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Background:High-risk human papillomavirus (HR-HPV) infection is a risk factor for anal cancer, yet no anal cancer screening guidelines exist for women with lower genital tract HPV-related disease. We sought to describe the prevalence of anal HR-HPV or cytologic abnormalities in such women.Methods:This cross-sectional study was performed between October 2018 and December 2021. Inclusion criteria were ≥21 years of age and a prior diagnosis of high-grade dysplasia/cancer of the cervix, vagina, or vulva. Participants underwent anal cytology and anal/cervicovaginal HR-HPV testing. Women with abnormal anal cytology were referred for high-resolution anoscopy (HRA).Results:324 evaluable women were enrolled. Primary diagnosis was high-grade dysplasia/cancer of the cervix (77%), vagina (9%), and vulva (14%). Anal HR-HPV was detected in 92 patients (28%) and included HPV-16 in 24 (26%), HPV-18 in 6 (7%), and other HR-HPV types in 72 (78%) patients. Anal cytology was abnormal in 70 patients (23%) and included atypical squamous cells of undetermined significance (80%), low-grade squamous intraepithelial lesion (9%), high-grade intraepithelial lesion (HSIL; 1%), and atypical squamous cells-cannot rule out HSIL (10%). Of these patients, 55 (79%) underwent HRA. Anal biopsies were performed in 14 patients: 2 patients had anal intraepithelial neoplasia (AIN) 2/3, 1 patient had AIN 1, and 11 patients had negative biopsies. Both patients with AIN 2/3 had a history of cervical dysplasia.Conclusions:Our results suggest an elevated risk of anal HR-HPV infection and cytologic abnormalities in women with lower genital tract dysplasia/cancer.Impact:These results add to the growing body of evidence suggesting the need for evaluation of screening methods for anal dysplasia/cancer in this patient population to inform evidence-based screening recommendations.

Supplementary Table from A Cross-Sectional Study of the Prevalence of Anal Dysplasia among Women with High-Grade Cervical, Vaginal, and Vulvar Dysplasia or Cancer: The PANDA Study

Women with Lynch syndrome have a 40% to 60% lifetime risk for developing endometrial cancer, a cancer associated with estrogen imbalance. The molecular basis for endometrial-specific tumorigenesis is unclear. Progestins inhibit estrogen-driven proliferation, and epidemiologic studies have shown that progestin-containing oral contraceptives (OCP) reduce the risk of endometrial cancer by 50% in women at general population risk. It is unknown whether they are effective in women with Lynch syndrome. Asymptomatic women ages 25 to 50 with Lynch syndrome were randomized to receive the progestin compounds Depo-Provera (depo-MPA) or OCP for three months. An endometrial biopsy and transvaginal ultrasound were conducted before and after treatment. Endometrial proliferation was evaluated as the primary endpoint. Histology and a panel of surrogate endpoint biomarkers were evaluated for each endometrial biopsy as secondary endpoints. A total of 51 women were enrolled, and 46 completed treatment. Two of the 51 women had complex hyperplasia with atypia at the baseline endometrial biopsy and were excluded from the study. Overall, both depo-MPA and OCP induced a dramatic decrease in endometrial epithelial proliferation and microscopic changes in the endometrium characteristic of progestin action. Transvaginal ultrasound measurement of endometrial stripe was not a useful measure of endometrial response or baseline hyperplasia. These results show that women with Lynch syndrome do show an endometrial response to short-term exogenous progestins, suggesting that OCP and depo-MPA may be reasonable chemopreventive agents in this high-risk patient population. Cancer Prev Res; 6(8); 774–81. ©2013 AACR.

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PDF file - 9245K, Supplementary Figure S1. Baseline endometrial abnormalities detected in the pre-treatment biopsies from these high-risk women.

Commentary on This Article from Prospective Multicenter Randomized Intermediate Biomarker Study of Oral Contraceptive versus Depo-Provera for Prevention of Endometrial Cancer in Women with Lynch Syndrome

XLSX file - 34K, Supplementary Table S1. Probes and primers for real-time PCR biomarker assays.

Purpose: The involvement of phosphatase and tensin homologue deleted on chromosome ten (PTEN) in endometrial carcinoma has implicated phosphatidylinositol 3-kinase signaling and mammalian target of rapamycin (mTOR) activation in this disease. Understanding the extent of mTOR involvement and the mechanism responsible for activation is important, as mTOR inhibitors are currently being evaluated in clinical trials for endometrial carcinoma. Although tuberous sclerosis complex 2 (TSC2) is the “gatekeeper” for mTOR activation, little is known about defects in the TSC2 tumor suppressor or signaling pathways that regulate TSC2, such as LKB1/AMP-activated protein kinase, in the development of endometrial carcinoma.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.

