Preexisting anticoagulation is an independent predictor of mortality in geriatric trauma patients - a retrospective cohort study.

Introduction: Preexisting anticoagulation is common among geriatric trauma patients. Geriatric trauma patients have a higher risk of mortality compared to younger patients. We sought to evaluate the association of preexisting anticoagulation with mortality in a group of geriatric trauma patients. Methods: A retrospective review of geriatric trauma patients was conducted for those admitted to a Level 1 trauma center from January 2018 to December 2020. Vital signs, demographics, injury characteristics, laboratory data, and mortality were all collected. Multivariable logistic regression analysis was performed for the association of preexisting anticoagulation and a primary endpoint of all-cause mortality. These groups were controlled for preexisting comorbidities, injury severity scores, and systolic blood pressure in the emergency department. Results: Four thousand four hundred thirty-two geriatric patients were admitted during the study period. This cohort was made up of 36.9% men and 63.1% women. Three thousand eight hundred fifty-nine (87.2%) were white; the average age was 81±8.5 years, and the median injury severity score (ISS) was 5. The mean systolic blood pressure was 150±32 mmHg, mean heart rate was 81±16 bpm, mean lactate was 2.3±1.3, mean hematocrit was 37.3±8.8, and mean international normalized ratio (INR) was 1.7±10.3. One thousand five hundred ninety-two (35.9%) patients were on anticoagulation (AC) upon presentation. One hundred and sixty-five (3.7%) mortalities were recorded. Multivariable logistic regression analysis results show that preexisting anticoagulation [ odds ratio (OR) 1.92, 95% CI 1.36-2.72] was independently predictive of death. The analysis was adjusted for systolic BP in the emergency department less than90 mmHg (OR 5.55, 95% CI 2.83-10.9), having more than 1 comorbidity (OR 2.30, 95% CI 1.57-3.38) and ISS (OR 1.13, 95% CI 1.10-1.15). Conclusion: Our study indicates that preexisting anticoagulation is associated with mortality among geriatric trauma patients.

Bioequivalence study of 20-mg and 100-mg temozolomide capsules (TOZ309 and Temodal®) in glioma patients in China.

BACKGROUND: Temozolomide is an alkylating agent approved by the U.S. Food and Drug Administration in 1999 for the treatment of patients with primary brain tumors. The aim of this study was to confirm the bioequivalence and safety of two strengths (20-100 mg) of generic temozolomide in the form of TOZ039 and Temodal® capsules administered to brain tumor patients. STUDY DESIGN: An open-label, randomized, two-phase, two-period, crossover pharmacokinetic study was performed in a single institution. The reference and test drugs were prescribed at a dose of 150 mg/m2 daily from days 1 to 5 of a 28-day cycle in the first phase; in the second phase, either a 150- or 200-mg/m2 dose was prescribed, depending on patient tolerance. On days 1 and 2 of each phase, a fixed 200-mg dose was administered either as ten 20-mg capsules in the first cycle or two 100-mg capsules in the second cycle. Drug administration in the first two days was randomized, i.e., if TOZ309 was administered on day 1, Temodal® was administered on day 2, and vice versa. The rest of the prescribed dose was administered in the form of Temodal® and spread equally over days 3-5. Blood samples were obtained for pharmacokinetic evaluation on days 1 and 2. Bioequivalence was demonstrated if the geometric means ratio of the three main pharmacokinetic parameters (mean maximum plasma concentration (Cmax), area under the concentration-time curve (AUC) 0-t, AUC 0-∞) fell within the equivalence boundary of 80-125%. RESULTS: Twenty-nine glioblastoma multiforme or anaplastic astrocytoma patients were enrolled and dosed with the test and reference formulations under fasting conditions. The 90% confidence interval of the geometric means ratio for Cmax (91.08%, 106.18%), AUC0-t (98.62%,102.18%), and AUC0-∞ (98.65%, 102.21%) was well within the 80%-125% range for the 20-mg capsule, as was the Cmax (90.49%, 113.32%), AUC0-t (99.89%, 104.63%) and AUC0-∞ (99.99%, 104.67%) for the 100-mg capsule drug product. Additionally, all the secondary pharmacokinetic parameters were not significantly different. After two cycles of treatment, there was no mortality among the 29 patients, treatment-related severe adverse events, or events that would require study discontinuation; however, one significant adverse effect (life-threatening seizures) occurred and was related to disease progression. Adverse events were reported in 82.8% (24/29) patients, and treatment emergent adverse events were reported in 72.4% (21/29) patients. CONCLUSION: It can be concluded that 20-mg and 100-mg capsules of TOZ309 are bioequivalent to Temodal® capsules of the same strength under fasting conditions. TRIAL REGISTRATION: https://www.chinadrugtrials.org.cn/index.html , CTR2017 0122.

