The predictive and prognostic significance of liquid biopsy in advanced epidermal growth factor receptor-mutated non-small cell lung cancer: A prospective study.

OBJECTIVE: To determine the predictive and prognostic roles of three blood-based biomarkers: circulating tumour DNA (ctDNA), circulating tumour cells (CTC) and carcinoembryonic antigen (CEA), in patients with advanced epidermal growth factor receptor-mutated (EGFR+) lung cancer. MATERIALS AND METHODS: We recruited 28 patients with 103 serial blood samples. We performed mutational analyses for EGFR mutations using droplet digital PCR (ddPCR) on ctDNA. We evaluated the accuracy of EGFR mutation detection in ctDNA compared with tissue biopsy. We also quantified CTCs, ctDNA and CEA in serially collected blood samples, and evaluated the baseline and changes in these blood-based biomarkers with clinical outcomes. RESULTS: EGFR mutation detection in plasma was highly concordant as compared with tissue biopsy. Detectable baseline ctDNA was associated with higher disease burden (p < 0.01). Early disappearance of ctDNA at 4 weeks was associated with radiological response at 12 weeks of treatment (p = 0.01) and improved progression free survival (PFS) (HR 5.47, 95%CI 1.32-22.72, p = 0.02) and overall survival (OS) (HR 5.46, 95%CI 1.28-23.22, p = 0.02). A decrease in CTC count at 4 weeks was associated with improved PFS (HR 3.81, 95%CI 1.13-12.79, p = 0.03) but not OS. 85% of patients with radiological progression had a ctDNA rise compared with 22% of patients with stable disease (p=0.01). ctDNA rise was seen on average 170 days prior to radiological progression. There is a significant association between the rise of CEA level with radiological progression (p=0.001). CONCLUSION: Early change in ctDNA, CTC and CEA levels may be long-term predictors of treatment benefit and failure prior to availability of radiological response data.

Circulating tumour cells and the epithelial mesenchymal transition in colorectal cancer.

Circulating tumour cells (CTCs) hold great potential as liquid biopsies to prognosticate disease and guide treatment in colorectal cancer. However, their emerging role in determining the molecular phenotype of tumour metastasis carries even more promising clinical use in the provision of comprehensive biomarker detection for targeted therapies and determination of drug resistance. The isolation of CTCs is technology dependent, and in the case of epithelial cell adhesion molecule-based platforms, the ability to detect cells that have undergone the epithelial to mesenchymal transition (EMT) is ineffective. CTCs displaying a mesenchymal phenotype are believed to have an increased metastatic potential. The rarity of CTCs provides another challenge in the enumeration of these cells. The future will likely involve the analysis of individual CTCs at any stage of the EMT in order to provide real-time phenotypic and molecular snapshots capable of tracking the dynamic evolution of tumour progression over time.

Circulating tumour cells--a bona fide cause of metastatic cancer.

Circulating tumour cells (CTCs) are emerging as important prognostic markers and have potential clinical utility as tumour biomarkers for targeted cancer therapy. Although CTCs were proposed more than 100 years ago as potential precursors that may form metastatic lesions, formal evidence that CTCs are indeed capable of initiating metastases is limited. Moreover, the process of CTCs shedding into the circulation, relocating to distant organ sites and initiating metastatic foci is complex and intrinsically inefficient. To partially explain the metastatic process, the concepts of CTCs as metastatic precursors or pre-metastatic conditioners have been proposed; however, it is questionable as to whether these are both variable pathways to metastasis or just markers of metastatic burden. This review explores the evidence for CTCs in the initiation and progression of metastatic cancer and the data supporting these different concepts in an attempt to better understand the role of CTCs in metastasis. A greater understanding of the metastatic potential of CTCs will open new avenues for therapeutic interventions in the future.

Mutant B-RAF-Mcl-1 survival signaling depends on the STAT3 transcription factor.

