Squamous Cell Carcinoma of the Vulva: A Subclassification of 97 Cases by Clinicopathologic, Immunohistochemical, and Molecular Features (p16, p53, and EGFR).

Squamous cell carcinomas (SCCs) of the vulva develop through human papilloma virus (HPV)-associated or HPV-independent pathways, but the relationship between pathogenesis, classification, and prognosis of these tumors is controversial. Therefore, we review the morphology, immunophenotype, and select molecular features of a consecutive series of 97 patients with vulvar SCC with a median clinical follow-up of 3.6 years. Tumors were histologically classified as basaloid (13), warty (11), mixed basaloid and warty (1), keratinizing (68), nonkeratinizing (3), and sarcomatoid (1). Diffuse p16 expression was associated with younger age at presentation (P<0.0001), basaloid and warty carcinoma subtypes (P<0.0001), and usual vulvar intraepithelial neoplasia (P<0.0001) and was negatively associated with p53 immunopositivity (P=0.0008). Five keratinizing SCCs showed p16 and p53 coexpression, but only 1 was positive for high-risk HPV by in situ hybridization. Among 8 of 36 tumors with EGFR gene amplification, 4 were p53 positive but none p16 positive. In a Cox regression model, early clinical stage (P<0.006), p16 expression (P=0.002), and absent p53 expression (P=0.02) were independent predictors of improved overall survival. These findings utilize morphologic and immunohistochemical analysis to support HPV-associated and HPV-independent pathogenesis of vulvar SCCs and support p16 and p53 immunohistochemistry as markers of disease biology and clinical outcome.

Decreased survival in EGFR gene amplified vulvar carcinoma.

OBJECTIVE: We undertook an extensive molecular characterization of the epidermal growth factor receptor (EGFR) gene in vulvar squamous cell carcinomas to investigate EGFR mutation and/or genomic amplification and its association with EGFR protein expression, high-risk human papillomavirus (HPV) status and clinical outcome. METHODS: A cohort of 51 vulvar cancer patients distributed across all FIGO stages was selected for immunohistochemistry (IHC) and fluorescence in situ hybridization. EGFR expression and gene amplification were correlated with high-risk HPV status, EGFR mutational status and clinical prognostic variables. Fisher's exact tests, Kaplan-Meier survival estimates and a Cox proportional-hazards model were utilized. RESULTS: EGFR gene amplification and chromosome 7 high polysomy were observed in 12% and 6% of cases, respectively. IHC of malignant tissue with 3+ staining demonstrated 100% sensitivity and 79% specificity to detect EGFR gene amplification, yielding a 39% positive predictive value. Decreased survival (p<0.025) was observed in patients with gene amplification, and was associated with a more statistically robust 3.3 hazard ratio (p<0.005) in the Cox proportional-hazards model that controlled for age at diagnosis, stage and lymph node metastasis. Univariate analysis confirmed that EGFR gene amplification was associated with the absence of high-risk HPV (p<0.001). Common activating EGFR gene mutations were not identified. CONCLUSION: A subset of patients with vulvar squamous cell carcinoma was identified with EGFR gene amplification that was HPV-independent and associated with poor prognosis. Given the association of EGFR amplification with response to targeted therapy in other tumor types, these patients may be candidates for therapeutic strategies that target the EGFR pathway.

Growth inhibitory effects of pegylated IFN-alpha2b and 5-fluorouracil in combination on renal cell carcinoma cell lines in vitro and in vivo.

We investigated the effects of pegylated IFN-alpha2b (PEG-IFN-alpha2b) alone and PEG-IFN-alpha2b plus 5-fluorouracil (5-FU) in vitro on the proliferation of renal cell carcinoma (RCC) cell lines. After the transplantation of RCC cells into nude mice, we administered IFN (PEG-IFN-alpha2b or IFN-alpha2b) alone, 5-FU alone, or IFN (PEG-IFN-alpha2b or IFN-alpha2b) plus 5-FU; and investigated tumor volume, tumor weight, the numbers of apoptotic cells and artery-like blood vessels, relative mRNA expression levels of enzymes which relate to 5-FU metabolism, angiogenesis factor, and type I interferon receptor. RCC cells in vitro were generally and relatively resistant to the anti-proliferative effects of PEG-IFN-alpha2b, but the addition of 5-FU augmented IFN-induced anti-proliferative effects with the induction of apoptosis. PEG-IFN-alpha2b in vivo presented stronger anti-tumor effects than IFN-alpha2b, and its combination with 5-FU augmented the effects. The significant anti-tumor effect of the combination treatment was the increase in apoptotic cell number, but there were no significant differences in the suppression of angiogenesis, expression of IFN receptor, and the actions of metabolic enzymes of 5-FU. In conclusion, PEG-IFN-alpha2b presents stronger anti-tumor effects than non-pegylated IFN, and the effects are augmented in the combination with 5-FU. Our findings suggest the clinical usefulness of PEG-IFN-alpha2b in the treatment of RCC.