Partially exhausted tumor-infiltrating lymphocytes predict response to combination immunotherapy.

BACKGROUND: Programmed death 1 (PD-1) inhibition activates partially exhausted cytotoxic T lymphocytes (peCTLs) and induces tumor regression. We previously showed that the peCTL fraction predicts response to anti-PD-1 monotherapy. Here, we sought to correlate peCTL and regulatory T lymphocyte (Treg) levels with response to combination immunotherapy, and with demographic/disease characteristics, in metastatic melanoma patients. METHODS: Pretreatment melanoma samples underwent multiparameter flow cytometric analysis. Patients were treated with anti-PD-1 monotherapy or combination therapy, and responses determined by Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST v1.1) criteria. peCTL and Treg levels across demographic/disease variables were compared. Low versus high peCTL (≤20% vs. >20%) were defined from a previous study. RESULTS: One hundred and two melanoma patients were identified. The peCTL fraction was higher in responders than nonresponders. Low peCTL correlated with female sex and liver metastasis, but not with lactate dehydrogenase (LDH), tumor stage, or age. While overall response rates (ORRs) to anti-PD-1 monotherapy and combination therapy were similar in high-peCTL patients, low-peCTL patients given combination therapy demonstrated higher ORRs than those who received monotherapy. Treg levels were not associated with these factors nor with response. CONCLUSION: In melanoma, pretreatment peCTL fraction is reduced in women and in patients with liver metastasis. In low-peCTL patients, anti-PD-1 combination therapy is associated with significantly higher ORR than anti-PD-1 monotherapy. Fewer tumor-infiltrating peCTLs may be required to achieve response to combination immunotherapy. TRIAL REGISTRATION: UCSF IRB Protocol 138510FUNDING. NIH DP2-AR068130, K08-AR062064, AR066821, and Burroughs Wellcome CAMS-1010934 (M.D.R.). Amoroso and Cook Fund, and the Parker Institute for Cancer Immunotherapy (A.I.D.).

Prognostic role of excision repair cross complementing-1 and topoisomerase-1 expression in epithelial ovarian cancer.

OBJECTIVE: Epithelial ovarian cancer is the most lethal gynecologic cancer worldwide and chemoresistance is one of the major causes of treatment failure. We investigated whether ERCC1, TAU, TOPO2A, TOPO1, P53, and C-MYC expression could be used as predictors for treatment outcomes. MATERIALS AND METHODS: Immunohistochemical staining was used to examine the expression of these biomarkers in resected tumor specimens from 38 patients treated in our institute. Clinicopathological data including demographics, staging, histological type, treatment response, expression of the biomarkers, and patient outcomes were analyzed. RESULTS: The median follow-up period was 47.5 months (range, 10-135 months) and the median overall survival was 56.0 months. Patients who did not have expression of ERCC1, and those who had expression of TOPO1 had significantly better overall survival. Cox regression analysis also confirmed that these two biomarkers were significant independent factors predicting survival (ERCC1, hazard ratio 5.51, 95% confidence interval: 2.02-14.00, p = 0.001; TOPO1, hazard ratio 0.22, 95% confidence interval: 0.06-0.77, p = 0.017). CONCLUSION: We concluded that poor overall survival was significantly associated with positive ERCC1 and negative TOPO1 expression. The results might be the consequence of chemoresistance to platinum and camptothecins, both of which are commonly used regimens in the treatment of epithelial ovarian cancer.