Approximately 50% of melanomas depend on mutant B-RAF for proliferation, metastasis and survival. The inhibition of oncogenic B-RAF with highly targeted compounds has produced remarkable albeit short-lived clinical responses in B-RAF mutant melanoma patients. Reactivation of signaling downstream of B-RAF is frequently associated with acquired resistance to B-RAF inhibitors, and the identification of B-RAF targets may provide new strategies for managing melanoma. Oncogenic B-RAF(V600E) is known to promote the stabilizing phosphorylation of the anti-apoptotic protein Mcl-1, implicated in melanoma survival and chemoresistance. We now show that B-RAF(V600E) signaling also induces the transcription of Mcl-1 in melanocytes and melanoma. We demonstrate that activation of STAT3 serine-727 and tyrosine-705 phosphorylations is promoted by B-RAF(V600E) activity and that the Mcl-1 promoter is dependent on a STAT consensus-site for B-RAF-mediated activation. Consequently, suppression of STAT3 activity disrupted B-RAF(V600E)-mediated induction of Mcl-1 and reduced melanoma cell survival. We propose that STAT3 has a central role in the survival and contributes to chemoresistance of B-RAF(V600E) melanoma.

The MAPK pathway functions as a redundant survival signal that reinforces the PI3K cascade in c-Kit mutant melanoma.

Stimulation of the c-Kit receptor tyrosine kinase has a critical role in the development and migration of melanocytes, and oncogenic c-Kit mutants contribute to the progression of some melanomas. c-Kit signalling activates the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways and their relative contribution to the activities of oncogenic and ligand-dependent c-Kit remains uncertain. We show that PI3K is a major regulator of MAPK activation in response to c-Kit activity and the dominant effector of c-Kit-driven melanocyte proliferation and melanoma survival. Nevertheless, inhibition of the PI3K pathway in c-Kit mutant melanoma cells did not replicate the apoptotic efficacy of the c-Kit inhibitor, imatinib mesylate. Instead, the simultaneous suppression of the PI3K and MAPK pathways promoted a strong synergistic apoptotic effect. These data indicate that MAPK functions as a redundant survival signal that reinforces the PI3K cascade in c-Kit mutant melanoma. Thus, the concurrent inhibition of PI3K and MAPK signalling is required to suppress oncogenic c-Kit activity and may provide an effective therapeutic strategy in c-Kit mutant melanomas.

Circulating tumour cells and circulating free nucleic acid as prognostic and predictive biomarkers in colorectal cancer.

The detection of circulating tumour cells or circulating free tumour nucleic acids can potentially guide treatment and inform prognosis in colorectal cancer using minimally invasive "liquid biopsies". Current literature supports the notion that high circulating tumour cell counts or presence of tumour nucleic acid correlate with inferior clinical outcomes for patients, but they are not yet part of routine clinical care. Future research evolves around the examination of the molecular phenotype of circulating tumour cells. The key unanswered areas include differentiating between circulating tumour cell presence and their proliferative capacity and dormancy, identifying tumour heterogeneity and understanding the epithelial-mesenchymal transition.

Apoptosis of human melanoma cells induced by inhibition of B-RAFV600E involves preferential splicing of bimS.

Bim is known to be critical in killing of melanoma cells by inhibition of the RAF/MEK/ERK pathway. However, the potential role of the most potent apoptosis-inducing isoform of Bim, Bim(S), remains largely unappreciated. Here, we show that inhibition of the mutant B-RAF(V600E) triggers preferential splicing to produce Bim(S), which is particularly important in induction of apoptosis in B-RAF(V600E) melanoma cells. Although the specific B-RAF(V600E) inhibitor PLX4720 upregulates all three major isoforms of Bim, Bim(EL), Bim(L), and Bim(S), at the protein and mRNA levels in B-RAF(V600E) melanoma cells, the increase in the ratios of Bim(S) mRNA to Bim(EL) and Bim(L) mRNA indicates that it favours Bim(S) splicing. Consistently, enforced expression of B-RAF(V600E) in wild-type B-RAF melanoma cells and melanocytes inhibits Bim(S) expression. The splicing factor SRp55 appears necessary for the increase in Bim(S) splicing, as SRp55 is upregulated, and its inhibition by small interfering RNA blocks induction of Bim(S) and apoptosis induced by PLX4720. The PLX4720-induced, SRp55-mediated increase in Bim(S) splicing is also mirrored in freshly isolated B-RAF(V600E) melanoma cells. These results identify a key mechanism for induction of apoptosis by PLX4720, and are instructive for sensitizing melanoma cells to B-RAF(V600E) inhibitors.

