Summary

Copy number variations (CNVs) have been shown to contribute substantially to disease susceptibility in several inherited diseases including cancer. We conducted a genome-wide search for CNVs in blood-derived DNA from 79 individuals (62 melanoma patients and 17 spouse controls) of 30 high-risk melanoma-prone families without known segregating mutations using genome-wide comparative genomic hybridization (CGH) tiling arrays. We identified a duplicated region on chromosome 4q13 in germline DNA of all melanoma patients in a melanoma-prone family with three affected siblings. We confirmed the duplication using quantitative PCR and a custom-made CGH array design spanning the 4q13 region. The duplicated region contains 10 genes, most of which encode CXC chemokines. Among them, CXCL1 (melanoma growth-stimulating activity α) and IL8 (interleukin 8) have been shown to stimulate melanoma growth in vitro and in vivo. Our data suggests that the alteration of CXC chemokine genes may confer susceptibility to melanoma.

Objective

Smoking and alcohol consumption explain little of the risk for upper-gastrointestinal (UGI) cancer in China, where over half of all cases in the world occur.

Methods

We evaluated questionnaire-based risk factors for UGI cancers in a case-control study from Shanxi Province, China, including 600 esophageal squamous cell carcinomas (ESCC), 599 gastric cardia adenocarcinomas (GCA), 316 gastric noncardia adenocarcinomas (GNCA), and 1514 age- and gender-matched controls.

Results

Ever smoking and ever use of any alcohol were not associated with risk of UGI cancer; only modest associations were observed between ESCC risk and highest cumulative smoking exposure, as well as GNCA risk and beer drinking. While several associations were noted for socioeconomic and some dietary variables with one or two UGI cancers, the strongest and most consistent relations for all three individual UGI cancers were observed for consumption of scalding hot foods (risk increased 150% to 219% for daily vs never users) and fresh vegetables and fruits (risk decreased 48% to 70% for vegetables and 46% to 68% for fruits, respectively, for high vs low quartiles).

Conclusion

This study confirms the minor role of tobacco and alcohol in UGI cancers in this region, and highlights thermal damage as a leading etiologic factor.

Objective

To identify the frequency of pregnancy and neonatal complications in pregnancies carrying fetuses affected with trichothiodystrophy (TTD).

Methods

We identified pregnancy and neonatal complications and serum screening results from mothers of TTD patients in a DNA repair diseases study from 2001 to 2011.

Results

Pregnancy reports of 27 TTD patients and their 23 mothers were evaluated and81% of the pregnancies had complications: 56% had preterm delivery, 30% had preeclampsia, 19% had placental abnormalities, 11% had HELLP syndrome, 4% had an emergency c-section for fetal distress; while44% had two or more complications. Only19% of the pregnancies delivered at term without complications. Eight of the 10 pregnancies tested had abnormal multiple marker results including elevated levels of human chorionic gonadotropin. Eighty-five percent of the neonates had complications: 70 % were low birth weight (<2500g), 35% had birth weight <10 centile for gestational age, 70% had NICU admission, 67% had a collodion membrane, and 31% of the 16 males had cryptorchidism. Cataracts were present in 54% of the TTD patients examined.

Conclusion

TTD is a multisystem disease that predisposes mothers of affected patients to substantial risks for pregnancy complications and TTD neonates have a high incidence of multiple abnormalities.

Affordable early screening in subjects with high risk of lung cancer has great potential to improve survival from this deadly disease. We measured gene expression from lung tissue and peripheral whole blood (PWB) from adenocarcinoma cases and controls to identify dysregulated lung cancer genes that could be tested in blood to improve identification of at-risk patients in the future. Genome-wide mRNA expression analysis was conducted in 153 subjects (73 adenocarcinoma cases, 80 controls) from the Environment And Genetics in Lung cancer Etiology (EAGLE) study using PWB and paired snap-frozen tumor and non-involved lung tissue samples. Analyses were conducted using unpaired t-tests, linear mixed effects and ANOVA models. The area under the receiver operating characteristic curve (AUC) was computed to assess the predictive accuracy of the identified biomarkers. We identified 50 dysregulated genes in stage I adenocarcinoma versus control PWB samples (False Discovery Rate ≤0.1, fold change ≥1.5 or ≤0.66). Among them, eight (TGFBR3, RUNX3, TRGC2, TRGV9, TARP, ACP1, VCAN, and TSTA3) differentiated paired tumor versus non-involved lung tissue samples in stage I cases, suggesting a similar pattern of lung cancer-related changes in PWB and lung tissue. These results were confirmed in two independent gene expression analyses in a blood-based case-control study (n=212) and a tumor-non tumor paired tissue study (n=54). The eight genes discriminated patients with lung cancer from healthy controls with high accuracy (AUC=0.81, 95% CI=0.74–0.87). Our finding suggests the use of gene expression from PWB for the identification of early detection markers of lung cancer in the future.