Clinical significance of ESR1 gene copy number changes in breast cancer as measured by fluorescence in situ hybridisation.

AIMS: The ESR1 gene encodes for oestrogen receptor (ER) α, which plays a crucial role in mammary carcinogenesis and clinical outcome in patients with breast cancer. However, the clinical significance of the ESR1 gene copy number change for breast cancer has not been clarified. METHODS: ESR1 gene copy number was determined by fluorescence in situ hybridisation (FISH) on tissue sections. A minimum of 20 tumour cells were counted per section, and a FISH ratio of ESR1 gene to CEP6 ≥ 2.0 was considered ESR1 amplification. A ratio >1.2 but <2.0 was considered ESR1 gain. The ESR1 copy number was further measured by quantitative real-time PCR (Q-PCR) with ASXL2 as a reference. RESULTS: FISH revealed ESR1 amplification in six cases (4.0%) and ESR1 gain in 13 cases (8.7%) from a total of 150 cases. ESR1 gain and amplification were more common in older patients (p<0.001), and correlated well with ER protein expression (p=0.03) measured by immunohistochemistry, and ESR1 copy number (p<0.001) measured by Q-PCR. Furthermore, the multivariate analysis revealed that ESR1 amplification was associated with a shorter disease-free survival (HR=5.56, p=0.03) and a shorter overall survival (HR=5.11, p=0.04). CONCLUSIONS: In general, the frequency of ESR1 amplification in breast cancer is low when measured by FISH in large sections. ESR1 gain and amplification in breast cancer may be associated with older age and poorer outcomes.

Anti-alpha-enolase autoantibodies are down-regulated in advanced cancer patients.

OBJECTIVE: Elevation of serum autoantibodies to alpha-enolase (ENO1) is often seen in inflammation diseases. However, it is unclear whether the levels of serum ENO1 autoantibodies could be affected during tumor progression. Hence, we attempted to determine the relative serum ENO1 autoantibody levels in healthy individuals and various stages of patients with lung and breast cancers. METHODS: Sera were obtained from 99 normal individuals, 21 patients with non-cancer-associated diseases and 178 cancer patients, including Stage I, II and IV non-small cell lung cancer, small cell lung cancer and breast cancer. The ENO1 autoantibody levels were determined by enzyme-linked immunosorbent assay. RESULTS: Compared with the healthy individuals, the levels of ENO1 autoantibodies were significantly decreased in Stage IV non-small cell lung cancer, small cell lung cancer and breast cancer patients. Consistently, this phenomenon was also observed in tumor-grafted mice. Using logistic regression analyses, data show that the titer status of ENO1 autoantibody level is highly associated with the late stage of lung and breast cancers when compared with those of healthy controls. In contrast, there were no statistic differences between healthy controls and early stages of non-small cell lung cancer patients, and total amounts of serum immunoglobulin A, immunoglobulin G and immunoglobulin M levels in Stage IV non-small cell lung cancer patients were not significantly distinct from those of the healthy controls. Thus, the decreased ENO1 autoantibody event in malignant stage of cancer patients is not contributed by reduction in total immunoglobulin. CONCLUSIONS: Marked decrease in the basal level of serum ENO1 autoantibodies is a common malignant event of lung and breast cancers, suggesting that ENO1 autoantibody may serve as a prognostic marker to monitor the disease progression of these cancer patients.

Molecular subtypes of breast cancer emerging in young women in Taiwan: evidence for more than just westernization as a reason for the disease in Asia.