Supplementary Data from Loss of Tuberous Sclerosis Complex-2 Function and Activation of Mammalian Target of Rapamycin Signaling in Endometrial Carcinoma

Background: Capecitabine is an established radiosensitizer in rectal and other cancers. We conducted a prospective single arm phase II study to examine the response rate of gross chemo-refractory breast cancer treated with concurrent capecitabine and radiotherapy. Methods: Patients who had inoperable or marginally operable gross disease in the breast and/or lymph node(s) after chemotherapy or gross disease on the chest wall or in the regional lymphatics after mastectomy were eligible. Patients 1-9 received capecitabine 825 mg/m2 BID daily beginning on the first day of radiotherapy. Excess grade 3 toxicity (%) was observed; the protocol was amended and subsequent patients received drug only on radiation treatment days. Radiation dose was at the discretion of the treating physician (50Gy-72 Gy, with no more than 2.5 Gy/fraction). Response was assessed by a single physician using paired radiation planning CTs (pretreatment and on-treatment after 45 Gy). Clinical correlation to all other available imaging was also made. Kaplan-Meier curves were used to estimate overall survival (OS) and local recurrence-free survival (LRFS). Circulating tumor cells (CTCs) in blood were examined in consenting patients. Results: The trial was stopped early after an unplanned interim analysis prompted by slow accrual suggested futility independent of response. From 2009-2012, 32 patients were accrued; 26 completed protocol specific treatment (17 post-mastectomy radiation with gross nodes, 4 pre-op, 5 aggressive palliation) and are included in this analysis. Median follow up was 7.3 months (interquartile range 6.7 – 17.4). Nineteen patients (73%) had a partial or complete response. Fourteen patients (53.9%) experienced at least one grade 3 non-dermatitis toxicity including 7/9 treated with continuous dosing. Four inoperable patients were treated with pre-op radiation therapy and 3 converted to operable. None achieved a pCR or near pCR. One-year actuarial OS was 52%. There was no difference in OS comparing among PMRT vs. preoperative or palliative RT (P = 0.90). One-year actuarial local recurrence free survival among PMRT patients was 38%. Ten patients had triple negative (TN) receptor status. There was no difference in radiation response by receptor status (P = 0.56); however, treatment was deemed subjectively futile (i.e., converted to operable but death secondary to new widespread M1 disease immediately post-op) in 9 of the 10 patients with TN disease versus 6 of the 16 patients with non-TN disease (P = 0.014). Median OS and 1-yr actuarial OS, among non-TN vs. TN patients were not reached vs. 6.1 months and 77% vs. 10% (P < 0.001), respectively. Eight/fifteen patients tested were positive for CTCs. CTCs did not correlate to receptor status, futility of RT or OS. Conclusions: Capecitabine can be safely administered as a daily concurrent chemoradiation regimen with weekend holidays. However, in this small, prospective and selected cohort, concurrent chemoradiation with capecitabine was futile among patients with TN breast cancer. Alternative strategies are urgently needed in TN patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-14-08.

Women with Lynch syndrome have a 40% to 60% lifetime risk for developing endometrial cancer, a cancer associated with estrogen imbalance. The molecular basis for endometrial-specific tumorigenesis is unclear. Progestins inhibit estrogen-driven proliferation, and epidemiologic studies have shown that progestin-containing oral contraceptives (OCP) reduce the risk of endometrial cancer by 50% in women at general population risk. It is unknown whether they are effective in women with Lynch syndrome. Asymptomatic women ages 25 to 50 with Lynch syndrome were randomized to receive the progestin compounds Depo-Provera (depo-MPA) or OCP for three months. An endometrial biopsy and transvaginal ultrasound were conducted before and after treatment. Endometrial proliferation was evaluated as the primary endpoint. Histology and a panel of surrogate endpoint biomarkers were evaluated for each endometrial biopsy as secondary endpoints. A total of 51 women were enrolled, and 46 completed treatment. Two of the 51 women had complex hyperplasia with atypia at the baseline endometrial biopsy and were excluded from the study. Overall, both depo-MPA and OCP induced a dramatic decrease in endometrial epithelial proliferation and microscopic changes in the endometrium characteristic of progestin action. Transvaginal ultrasound measurement of endometrial stripe was not a useful measure of endometrial response or baseline hyperplasia. These results show that women with Lynch syndrome do show an endometrial response to short-term exogenous progestins, suggesting that OCP and depo-MPA may be reasonable chemopreventive agents in this high-risk patient population. Cancer Prev Res; 6(8); 774–81. ©2013 AACR.

