture and animal models (3)(4)(5)(6)(7)(8)(9)(10). Because Rb inactivation is pivotal for carcinogenesis, this poses the problem of how cancer cells escape apoptosis when Rb function is disrupted.Inherited cancers and cells in which Rb is inactivated by mutation have increased rates of both proliferation and apoptosis (11). Most sporadic cancers preferentially inactivate Rb by hyperphosphorylation, which may occur through mutation of cyclin D, cdk4, or p16. Such cancers are generally slow growing and resistant to apoptosis induced by chemotherapy or radiation (12). It is possible that in these cancers the tumor suppressor function of Rb is inhibited, whereas the anti-apoptotic function remains intact (13). Inactivation by hyperphosphorylation might promote proliferation by increasing free E2F1, as well as inhibit apoptosis by retaining the anti-apoptotic function of Rb. This is consistent with evidence suggesting that it is the hyperphosphorylated form of Rb rather than Rb per se that protects cells from apoptosis (14). The induction of apoptosis in various cell lines is accompanied by a shift in Rb from the hyperphosphorylated to the hypophosphorylated form (15,16). Rb dephosphorylation, which has been shown to be required for apoptosis, occurs in the early stage of apoptosis (17,18). Inhibition of Rb dephosphorylation prevents apoptosis, whereas induction of dephosphorylation leads to apoptosis (19). In DBA/2 mice, increased levels of hyperphosphoryla...

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Loss of fragile histidine triad (FHIT) gene expression is seen in approximately 50% of hepatocellular carcinomas in China. However, little information is available on FHIT expression in hepatocellular carcinoma in the United States, where carcinogen exposure is generally lower. Investigations of FHIT mRNA in hepatocellular neoplasms and paired non-neoplastic tissues demonstrated normal-sized FHIT transcripts in all non-neoplastic tissues and in all neoplasms including 11 hepatocellular carcinomas, two fibrolamellar carcinomas, and four benign proliferative lesions. In addition, all but one malignant and all benign neoplasms showed aberrant smaller transcripts. The smaller aberrant transcripts were overexpressed in 6/11 hepatocellular carcinomas and 1/2 fibrolamellar carcinomas. An additional 79 hepatocellular carcinomas, 12 fibrolamellar carcinomas and 15 hepatic adenomas were examined for FHIT expression by immunohistochemistry. No loss of immunostaining was seen in 67/79 (85%) of hepatocellular carcinomas, while a moderate or marked decrease was seen in 12/79 (15%). Fibrolamellar carcinomas and hepatic adenomas showed no loss of FHIT expression. In conclusion, hepatocellular carcinomas retained expression of normal FHIT mRNA transcripts, but also showed universal expression of smaller sized aberrant transcripts and commonly overexpressed these aberrant transcripts. Loss of FHIT protein expression is relatively uncommon in this cohort from the United States, where exposure to hepatic carcinogens is generally low.

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8122 Background: First-line chemotherapy cures ∼50% of pts with DLBCL, while salvage therapy followed by ASCT can cure another ∼30%. R significantly improves response rates to 1st line therapy. This retrospective study was designed to test the hypotheses that: 1) ASCT is effective in pts relapsed after R-chemotherapy and 2) addition of R to salvage improves outcomes after ASCT. Methods: We identified 84 pts with relapsed/refractory DLBCL who underwent ASCT at our institution between 1990 and 2006. In all, 32% received a R-chemo 1st line regimen and 27% received R with salvage. The median age at ASCT was 49 yrs and the median time from diagnosis to ASCT was 16 mos. High-dose regimens included BCV (48%), BEAM (8%) and alkylator/TBI (20%). Results: Overall response rate (ORR) after ASCT was 52%, with 37% of pts in CR by day 100. Among those in CR, 16% had a CR pre-ASCT, 72% had a lesser response, and 9% were chemo-resistant. The addition of R to salvage (23/84 pts) was favorably associated with ORR after ASCT (OR: 5.2, 95% CI: 1.1 - 25, p=0.029), even in pts who had failed a prior R regimen (p=0.013). Other factors favorably associated with ORR were response to salvage (p=0.046) and time to ASCT >12 mos (p=0.017). At last f/u (med: 22 mos, iqr: 7 - 55 mos), event-free (EFS) and overall survival (OS) were both 35%. The only factor associated with EFS and OS in univariate and multivariate analyses was ORR after ASCT (HR: 0.16, 95% CI: 0.07 - 0.37, p<0.001 and HR: 0.12, 95% CI: 0.05 - 0.28, p<0.001 respectively). Age at ASCT, time to ASCT, year of ASCT, mobilization/conditioning regimen, and failure of a R-chemo regimen were not associated with EFS or OS. Conclusions: Pts with DLBCL who have failed a R-chemo first-line regimen derive an equal benefit from ASCT as pts who are R-naïve, with significant long-term EFS and OS. Additionally, inclusion of R in salvage therapy prior to ASCT provides superior response rates, even after a failed prior R-chemo regimen. These results confirm the benefit of ASCT for pts with DLBCL in the rituximab era and argue for the incorporation of R and related agents in studies of high-dose therapy and ASCT. No significant financial relationships to disclose.