BACKGROUND: In the past two decades, the incidence of breast cancer in young Taiwanese females has been rapidly increasing, approaching the risk level of western countries. As a first step to investigate the possible etiology, we examined the molecular subtypes of female breast cancer in Taiwan. METHODS: This study included 1,028 consecutive patients with breast cancer diagnosed in National Taiwan University Hospital between 2004 and 2006. Estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor-2, cytokeratin 5/6, and epidermal growth factor receptor expression and/or gene amplification were analyzed. RESULTS: Younger (50 years) patients. The higher prevalence of luminal A subtype was mainly attributed to a higher ER (75% versus 63%; P < 0.001) and PR (47% versus 33%; P < 0.001) expression rate in younger patients than older patients. Tumors with histologic grade 3 were less prevalent in younger patients than in older patients (23% versus 30%; P = 0.01). For very young (<35 years) patients, the molecular subtype distribution, ER and/or PR expression rate, and histologic grade were not significantly different from those of less young (35-50 years) patients. CONCLUSIONS: Young breast cancer patients in Taiwan are characterized by a high prevalence of luminal A subtype and low prevalence of histologic grade 3 tumor and/or basal-like subtype. These features are distinct from young breast cancer patients in western countries.

Prognostic significance of beta-catenin and topoisomerase IIalpha in de novo acute myeloid leukemia.

The Wnt/beta-catenin signaling is important for controlling self-renewal of hematopoietic stem cells and its constitutive activation has recently been documented in a significant proportion of acute myeloid leukemia (AML) cases. Topoisomerase IIalpha (Topo IIalpha) is a marker of cell proliferation and a crucial target for anthracycline cytotoxicity, the mainstay of management employed in AML. We retrospectively investigated the prognostic roles of beta-catenin and topo IIalpha in a cohort of 59 patients with newly diagnosed AML by immunohistochemistry. Aberrant beta-catenin expression was demonstrated in 13 patients (22%), and it was more likely to occur in those with unfavorable karyotypes. Advanced age and poor performance status adversely influenced the achievement of complete remission, while neither aberrant beta-catenin expression nor enhanced topo IIalpha activity did. On multivariate survival analysis, four factors independently predicted a shortened overall survival: aberrant beta-catenin expression, high topo IIalpha activity, poor-risk cytogenetics, and presence of at least one comorbidity factor. Our results suggest that both beta-catenin and topo IIalpha independently predicted an adverse prognosis and might serve as new markers for risk stratification in AML patients.

Phase II randomized study of daily gefitinib treatment alone or with vinorelbine every 2 weeks in patients with adenocarcinoma of the lung who failed at least 2 regimens of chemotherapy.

BACKGROUND: The objective of this study was to assess the efficacy of adding chronic, intermittent, low-dose vinorelbine to gefitinib treatment for patients who had adenocarcinoma of the lung who failed>or=2 regimens of chemotherapy. METHODS: Patients were randomized into 2 arms: Oral gefitinib 250 mg daily (the G arm) or vinorelbine 15 mg/m2 as an intravenous infusion on Day 1 and oral gefitinib 250 mg daily on Days 2 through 14 every 2 weeks (the GV arm). From August 2004 to October 2005, 48 patients were enrolled. Epidermal growth factor receptor (EGFR) exon 18 through 21 nucleotide sequence analysis and fluorescence in situ hybridization were performed in patients who had tumor tissue specimens available for analysis. RESULTS: After randomization, each arm had 24 patients. However, 3 patients refused vinorelbine treatment and were given gefitinib treatment only. Thus, 27 patients received G treatment, and 21 patients received GV treatment. Objective response rates were 55.6% in the G arm and 52.4% in the GV arm. All toxicities in both arms were mild. The 1-year progression-free survival rate was 57.1% in the GV arm and 21.2% in the G arm (P=.008). The median survival was 13.3 months in the G arm and 23.4 months in the GV arm (P=.1231). Three of 6 patients (50%) had an exon 19 in-frame deletion, and 2 of 10 patients had EGFR gene high polysomy or amplification (20%). CONCLUSIONS: Gefitinib was highly effective in ethnic Chinese patients with adenocarcinoma of the lung who failed previous platinum and taxane treatment. The addition of low-dose vinorelbine every 2 weeks produced a significantly better 1-year progression-free survival rate.