Background: Male breast cancer (MBC) is an uncommon malignancy with approximately 2000 American men diagnosed per year. It has been observed that the side effect profiles and tolerability with anti-hormonal treatments such as tamoxifen differ between women and men. There is a paucity of data specifically evaluating the side effect profiles of antihormonal treatments in MBC patients (pts). Objective: To specifically study the side effects of tamoxifen in a cohort of MBC pts and to determine toxicities in this population. Methods: We retrospectively reviewed all MBC pts evaluated at our institution from 1999-2009. Out of a total of 126 MBC pts, 64 met the following inclusion criteria: stage I-III, treated with tamoxifen, and at least one follow-up visit after starting tamoxifen. A descriptive analysis of side effects was performed on these 64 pts. Patient Characteristics and Results: Median follow-up time at our institution was 4.2 years (range 0.3-10.9). Median follow-up from the start of tamoxifen therapy was 4.0 years (range 0.3-19.4). The median age at diagnosis was 61 years (range 30-79). Breakdown by stage included: 29.7% Stage I, 54.7% stage II, and 15.6% stage III. Median initial tumor size was 2.0 cm (range 0.5-6.5). 100% were hormone-receptor positive and 7 tumors (11%) were HER2/neu-positive. Two pts had history of prior therapeutic radiation, and 11 pts (17%) had a prior or concomitant cancer diagnosis, including 6 pts with history of prostate cancer and 2 pts with history of renal cell carcinoma. Sixteen (25%) developed metastatic disease or recurrence. Thirty (47%) pts experienced at least one or more side effect while taking tamoxifen. There were a total of 101 documented side effects (more than one side effect in some pts). The most common toxicity was weight gain (14 out of 64 pts, 22%) and sexual dysfunction/loss of libido (14pts, 22%), followed by hot flashes (8pts, 13%), neurocognitive deficits (6 pts, 1%), thromboembolic events (4 pts, 1%) ocular events (3pts), mood alterations (2pts), depression (2 pts), GI disturbance(2 pts), insomnia (1) and leg cramps (1). Importantly, 13 out of the 64 (20.3%) pts discontinued tamoxifen due to toxicity: 1 for ocular, 2 for neurocognitive deficits, 4 for thromboembolic events, 2 for bone pain, 3 for loss of libido, and 1 for leg cramps. Conclusions: To our knowledge, this is the largest retrospective study specifically examining tamoxifen-related side effects among MBC pts. Among MBC pts, there is a high rate of discontinuation of tamoxifen. Further prospective work regarding better understanding of male hormonal biology as well as novel anti-hormonal drug development may be warranted in this unique population. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-16-02.

Purpose: The involvement of phosphatase and tensin homologue deleted on chromosome ten (PTEN) in endometrial carcinoma has implicated phosphatidylinositol 3-kinase signaling and mammalian target of rapamycin (mTOR) activation in this disease. Understanding the extent of mTOR involvement and the mechanism responsible for activation is important, as mTOR inhibitors are currently being evaluated in clinical trials for endometrial carcinoma. Although tuberous sclerosis complex 2 (TSC2) is the “gatekeeper” for mTOR activation, little is known about defects in the TSC2 tumor suppressor or signaling pathways that regulate TSC2, such as LKB1/AMP-activated protein kinase, in the development of endometrial carcinoma. 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.

Purpose: Epidermal growth factor receptor (EGFR) is an attractive target for the treatment of urothelial carcinoma, but a clinical response can be expected in only a small proportion of patients. The aim of this study was to define molecular markers of response to cetuximab therapy in a panel of urothelial carcinoma cell lines.Experimental Design: Eleven cell lines were investigated for antiproliferative response to cetuximab based on [3H]thymidine incorporation. A variety of markers, including EGFR expression, phosphorylation, and gene amplification, as well as the expression of other growth factor receptors, their ligands, and markers of epithelial-to-mesenchymal transition were investigated. Cohen's κ statistic was used to estimate the agreement between response and expression of these markers. E-cadherin was silenced by small interfering RNA in two sensitive cell lines, and the effect on the response to cetuximab was measured.Results: We were able to identify a panel of relevant markers pertaining especially to alternate growth factor receptor expression and epithelial-to-mesenchymal transition that predicted response to cetuximab. The data suggested that expression of intact HER-4 (κ, 1.00; P = 0.008), E-cadherin (κ, 0.81; P = 0.015), and β-catenin (κ, 0.81; P = 0.015) and loss of expression of platelet-derived growth factor receptor β (κ, 0.57; P = 0.167) were associated with response to cetuximab therapy. Silencing E-cadherin in two sensitive cell lines reduced responsiveness to cetuximab in both (P < 0.001).Conclusions: A panel of predictive markers for cetuximab response has been established in vitro and is currently being evaluated in a prospective clinical trial of neoadjuvant EGFR-targeted therapy. Most importantly, E-cadherin seems to play a central role in modulation of EGFR response in urothelial carcinoma.