Impaired inhibition of NF-kappaB activity by melanoma-associated p16INK4a mutations.

The cyclin-dependent kinase inhibitor p16INK4a has been identified as tumor suppressor and melanoma predisposition gene. While its cell cycle inhibitory ability is important in protecting cells from uncontrolled growth and possible tumor formation, other functions of p16INK4a are likely to contribute to its nature as a tumor suppressor. p16INK4a binding and inhibition of the transcription factor NF-kappaB has been shown and is consistent with the reports of abnormally increased NF-kappaB activity in various cancers including melanoma. Here, we present evidence that wild type p16INK4a binds to the NF-kappaB subunit RelA more efficiently than melanoma-associated p16INK4a mutations. Furthermore, whereas wild type p16INK4a strongly inhibits NF-kappaB transcriptional activity, a subset of melanoma-associated p16INK4a mutants show reduced NF-kappaB inhibitory function. p16INK4a does not affect NF-kappaB nuclear translocation or DNA binding, suggesting a mechanism that reduces NF-kappaB transactivation activity.

Serum carotenoids and risk of cervical intraepithelial neoplasia in Southwestern American Indian women.

The objective of this research was to evaluate the association between serum carotenoids and cervical intraepithelial neoplasia (CIN) among Southwestern American Indian women. Cases were American Indian women with biopsy-proven CIN II/III cervical lesions (n = 81) diagnosed between November 1994 and October 1997. Controls were American Indian women from the same clinics with normal cervical epithelium (n = 160). All of the subjects underwent interviews and laboratory evaluations. Interviews evaluated demographic information, sexual history, and cigarette smoking. Serum concentrations of alpha-carotene, beta-carotene, beta-cryptoxanthin, lycopene, and lutein/zeaxanthin were measured by high performance liquid chromatography. Cervical human papillomavirus infection was detected using a PCR-based test. Increasing levels of alpha-carotene, beta-cryptoxanthin, and lutein/zeaxanthin were associated with decreasing risk of CIN II/III. In addition, the highest tertiles of beta-cryptoxanthin (odds ratio = 0.39, 95% confidence interval = 0.17-0.91) and lutein/zeaxanthin (odds ratio = 0.40, 95% confidence interval = 0.17-0.95) were associated with the lowest risk of CIN. In conclusion, specially targeted intervention efforts to increase consumption of fruits and vegetables may protect Southwestern American Indian women from developing CIN.

Functional impairment of melanoma-associated p16(INK4a) mutants in melanoma cells despite retention of cyclin-dependent kinase 4 binding.

PURPOSE: Melanoma-associated germ-line mutations affecting the tumor suppressor and cyclin-dependent kinase (CDK) inhibitor, CDKN2A/p16(INK4a) have been identified in >100 melanoma-prone families. To predict the melanoma risk for carriers of specific mutations, it is useful to test the function of the mutant proteins in biochemical assays; however, it is unclear how well these results correlate with their cellular effects. We examined the relationship between loss of CDK binding by mutant proteins and various measures of cellular growth in melanoma cells. EXPERIMENTAL DESIGN: The cellular activities of four melanoma-associated p16(INK4a) mutations (Arg24Pro, Ala36Pro, Met53Ile, and Val126Asp) were compared by use of inducible expression in stably transfected melanoma cells, deficient in expression of the endogenous protein, and compared with their ability to bind CDK4. RESULTS: The cell cycle-inhibitory activity of all of the mutants was profoundly reduced, and partially retained capacity for CDK4 binding in functional assays did not correlate with significant preservation of cell cycle-regulatory function. CONCLUSION: Testing of p16(INK4a) interactions with CDKs in protein-binding assays is an unreliable predictor of mutant p16(INK4a) function in cells. In addition to exhibiting reduced stability, these mutant proteins may also be defective in interaction with cellular targets other than CDKs.

Contraceptive and reproductive risk factors for cervical intraepithelial neoplasia in American Indian women.