Overexpression of the myc target gene Mina53 in advanced renal cell carcinoma.

The myc target gene Mina53 was reported to be overexpressed in esophageal cancer with a poor prognosis. The purpose of the present study was to examined Mina53 expression and its relationship to clinicopathological parameters in human renal cell carcinoma (RCC). Mina53 and Ki-67 expression was examined on immunohistochemistry for 64 surgically resected RCC and non-cancerous tissue. In addition, the relationship between Mina53 expression and clinicopathological prognostic factors of RCC such as age, stage, microvenous invasion (MVI), histological subtype, Ki-67 labeling index (LI), and prognosis, was examined. Mina53 was expressed in the nuclei of tumor cells and tubular nuclei of normal renal tissue. The expression level of Mina53 was significantly higher in patients with poor prognostic factors (stage IV, MVI-positive, and sarcomatoid RCC, and high Ki-67 LI). The prognosis of high Mina53-expressing tumors was significantly poorer than that of non-Mina53-high tumors (P < 0.0001). In conclusion, Mina53 is overexpressed in RCC tissue from patients with poor prognostic factors, suggesting that Mina53 overexpression is one of the factors for poor prognosis in RCC.

Growth inhibitory effects of IFN-beta on human liver cancer cells in vitro and in vivo.

We investigated the effects of interferon-beta (IFN-beta) on the growth of human liver cancer cells. The effects of IFN-beta with or without 5-fluorouracil (5-FU) on the proliferation of 13 liver cancer cell lines were investigated in vitro. Chronologic change in IFN-alpha receptor 2 (IFNAR-2) expression was monitored in hepatocellular carcinoma (HCC) cells (HAK-1B) cultured with IFN-beta. After HAK-1B cells were transplanted into nude mice, various doses of IFN-beta were administered, and the tumor volume, weight, histology, tumor blood vessel, and angiogenesis factor expression were examined. IFN-beta inhibited the growth of 11 cell lines with apoptosis in a dose-dependent and time-dependent manner. With IFN-beta, IFNAR-2 expression in HAK-1B cells was significantly downregulated from 6 to 12 h. IFN-beta induced a dose-dependent decrease in tumor volume and weight and a significant increase of apoptosis in the tumor. Both basic fibroblast growth factor (bFGF) and blood vessel number in the tumor decreased only in mice receiving the lowest dose (1000 IU) of IFN-beta. IFN-beta with 10 muM of 5-FU frequently induced synergistic antiproliferative effects. IFN-beta with or without 5-FU induces strong antitumor effects in HCC cells, and we conclude that IFN-beta is useful for the prevention and treatment of HCC.

Effects of IFN-alpha on alpha-fetoprotein expressions in hepatocellular carcinoma cells.

We investigated the effects of pegylated (PEG)-IFN-alpha2b on alpha-fetoprotein (AFP) expression as demonstrated by protein and mRNA levels in six human hepatocellular carcinoma (HCC) cell lines. The number of KIM-1 cells in culture with PEG-IFN-alpha2b decreased between 24 amd 240 h, whereas the levels of intracellular and secreted AFP per cellular protein increased (except at 192 h), with levels 1.9-fold and 2.9-fold higher at maximum, respectively, than cells without PEG-IFN-alpha2b (control). The mRNA level increased between 72 and 192 h, when the level was 3-fold higher than that of the control. In the 72-h culture with 40-5000 IU/mL PEG-IFN-alpha2b, there were dose-dependent increases in AFP protein and mRNA expression and dose-dependent decrease in cell number resulting from apoptosis and blockage of the cell cycle at the S-phase. The rate of fucosylated AFP in the cell lysate decreased in a dose-dependent and time-dependent manner. In the PEG-IFN-alpha2b culture of the other five HCC cell lines, cell proliferation was suppressed, but the expressions of AFP protein and mRNA increased in only two cell lines, and suppression of cell proliferation was not related to the increase in AFP expressions. Our findings demonstrated that PEG-IFN-alpha2b induces an increase in AFP expression at both the protein and mRNA levels.