The chemokine receptor CXCR4 mediates the migration of hematopoietic cells to the stroma-derived factor 1α (SDF-1α)–producing bone marrow microenvironment. Using peptide-based CXCR4 inhibitors derived from the chemokine viral macrophage inflammatory protein II, we tested the hypothesis that the inhibition of CXCR4 increases sensitivity to chemotherapy by interfering with stromal/leukemia cell interactions. First, leukemic cells expressing varying amounts of surface CXCR4 were examined for their chemotactic response to SDF-1α or stromal cells, alone or in the presence of different CXCR4 inhibitors. Results showed that the polypeptide RCP168 had the strongest antagonistic effect on the SDF-1α– or stromal cell–induced chemotaxis of leukemic cells. Furthermore, RCP168 blocked the binding of anti-CXCR4 monoclonal antibody 12G5 to surface CXCR4 in a concentration-dependent manner and inhibited SDF-1α–induced AKT and extracellular signal-regulated kinase phosphorylation. Finally, RCP168 significantly enhanced chemotherapy-induced apoptosis in stroma-cocultured Jurkat, primary chronic lymphocytic leukemia, and in a subset of acute myelogenous leukemia cells harboring Flt3 mutation. Equivalent results were obtained with the small-molecule CXCR4 inhibitor AMD3465. Our data therefore suggest that the SDF-1α/CXCR4 interaction contributes to the resistance of leukemia cells to chemotherapy-induced apoptosis. Disruption of these interactions by the peptide CXCR4 inhibitor RCP168 represents a novel strategy for targeting leukemic cells within the bone marrow microenvironment. [Mol Cancer Ther 2006;5(12):3113–21]

HER2 overexpression is one of the most recognizable molecular alterations in breast tumors known to be associated with a poor prognosis. In the study described here, we explored the effect of HER2 overexpression on the sensitivity of breast cancer cells to the growth-inhibitory effects of 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), a synthetic triterpenoid, both in vitro and in vivo in a xenograft model of breast cancer. Both cell growth and colony formation in the soft agar assay, a hallmark of the transformation phenotype, were preferentially suppressed in HER2-overexpressing cell lines at low concentrations of CDDO, whereas growth-inhibitory effects at high concentrations did not correlate with the expression level of HER2. CDDO dose-dependently inhibited phosphorylation of HER2 in HER2-overexpressing cells and diminished HER2 kinase activity in vitro. CDDO induced the transactivation of the nuclear receptor peroxisome proliferator-activated receptor-γ in both vector control and HER2-transfected MCF7 cells. Dose-response studies showed that the growth inhibition seen at lower concentrations of CDDO correlated with induction of the tumor suppressor gene caveolin-1, which is known to inhibit breast cancer cell growth. CDDO also reduced cyclin D1 mRNA and protein expression. In vivo studies with liposomally encapsulated CDDO showed complete abrogation of the growth of the highly tumorigenic MCF7/HER2 cells in a xenograft model of breast cancer. These findings provide the first in vitro and in vivo evidence that CDDO effectively inhibits HER2 tyrosine kinase activity and potently suppresses the growth of HER2-overexpressing breast cancer cells and suggest that CDDO has a therapeutic potential in advanced breast cancer. [Mol Cancer Ther 2006;5(2):317–28]

In acute myeloid leukemia (AML), resistance to chemotherapy is associated with defects in both the extrinsic and intrinsic pathways of apoptosis. Novel agents that activate endogenous apoptosis-inducing mechanisms directly may be potentially useful to overcome chemoresistance in AML. We examined the mechanisms of apoptosis induction by the novel synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) in AML cells. CDDO-induced apoptosis was associated with the loss of mitochondrial inner transmembrane potential, caspases activation, the translocation of apoptosis-inducing factor to the nucleus, and DNA fragmentation in AML cells. Apoptosis was equally evident in cells deficient in caspase-9 or caspase-8 after exposure to CDDO, suggesting caspase-independent cell death. The use of small interfering RNA to reduce the expression of apoptosis-inducing factor partially inhibited CDDO-induced apoptosis in AML cells. Cells overexpressing Bcl-2 were markedly resistant to CDDO-induced apoptosis. Moreover, CDDO promoted the release of cytochrome c from isolated mitochondria, suggesting that CDDO targets the mitochondria directly to trigger the intrinsic pathway of cell death in intact cells. Together, these results suggest that CDDO functions by activating the intrinsic pathway of apoptosis and initiates caspase-dependent and independent cell death. The direct modulation of mitochondrial-mediated, caspase-independent apoptosis by CDDO may be advantageous for overcoming chemoresistance in AML.