OBJECTIVES: To evaluate contraceptive and reproductive risk factors for cervical intraepithelial neoplasia (CIN) in southwestern American Indian women. METHODS: We conducted a clinic-based case-control study. Cases were American Indian women with biopsy-proven CIN I, CIN II or CIN III. Controls were from the same clinics and had normal cervical epithelium. All subjects underwent structured interviews focused on contraceptive and reproductive factors. Laboratory assays included polymerase chain reaction (PCR)-based tests for cervical human papillomavirus (HPV) infection. RESULTS: We enrolled 628 women in the study. The strongest risk factors for CIN II/III included HPV infection (adjusted odds ratio [OR] = 7.9, 95% CI : 4.7-13.2), and low income (OR = 3.1, 95% CI : 1.7-5.7). The use of an intrauterine device (IUD) ever (OR = 3.0, 95% CI : 1.4-6.1) and currently (OR = 4.1, 95% CI : 1.1-14.6), and > or = 3 vaginal deliveries (OR = 5.2, 95% CI : 2.4-11.1) were associated with CIN II/III. History of infertility was also associated with CIN II/III (OR = 2.1, 95% CI : 1.0-4.2). CONCLUSIONS: The data suggest that history of infertility, IUD use and vaginal deliveries were associated with CIN among American Indian women.

Risk factors for cervical intraepithelial neoplasia in southwestern American Indian women.

The authors assessed risk factors for cervical intraepithelial neoplasia (CIN) among southwestern American Indian women using case-control methods. Cases were New Mexico American Indian women with biopsy-proven grade I (n = 190), grade II (n = 70), or grade III (n = 42) cervical lesions diagnosed between November 1994 and October 1997. Controls were American Indian women from the same Indian Health Service clinics with normal cervical epithelium (n = 326). All subjects underwent interviews and laboratory evaluations. Interviews focused on history of sexually transmitted diseases, sexual behavior, and cigarette smoking. Laboratory assays included polymerase chain reaction-based tests for cervical human papillomavirus infection, tests for gonorrhea and chlamydia, wet mounts, and serologic assays for antibodies to Treponema pallidum, herpes simplex virus, and hepatitis B and C viruses. In multiple logistic regression analysis, the strongest risk factors for CIN II/III among American Indian women were human papillomavirus type 16 infection (adjusted odds ratio (OR) = 7.6; 95% confidence interval (CI): 2.4, 23.2), any human papillomavirus infection (OR = 5.8; 95% CI: 3.3, 10.0), low income (OR = 3.3; 95% CI: 1.7, 6.2), and history of any sexually transmitted disease (OR = 2.0; 95% CI: 1.1, 3.5). Unlike previous research, this study found no strong associations between CIN and sexual activity or cigarette smoking.

Data model for the elimination of matrix effects in enzyme-based flow-injection systems.

This contribution presents a new conceptional enzyme-based flow injection analysis (FIA) system for the process and quality control of food processing and biotechnological systems. It provides the determination of different analytes in distinct process media on the base of a common experimental set-up. In contrast to known comparable systems, analysis is performed without the commonly used sample preparation and dilution steps. Instead, the adaptation to the necessary measurement range is realized by optimization of intrinsic system parameters. The central principle of the work presented is the elimination of occurring interferences by the heterogeneous matrix of the process sample. Based on a particular injection mode, the application of dehydrogenases as indicator enzymes and a specially developed data model using cognitive methods, cross sensitivities of the detector as well as disturbed reaction rates of the enzymes could be almost completely compensated. Two applications are presented, the analysis of ethanol in non-alcoholic beer and the online determination of D-/L-lactate during a lactic acid fermentation, which reveal the advantage of the developed system.

Serum micronutrients and cervical dysplasia in Southwestern American Indian women.