Effect of age on pulmonary metastases and immunotherapy in young and middle-aged mice.

BACKGROUND: We used B16-F10 (B16) melanoma tumor cells and syngeneic C57BL/6 (B6) mice as a study model for pulmonary metastases, to better understand whether or not there exist differences in tumorigenicity and in the effectiveness of immunotherapy as a function of host age (1-, 3-, 12- and 24-month-old). METHODS: Intravenous injection of B16 melanoma cells were administered to B6 mice of different ages with/without interleukin (IL)-2 and IL-12 daily treatment. Tumor growth, splenocyte function, serum cytokines (IL-10, interferon-gamma, vascular endothelial growth factor) and survival were compared. RESULTS: The study showed that, without IL-2 and IL-12 treatment, middle-aged mice suffering from pulmonary metastases had fewer pulmonary metastases and better survival than younger mice suffering from pulmonary metastases. Three days' IL-2 plus IL-12 treatment could not prolong mice survival, but prolonged treatment significantly improved the survival of both the younger and older tumor-bearing mice, especially the older mice, despite the fact that the younger mice had a better serum cytokine and splenocyte cellular immune response to cytokine treatment. CONCLUSION: The young B6 mice that suffered from B16 pulmonary metastases had a poorer prognosis than the middle-aged mice. Short-term IL-2 plus IL-12 treatment is ineffective in prolonging survival, and a longer duration of treatment is needed. This kind of immunotherapy was effective in both the young and middle-aged mice, but it was more effective in the middle-aged mice.

Effects of paclitaxel and doxorubicin in histocultures of hepatocelular carcinomas.

Cancer has been the leading cause of death in Taiwan over the past two decades and liver cancer is the leading cause of all cancer deaths in Taiwan with a trend of increase in incidence. Therapeutic options and efficacy for liver cancer have been limited and the 5-year survival rate is less than 7% in the Unite States. The study was conducted to establish a histoculture system of human hepatocellular carcinomas (HCC) for biological and pharmacological studies and to determine the efficacy of anticancer drugs with the established HCC histocultures. Patient HCC tissues freshly obtained after surgeries were prepared and histocultured. The histocultured HCC were treated with doxorubicin and paclitaxel of various concentrations for 96-h. Upon drug treatments, the activity of tumor cell proliferation and extent of cell death induction were measured and changes of the alpha-fetoprotein levels in the culture medium were determined. We demonstrated that human HCC can be successfully cultured in a 3-dimensional histoculture system and used for pharmacological studies. Doxorubicin and paclitaxel showed concentration-dependent activities in anti-proliferation and cell death induction against the human HCC. Inhibitory effects of both drugs on alpha-fetoprotein production of the cultured HCC were in agreement with their anti-proliferative effects. Exposure time-dependent antitumoral effects of paclitaxel treatments at 3-, 24-, and 96-h against the histocultured HCC PLC/PRF/5 xenograft tumors were also observed. In conclusion, we have demonstrated a histoculture system for patient HCC and it can be utilized in selection of active drugs prior to treatments in patients and in evaluation of new agents against HCC, for which therapeutic agents are in desperate needs worldwide.

The sensitivity of human mesenchymal stem cells to ionizing radiation.