Growth inhibitory effects of interferon-alpha subtypes vary according to human liver cancer cell lines.

BACKGROUND: Interferon (IFN)-alpha preparations used in the treatment of viral and neoplastic disease consist of single or multiple IFN-alpha subtypes that may possess different biological activity, but there are no data on liver cancer cells. METHODS: Antiproliferative effects and the mechanisms of growth inhibition of five IFN-alpha subtypes (alpha1, alpha2, alpha5, alpha8 and alpha10) were examined in vitro using 13 human liver cancer cell lines. RESULTS: The antiproliferative effect of each IFN-alpha subtype was different in each cell line. The 50% growth inhibitory concentration (IC50) on an antiviral unit basis showed that alpha5 presented the most potent antiproliferative effects in 11 of the 13 cell lines, and alpha8 in two cell lines. On average, the antiproliferative effects were strong in descending order from alpha5, alpha8, alpha10, alpha2 to alpha1. On weight basis, the most potent antiproliferative effect was shown by alpha8 in nine of the 13 cell lines, alpha5 in four cell lines, and the potency of the effects on average in descending order was alpha8, alpha5, alpha10, alpha2 and alpha1. No significant difference was observed between natural and recombinant alpha2. The mechanism of growth inhibition of each subtype in HAK-1B and KMCH-1 cell lines were apoptosis and S-phase arrest, and their induction levels were related to a certain degree to the antiproliferative effects. CONCLUSIONS: Our findings show that the antiproliferative effect of each IFN-alpha subtype varies according to the cell line, but that the cells are relatively or absolutely responsive to alpha5 and alpha8 subtypes.

Growth inhibitory effects of pegylated IFN alpha-2b on human liver cancer cells in vitro and in vivo.

PURPOSE: We investigated the effects of pegylated IFN-alpha2b (PEG-IFN-alpha2b) on the growth of human liver cancer cells. METHODS: The effect of PEG-IFN-alpha2b on the proliferation of 13 liver cancer cell lines was investigated in vitro. Chronological changes in growth and IFN-alpha receptor-2 (IFNAR-2) expression were monitored in hepatocellular carcinoma (HCC) cells (HAK-1B) cultured with PEG-IFN-alpha2b. After HAK-1B cells were transplanted into nude mice, various doses of PEG-IFN-alpha2b or IFN-alpha2b were administered, and tumor volume, weight, histology, and IFNAR-2 expression were examined. RESULTS: PEG-IFN-alpha2b inhibited the growth of nine cell lines with apoptosis in a dose- and time-dependent manner. Continuous contact with PEG-IFN-alpha2b induced time-dependent growth inhibition and down-regulation of IFNAR-2 expression. PEG-IFN-alpha2b induced a dose-dependent decrease in tumor volume and weight, a significant increase of apoptotic cells, and a decrease in IFNAR-2 expression in the tumor. The clinical dose for chronic hepatitis C was also effective. The antitumor effect of PEG-IFN-alpha2b was significantly stronger than that of non-PEG-IFN-alpha2b in vivo. CONCLUSIONS: Continuous contact with PEG-IFN-alpha2b induces strong antitumor effects and the down-regulation of IFNAR-2 in HCC cells. The data suggest potential clinical application of PEG-IFN-alpha2b for the prevention and treatment of HCC.

Antiproliferative effects of 5-fluorouracil and interferon-alpha in combination on a hepatocellular carcinoma cell line in vitro and in vivo.

BACKGROUND AND AIM: We investigated the antiproliferative effects of interferon-alpha (IFN-alpha) and 5-fluorouracil (5-FU) in combination on a hepatocellular carcinoma (HCC) cell line. METHOD: In the in vitro study, IFN-alpha and/or 5-FU was added to the culture of the poorly differentiated-type HCC cell line, HAK-1B, and their antiproliferative effects and additional or synergic effects in combination treatment were examined. In the in vivo study, HAK-1B cells were transplanted into nude mice and the changes in tumor volume and weight, apoptosis, BrdU and cyclin A positive cells, and artery-like blood vessels were investigated. Expressions of angiogenesis factors and IFN-alpha receptor (IFNAR-2) were examined in the developed tumors. RESULTS: In vitro growth of HAK-1B cells was suppressed dose-dependently to 5-FU, but the addition of IFN-alpha did not induce additional or synergic effects. In vivo growth in terms of tumor diameter and weight was suppressed at most in the IFN-alpha + 5-FU (combination) group, that is, the tumor volume became 29.3% and the tumor weight became 54.7% of the control. In the combination group, numbers of BrdU-positive S-phase cells and cyclin A positive cells increased together with the increase in apoptotic cells, but there was no significant relation between the tumor shrinkage effects and angiogenesis factors or artery-like blood vessels. In the combination group, INFAR-2 decreased significantly in comparison to the other groups. CONCLUSION: The synergic growth-suppression effects in the current in vivo study using the combination treatment are attributable to the enhanced induction of S-phase arrest and of apoptosis.