We carried out a clinic-based case-control study to assess serum micronutrients as risk factors for cervical dysplasia among Southwestern American Indian women, a group with high rates of cervical preinvasive lesions. Cases were American Indian women with biopsy-proven cervical intraepithelial neoplasia (CIN I or CIN II/III). Controls were from the same Indian Health Service clinics with normal cervical epithelium. We interviewed women about histories of sexually transmitted diseases, sexual behavior, diet, hygienic practices, cigarette smoking, and reproductive factors. Laboratory assays included serum for retinol (vitamin A), ascorbic acid (vitamin C), alpha-tocopherol (vitamin E), and red blood cell folate levels, DNA for human papillomavirus (HPV) typing, and tests for other sexually transmitted diseases. The strongest risks for cervical dysplasia were associated with cervical HPV infection [odds ratio (OR) = 3.2, 95% confidence interval (CI) = 2.2-4.6 and OR = 7.9, 95% CI = 4.8-13.1 for CIN I and CIN II/III, respectively]. With adjustments made for HPV infection and other relevant confounders, subjects in the lowest serum retinol quartile were at increased risk of CIN I compared with women in the highest quartile (OR = 2.3, 95% CI = 1.3-4.1). The data suggest that low serum alpha-tocopherol was associated with CIN I/III, although the adjusted OR was not statistically significant (OR = 2.0, 95% CI = 0.9-4.8). Low serum ascorbic acid and red blood cell folate were not associated with cervical dysplasia.

Effect of cervical carcinoma in situ and its management on pregnancy outcome.

OBJECTIVE: To assess the effect of cervical carcinoma in situ (CIS) and its management on subsequent pregnancy outcome. METHODS: We used a population-based retrospective cohort design that included record linkage between cancer data and birth records. The Cancer Surveillance System records of women with CIS (n = 1851, 312 diagnosed during pregnancy) diagnosed between 1984 and 1992, were linked to birth certificates of their first subsequent deliveries after CIS diagnosis. The comparison group (n = 9201) was a random sample of women without CIS who gave birth during the same years. The outcome measures were preterm and low birth weight infants subsequent to CIS diagnosis and treatment. Treatments included no therapy, dilation and curettage or endocervical curettage, cryosurgery or laser vaporization, and conization. RESULTS: Women with CIS who were not treated with conization had a small increased risk of preterm delivery (odds ratio [OR] 1.4, 95% confidence interval [CI] 1.0, 2.0) and no increased risk of low birth weight infant (OR 1.0, 95% CI 0.7, 1.6), compared with women without CIS, after adjusting for maternal smoking, race, parity, marital status, and history of induced pregnancy termination. Women with CIS who had conization were more likely to deliver premature infants (OR 1.6, 95% CI 1.2, 2.0) than women without CIS, after adjusting for the same confounding factors. The apparently increased risk of low birth weight (OR 1.8, 95% CI 1.4, 2.4) seemed to be a reflection of premature delivery. CONCLUSION: The risk of prematurity increased after conization for CIS and did not increase when women with CIS had other procedures.

Risk factors for cervical intraepithelial neoplasm in Alaska Native women: a pilot study.

Although rates for invasive cervical cancer have declined over the past twenty years among Alaska Native women, they continue to show high rates of pre-invasive cervical lesions (cervical intraepithelial neoplasia, or CIN). We investigated risk factors for CIN II/III among Alaska Native women in a pilot case-control study. Cases (n = 26) included women with biopsy-proven CIN II/III, while controls (n = 52) had normal cervical epithelium. The strongest risks associated with CIN II/III were HPV infection of any type (Crude Odds Ratio [OR] 8.4, 95% Confidence Interval [CI]: 2.9-29.4), HPV 16 infection (OR 40.8, 95% CI: 9.4-176.4), and a family history of cervical dysplasia (OR 3.9, 95% CI: 1.3-11.3). We also found that use of depot-medroxy progesterone acetate was associated with CIN (OR 3.0, 95% CI: 1.1-8.5). A larger investigation would be necessary to allow adequate evaluation of these, and other, risk factors for CIN among Alaska Native women.

Colorectal cancer incidence and survival among Alaska Natives, 1969-1993.