PURPOSE: Recent studies have shown that mesenchymal stem cells (MSCs) obtained from bone marrow transplantation patients originate from the host. This clinical observation suggests that MSCs in their niches could be resistant to irradiation. However, the biologic responses of bone marrow MSCs to irradiation have rarely been described in the literature. METHODS AND MATERIALS: In this study, human bone marrow-derived, clonally expanded MSCs were used to investigate their sensitivity to irradiation in vitro, and the cellular mechanisms that may facilitate resistance to irradiation. The human lung cancer cell line A549 and the breast cancer cell line HCC1937 were used as controls for radiosensitivity; the former line has been shown to be radioresistant and the latter radiosensitive. We then examined their in vitro biologic changes and sensitivities to radiation therapy. RESULTS: Our results suggest that MSCs are characterized as resistant to irradiation. Several cellular mechanisms were demonstrated that may facilitate resistance to irradiation: ATM protein phosphorylation, activation of cell-cycle checkpoints, double-strand break repair by homologous recombination and nonhomologous end joining (NHEJ), and the antioxidant capacity for scavenging reactive oxygen species. CONCLUSIONS: As demonstrated, MSCs possess a better antioxidant reactive oxygen species-scavenging capacity and active double-strand break repair to facilitate their radioresistance. These findings provide a better understanding of radiation-induced biologic responses in MSCs and may lead to the development of better strategies for stem cell treatment and cancer therapy.

Comprehensive breast cancer adjuvant digital summary.

BACKGROUND: Patients with breast cancer often fail to recall the details of their original diagnosis and adjuvant therapy with the passage of time. Subsequent follow-up and treatment at a later time and a different institution wastes valuable time and effort to retrieve the original data. PATIENTS AND METHODS: Twenty-five consecutive patients with breast cancer of all stages admitted for adjuvant/neoadjuvant treatment and surgical excision were entered on study. An individualized comprehensive visual evaluation summary sheet (VESS) was created that detailed initial diagnosis, preceding relevant investigations, drug scheduling, and dosages of adjuvant therapy. Completion of a VESS required a computer, a digital camera with connection to a microscope, and radiology images over the PACS system. The completed one-page summary can be printed or stored. RESULTS: A VESS takes up an average of 4.4 MB (1.24-8 MB), each containing 11.5 images (range, 4-23 images), spanning a time period of around 216 days (range, 125-558 days). CONCLUSIONS: Patients received a complete summary of pertinent information concerning their diagnosis and adjuvant therapy.

Tumor beta-1,4-galactosyltransferase IV overexpression is closely associated with colorectal cancer metastasis and poor prognosis.

PURPOSE: To elucidate the significance of beta-1,4-galactosyltransferase IV (beta-1,4-GT-IV) in the clinical presentation and prognostication of colorectal cancer. EXPERIMENTAL DESIGN: Tissue lysates from paired tumor and nontumor tissues of a colon cancer patient were labeled separately with fluorescent dyes Cy5 and Cy3 for two-dimensional difference in-gel electrophoresis. Subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and immunoblot analyses identified a down-regulated level of beta-1,4-GT-IV in the tumor tissue. In the follow-up study, paired tissue lysates were obtained from 100 colorectal cancer patients with immunoblot analyses done to compare the levels of beta-1,4-GT-IV expression in these patients. RESULTS: Of 100 colorectal patients studied, 48% had down-regulated expression of beta-1,4-GT-IV in the tumor tissue but 28% of patients exhibited elevated beta-1,4-GT-IV levels. Increased beta-1,4-GT-IV in the tumor tissue was significantly coexistent with raised serum level of CA-199 and the presence of tumor metastasis (P=0.006 and P<0.001, respectively) but was independent of age and gender of patient, tumor site, tumor size, serum level of carcinoembryonic antigen, grade of tumor cell differentiation, and depth of tumor invasion. The results of logistic regression analyses suggested that tumor beta-1,4-GT-IV overexpression and tumor invasion, but not other patient variables such as tumor size and serum levels of carcinoembryonic antigen and CA19-9, were significantly correlated with the occurrence of metastases (P<0.05). In a multivariate regression analysis, the patient group with tumor beta-1,4-GT-IV overexpression strongly predicted for tumor metastasis (odds ratio, 10.009; 95% confidence interval, 2.992-33.484; P<0.001). Likewise, tumor beta-1,4-GT-IV overexpression was significantly associated with poor overall survival (P<0.01). By Cox regression analysis, this association remained significant even after adjustment for tumor metastasis (P=0.048). CONCLUSION: Increased beta-1,4-GT-IV expression in tumor tissue was strongly associated with tumor metastases and poor prognosis in colorectal cancer.