Expression and activation of apoptosis-related molecules involved in interferon-alpha-mediated apoptosis in human liver cancer cells.

Interferon (IFN)-alpha directly inhibits proliferation of liver cancer cells by inducing apoptosis, but the molecular mechanisms by which IFN-alpha induces apoptosis in these cells are not fully understood. We examined the effect of broad spectrum caspase inhibitor, Z-VAD-fmk, and the caspase activation in IFN-alpha-mediated apoptosis by using 4 liver cancer cell lines that were sensitive or resistant to IFN-alpha-mediated apoptosis. Involvement of apoptosis-related mitochondrial proteins and Bcl-2 family proteins in IFN-alpha-mediated apoptosis was further examined in 1 sensitive cell line (KIM-1). The Z-VAD-fmk completely or moderately inhibited IFN-alpha-mediated apoptosis in the sensitive cells. IFN-alpha induced time-dependent activation of caspase-3 in the sensitive cells, while the resistant cells showed mild or no activation. Activation of caspase-9, caspase-8, and caspase-7, and the cleavage of poly(ADP-ribose)polymerase were identified in either or both of the sensitive cell lines, but not in the resistant cells. In KIM-1 cells, the release of cytochrome c and Smac/DIABLO from mitochondria to cytosole was confirmed. Meanwhile, Bcl-xL was upregulated, and Bid activation or translocation, or conformational changes of Bax were not identified. In conclusion, our results suggest IFN-alpha-mediated apoptosis in liver cancer cells involves the mitochondrial apoptotic pathway and is induced by activating various caspases.

Expression of matrix metalloproteinases (MMPs) in cultured hepatocellular carcinoma (HCC) cells and surgically resected HCC tissues.

Matrix metalloproteinases (MMPs) relate to the growth and infiltration of cancer cells, but the frequency and amount of their expression are not yet fully examined in hepatocellular carcinoma. Expression of MMPs (MMP-2, MMP-7, MMP-9, MT1-MMP, MT2-MMP, MT3-MMP) and tissue inhibitors of metalloproteinase (TIMP: TIMP-1, TIMP-2) was investigated on cultured hepatocellular carcinoma (HCC) cells and surgically resected HCC tissues. The cultured cells and tissues expressed MMPs and TIMPs at various degrees, and high expression was observed for MMP-2, MMP-9, MT1-MMP and TIMP-2. Expression of MMP-7, MT2-MMP and TIMP-1 was found at a low frequency and a low amount in both the cells and the tissues. MMP-2 was expressed in various cells: HCC cells, vascular wall and sinusoidal endothelial cells in the cancer area of surgically resected tissues; and hepatocytes, bile duct cells, vascular wall, macrophages and Kupffer cells in the non-cancerous area. MMPs and TIMPs were expressed at a relatively high frequency in hepatocytes of the cancerous area and surrounding non-cancerous area as well as in the other cells and tissues. MMPs and TIMPs may be involved in the progression of hepatocellular carcinoma including the infiltration of cancer cells.

Antiproliferative effects of interferon-alphaCon1 on ovarian clear cell adenocarcinoma in vitro and in vivo.

PURPOSE: We examined the antiproliferative effect of IFN-alphaCon1 and its mechanism on ovarian clear cell adenocarcinoma in vitro and in vivo. EXPERIMENTAL DESIGN: (a) The effects of IFN-alphaCon1 on growth, morphology, cell cycle, and type I IFN-alpha receptor (IFNAR-2) expression were examined on two ovarian clear cell adenocarcinoma cell lines (KOC-5C and KOC-7C) in vitro. (b) KOC-5C or KOC-7C cells were transplanted into nude mice, and changes in tumor volume, tumor weight, apoptosis, necrosis, and microvessel density were investigated. The expression of angiogenesis factors was examined in the serum and the developed tumors. RESULTS: Both cell lines expressed IFNAR-2 mRNA, but its protein was detected only in KOC-7C. In KOC-7C cells, antiproliferative effects were observed in a time- and dose-dependent manner and cell division was blocked at the S phase. The KOC-7C tumors showed decreases in tumor volume and weight; a decreasing tendency in basic fibroblast growth factor (bFGF), vascular endothelial growth factor, and interleukin (IL)-8 protein expression in the tumor; a significant decrease in bFGF and IL-8 protein expression in the serum, and of microvessel density; and significant increase in apoptosis and necrosis in the tumor. In the KOC-5C tumors, these in vitro and in vivo changes were not apparent, and the antiproliferative effects of IFN-alphaCon1 were not obvious. CONCLUSIONS: IFN-alphaCon1 suppresses tumor proliferation by inducing apoptosis, blocking the cell cycle, and inhibiting tumor angiogenesis. Our findings show that the clinical efficacy of IFN-alphaCon1 can be predicted by examining IFNAR-2 expression on tumor cells, and the efficacy of IFN-alphaCon1 treatment can be evaluated by measuring serum bFGF and IL-8 levels.