BACKGROUND: Although colorectal cancer rates are low among most groups of Native Americans in North America, rates for Alaska Natives have been substantially elevated compared with US rates for all races combined. METHODS: To better describe the epidemiology of colorectal cancer incidence and survival among Alaska Natives, stratified by gender and tribal/ethnic affiliation, we examined data collected by the Alaska Native Cancer Registry 1969-1993. We calculated age-adjusted and age-specific incidence as well as actuarial survival rates, and examined histological type, site, stage at diagnosis, and treatment. We compared these data to colorectal cancer data from whites living in western Washington. RESULTS: In all, 587 colorectal cancer cases were identified among Alaska Natives over the 25-year period, for an age-adjusted annual incidence rate of 71.4/100000 in women, and 69.3/100000 in men. Compared to Alaska Indians, colon cancer rates were significantly higher in Aleuts (relative risk [RR] = 1.6, 95% CI: 1.2-2.2) and in Eskimos (RR = 1.5, 95% CI: 1.2-1.8), while rectal cancer rates did not differ by race/ethnicity. Alaska Natives experienced a 50% higher incidence rate of colorectal cancer overall compared to western Washington whites (RR = 1.5, 95% CI: 1.3-1.6), although rectal cancer rates were similar in the two populations. The highest RR were seen among Alaska Native women; Aleuts and Eskimos had colon cancer rates more than twice that of western Washington white women. No unusual qualitative features were found in the cancers occurring in Alaska Natives. Actuarial colorectal cancer survival rates for Alaska Natives overall were 74% at one year and 42% at 5 years; these rates were very similar to those observed for the western Washington population. Both one and 5-year survival rates showed a significant trend towards improvement over time. CONCLUSIONS: Alaska Natives had substantially higher colorectal cancer incidence rates compared to western Washington whites. Rates were particularly high for Aleut and Eskimo women. These data suggest a need for intensified secondary prevention strategies for this high-risk population, while further research is needed to identify modifiable risk factors.

Increasing incidence of colon and rectal cancer among Hispanics and American Indians in New Mexico (United States), 1969-94.

OBJECTIVES: This study evaluates time trends in colon and rectal cancer incidence and mortality among the three major race/ethnic groups (Hispanics, American Indians, and non-Hispanic Whites) in New Mexico (United States). METHODS: We used data from the New Mexico Tumor Registry (NMTR) and computed average annual age-standardized incidence and mortality rates. Colon cancer incidence rates were further examined by anatomical subsite. Estimated annual percent change (EAPC) in incidence and mortality over time were computed using Poisson regression. RESULTS: Invasive colorectal cancer incidence rates increased from 1969-89 in all three race/ethnic groups, but decreased among non-Hispanic Whites in 1990-94, while rates continued to increase among minority populations, especially among minority men. Over the 26-year period, EAPC in colon cancer incidence among men was 3.6 percent for Hispanics, 4.7 percent for American Indians, and 0.7 percent for non-Hispanic Whites. Right-sided colon cancers were more common among American Indian women, and among all women aged 65 years and older. Mortality rates decreased steadily among non-Hispanic Whites over the study period, especially among women. CONCLUSIONS: Studies are needed to identify important modifiable risk factors and to develop strategies to increase the use of colorectal cancer screening-procedures among the minority populations.

Stroke risk factor knowledge in Hispanic and non-Hispanic white women in New Mexico: implications for targeted prevention strategies.

Hispanic women in New Mexico have recently experienced an increase in age-adjusted mortality compared with non-Hispanic white women. Since patients' knowledge of stroke risk factors may affect risk factor control, the present study was undertaken to characterize stroke risk factor understanding in Hispanic and non-Hispanic white women in New Mexico. We administered a stroke risk factor knowledge survey to 215 women hospitalized in Albuquerque, New Mexico. Patients were classified by each of three dichotomous groupings: stroke or nonstroke diagnosis; Hispanic or non-Hispanic white ethnicity; history of cardiovascular risk factors. The frequency of specific item responses was determined for each patient grouping. Two-way analysis of variance was used to determine whether composite knowledge score differed among patient groups. Stress was the attribute most commonly thought to be a risk factor for stroke. Although no ethnic differences were found on composite knowledge score, Hispanic women were significantly less likely to report hypertension as a stroke risk factor than non-Hispanic white women. We suggest that stroke risk factor understanding in Hispanic and non-Hispanic white women in New Mexico is inadequate. Insufficient understanding of the consequences of hypertension, including stroke, may diminish the degree of hypertension control that patients achieve. Further study of the relationship between stroke risk factor understanding and health behavior could enhance prevention efforts.