Novel rapid tissue lysis method to evaluate cancer proteins: correlation between elevated Bcl-X(L) expression and colorectal cancer cell proliferation.

AIM: We optimized a rapid and efficient tissue lysis method using the MagNA Lyser (Roche, Germany). Using this novel method combined with immunoblot analysis, we investigated the correlation between abnormal Bcl-X(L) expression and clinicopathological characteristics in colorectal cancer. METHODS: Tissue samples from Sprague-Dawley rats were tested to determine optimal lysis conditions for use with MagNA Lyser. We next used the new method to extract tissue proteins from the tumor tissue of a colorectal cancer patient. The availability of extractable tissue proteins for proteomic study was demonstrated by two-dimensional (2D) gel electrophoresis and subsequent matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. In addition, we prepared tissue lysates from paired tumor tissues and adjacent nontumor tissues of 50 colorectal carcinoma patients. Ensuing immunoblot analyses were performed to detect the level of Bcl-X(L) expression. RESULTS: The optimal sample sizes processed were found to be around 200 mg, with oscillation frequency of 6500 r/min for 80 s. Test of the first human tissue lysate confirmed that the MagNA Lyser method was adequate for protein extraction and subsequent identification by current proteomic protocols. The method was also applicable to immunoblot analysis. Thirty of 50 (60%) colorectal patients exhibited higher level of Bcl-X(L) expression in their tumor tissues. Raised level of Bcl-X(L) expression correlated with patients' gender and tumor cell proliferation index (P = 0.037 and P<0.001, respectively), but was independent of clinicopathological characteristics and overall survival. CONCLUSION: We report a novel tissue lysis method applicable to proteomic and immunoblot analyses, which can facilitate the discovery and detection of cancer protein alterations.

erb-b2 amplification by fluorescence in situ hybridization in breast cancer specimens read as 2+ in immunohistochemical analysis.

We conducted this study to ascertain the prevalence of erb-b2 gene amplification in breast cancer specimens read as 2+ in immunohistochemical analysis. Slides from patients with metastatic or recurrent breast cancer were eligible for fluorescent in situ hybridization (FISH) study if they were read as 2+ immunohistochemically for erb-b2 by a certified pathologist. The PathVysion kit (Vysis, Downers Grove, IL) was used for FISH studies. Amplification of the erb-b2 gene was defined as an erb-b2/CEP17 (chromosome 17 centromere) ratio of 2 or more in 30 tumor cells counted. From May 2003 to June 2004, 221 slides were submitted from 24 hospitals around the island. Of 216 successful hybridizations, 96 (44.4%) were determined to be erb-b2 amplified. In addition, the topoisomerase IIa gene was coamplified in 11 (21%) of 53 and deleted in 8 (15%) of 53 erb-b2 amplified cases. The erb-b2 gene amplification rate was very high in cases determined to be 2+ by immunohistochemical analysis; therefore, determination of erb-b2 status by FISH in cases scored 2+ immunohistochemically is strongly recommended.

A simple method for the detection and genotyping of high-risk human papillomavirus using seminested polymerase chain reaction and reverse hybridization.