Interferon-alphaCon1 suppresses proliferation of liver cancer cell lines in vitro and in vivo.

BACKGROUND/AIMS: We investigated the effects of consensus interferon (IFN-alphaCon1), a nonnaturally occurring type I interferon with higher specific activity than other type I IFNs, on the growth of human liver cancer cells. METHODS: The effect of IFN-alphaCon1 on the proliferation of 13 liver cancer cell lines was investigated in vitro. Hepatocellular carcinoma (HCC) cells (KIM-1 and HAK-1B) were transplanted subcutaneously into the back of nude mice, then IFN-alphaCon1 was subcutaneously administered to the mice once a day for 2 weeks, and tumor volume and histology were examined. RESULTS: IFN-alphaCon1 expressed a dose-dependent growth inhibitory effect in all cell lines in vitro. KIM-1 tumor volume in mice that received 0.01 microg (10(4)IU)/mouse/day of IFN-alphaCon1 (similar to the clinical dose for chronic hepatitis C) was 62% of the control, 0.1microg/mouse/day resulted in 26%, and 1 microg/mouse/day resulted in 10%. HAK-1B tumor volume under the same treatment was 61, 24 and 0% of the control, respectively. The number of apoptotic cells significantly increased and the number of blood vessels significantly decreased with the increase in IFN-alphaCon1 dose. CONCLUSIONS: IFN-alphaCon1 suppressed HCC growth in nude mice. These data indicate the potential clinical application of IFN-alphaCon1 in the prevention and treatment of HCC.

Metastatic carcinoid tumor in the orbit.

In 1991, a carcinoid tumor of the duodenum with metastasis in the regional lymph nodes was resected in a 39-year-old-man. A metastasis in the mediastinum was found and was resected in 1996. In 1999, a metastasis in the left lung was discovered and was excised. In 2001, he noticed right-sided diplopia, and a computed tomography (CT) scan disclosed a tumor of 2 cm diameter in the right orbit with infiltration to the frontal sinus. The tumor was resected, and was diagnosed metastatic carcinoid tumor. An orbital metastasis of carcinoid tumor is rare, and only about 30 cases have been reported in the English literature. The rarity metastasis of carcinoid tumors in the orbit prompted us to report this case.

Primary leiomyoma of the ovary--a report of 2 resected cases.

Leiomyoma of the ovary is relatively rare and its origin is still controversial. Here, we report 2 cases of ovarian leiomyoma. Case 1, a 59-year-old woman who complained of abdominal distention. Ultrasonography and computed tomography revealed a large solid mass in the lower abdomen. The mass was diagnosed as uterine myoma preoperatively, but it turned out to be a left ovarian tumor at laparotomy. The tumor was about an adult-head size, grayish, elastic hard, and solid. In case 2, a fist-sized mass was accidentally detected in the right ovary of a 84-year-old woman during laparotomy for rectal cancer. Histologically both tumors were composed of solid proliferation of short spindle-shaped cells, that formed interlacing fascicles and showed varying degrees of hyalinization. Both tumors were diagnosed as leiomyoma. Although this tumor is rather infrequent, it is necessary to consider leiomyoma for differential diagnosis, when a solid tumor is detected in the ovary.

Intravenous leiomyomatosis with uterine leiomyoma and adenomyosis: a case presentation and brief comment on the histogenesis.

Uterine intravenous leiomyomatosis a rather rare pathologic condition, and a total of 139 cases have appeared in the English literature. Although two proposals for the histologic origin, uterine lyomyoma itself and/or vascular smooth muscle, have been widely accepted, the precise histogenesis still remains unclear. The additional 140th case of intravenous leiomyomatosis is described, and a third postulation for the histogenesis is proposed.