BACKGROUND: To develop a simple and cost-effective method for the detection and genotyping of high-risk human papillomaviruses (HPV) using seminested polymerase chain reaction (PCR) and reverse hybridization. METHODS: Cervical swabs for HPV testing were collected from 127 women with normal cervical cytology and 57 patients with cervical lesions of various degrees. After DNA isolation, PCR amplification was first carried out using MY11 and MY09/HMB01 primers, then labeled by seminested PCR using the first PCR products and MY11/bioGP6+ primers. One fifth of the second PCR products were resolved by gel electrophoresis. Genotyping for high-risk HPV was done separately, using the remaining products, by a high-risk HPV chip, which contained 13 type-specific oligonucleotides on a nylon membrane. The final result was detected by colorimetric change on the chip under direct visualization. RESULTS: High-risk HPV DNA was detected in 19 (15%) of 127 women with normal cervical smear cytology, in 26 (89.7%) of 29 patients with cervical intraepithelial neoplasia (CIN), and in 27 (96.4%) of 28 patients with invasive cervical carcinoma. Multiple high-risk HPV infections were detected in five cases. HPV type 16 was the most frequent type of infection, comprising 34.5% and 53.6% of the patients with CIN and invasive carcinoma, respectively. The samples without a visible 190-bp band on electrophoresis exclusively showed negative hybridization results. This method could detect one to two copies of the HPV-16 genome derived from one SiHa cell. The overall sensitivity of HPV detection was 25 to 50 copies of HPV genome for each specimen. Thirteen high-risk types and twenty-four different types of HPV DNA showed specific hybridization without any cross-reaction. CONCLUSIONS: Our results demonstrated the feasibility and optimistic prospects for this simple and cheap method of high-risk HPV genotyping. This technology can be easily set up in a routine molecular laboratory and would probably be of great value in cervical cancer prevention programs.

Impact of circulating free tumor cells in the peripheral blood of colorectal cancer patients during laparoscopic surgery.

Despite widespread use of laparoscopic surgery for colorectal operations, its application for curative resection of colorectal cancer is still controversial. One of the major concerns is the impact of the laparoscopic procedure on dissemination of tumor cells. The main purpose of this study was to investigate the impact of laparoscopic surgery on circulating tumor cells in colorectal cancer patients. Quantitation of circulating free tumor cells (FTCs) was performed preoperatively, during the operation, and 14 days later by means of real-time quantitative reverse transcriptasepolymerase chain reaction (RT-PCR) targeting guanylyl cyclase C (GCC) mRNA in 42 colorectal cancer patients undergoing laparoscopic resections. Despite an increasing trend of FTC detection in patients with advancing stage, there is no significant difference in the preoperative FTC level by disease stage. No elevation in FTC level was found during the laparoscopic procedure in most patients compared with their preoperative FTC value. Patients with a persistently high FTC load [per nucleated blood cells (NBCs)] (> 10(2) FTCs/10(6) NBCs) 2 weeks postoperatively portends a poor prognosis regarding disease recurrence and tumor-related mortality when compared to those with an undetectable or low FTC load (< or = 10(2) FTCs/10(6) NBCs). We concluded that the laparoscopic procedure itself had no significantly deleterious effect on circulating FTCs and that the detection of FTCs by real-time qRT-PCR might be of clinical importance during the postoperative follow-up for colorectal cancer patients.

Sequence-dependent effect of a cyclooxygenase-2 inhibitor on topoisomerase I inhibitor and 5-fluorouracil-induced cytotoxicity of colon cancer cells.

Selective cyclooxygenase-2 (COX-2) inhibitors have been found to induce anti-proliferative and apoptotic activity in many cancer cells. However, interaction between COX-2 inhibitors and other chemotherapeutic agents remains to be determined. We investigated the interactive effects of a selective COX-2 inhibitor, etodolac, in combination with 5-fluorouracil (5-FU) or SN-38 (active metabolite of irinotecan) on colon cancer cell lines, HT29 and SW620, in simultaneous and sequential administration schedules. Isobologram analysis demonstrated that etodolac in combination with 5-FU or SN-38 according to a simultaneous schedule resulted in only an additive effect; however, synergism was achieved in a sequential schedule. Apoptosis induction in both cell lines was also significantly increased after sequential treatment with etodolac followed by either 5-FU or SN-38 compared to that after simultaneous treatment with etodolac and either 5-FU or SN-38. Our study suggests apoptosis-inducing synergism resulted from administration of etodolac and either 5-FU or SN-38 sequentially, but not simultaneously.