Background

Chordoma, an age-dependent rare cancer, arises from notochordal remnants. Fewer than 5% of chordomas occur in children. Tuberous sclerosis complex (TSC) is an autosomal dominant neurocutaneous syndrome characterized by abnormal tissue growths in multiple organ systems. Reports of chordoma in patients with TSC suggest that TSC1 and TSC2 mutations may contribute to chordoma etiology.

Methods

To determine whether the 10 TSC-associated chordomas reported in the literature are representative of chordoma in the general pediatric population, we compared age at diagnosis, primary site and outcome in them to results from a systematic assessment of 65 pediatric chordoma cases reported to the US population-based cancer registries contributing to the SEER Program of the National Cancer Institute.

Results

TSC-associated chordomas differed from chordomas in the general pediatric population: median age at diagnosis (6.2 months, TSC vs. 12.5 years, SEER); anatomic site (40% sacral, TSC vs. 9.4% sacral, SEER); and site-specific age at diagnosis (all 4 sacral chordomas diagnosed during the fetal or neonatal period, TSC vs. all 6 sacral chordomas diagnosed at > 15 years, SEER). Finally, 3 of 4 patients with TSC-associated sacral chordoma were alive and tumor-free at 2.2, 8 and 19 years following diagnosis vs. a median survival of 36 months among pediatric sacral chordoma patients in SEER.

Conclusions

These results strengthen the association between pediatric chordoma and TSC. Future clinical and molecular studies documenting the magnitude and clinical spectrum of the joint occurrence of these two diseases should provide the basis for delineating the biological relationship between them.

Purpose

Epstein–Barr virus (EBV) infection and a family history of nasopharyngeal carcinoma (NPC) are associated with NPC risk. We examined the risk associated with EBV markers and their clinical utility to identify NPC susceptibles within high-risk NPC families.

Experimental Design

We evaluated antibody titers against viral capsid antigen (VCA) IgA, EBV nuclear antigen-1 (EBNA1) IgA, and DNase among unaffected relatives of NPC cases from 358 multiplex families in Taiwan. Incident NPC cases were identified via linkage to the National Cancer Registry. Clinical examinations of 924 individuals were also done to identify occult, asymptomatic NPC. Baseline EBV serology was used to estimate NPC risk using rate ratios with 95% CI. Associated sensitivity/specificity and receiver operating characteristic (ROC) curves were calculated.

Results

A total of 2,444 unaffected individuals with 15,519 person-years (6.5 years median follow-up) yielded 14 incident NPC cases (nearly 11 times the general population rate). The absolute rate of NPC among anti-EBV EBNA1 IgA seropositives using a standard positivity cutoff versus an optimized cutoff point defined by ROC analyses was 265/100,000 person-years with a 4.7-fold increased risk of NPC (95% CI: 1.4–16) and 166/100,000 person-years with a 6.6-fold increase (95% CI: 1.5–61), respectively. Sensitivity and specificity using the optimized positivity cutoff points were 85.7% and 51.2%, respectively. It is estimated that active evaluation of 49% of individuals from high-risk NPC families seropositive for this marker could lead to earlier detection of up to 86% of NPC cases. Risks associated with the other three EBV markers were weaker.

Conclusions

Future efforts are needed to identify susceptibility markers among high-risk NPC families that maximize both sensitivity and specificity.

Synopsis

Hereditary genodermatoses with cancer predisposition are reviewed, including Nevoid Basal Cell Carcinoma Syndrome, Neurofibromatosis Types 1 and 2, Tuberous Sclerosis Complex, Xeroderma Pigmentosum, and Dyskeratosis Congenita. Hereditary melanoma is also included, though it differs from the others in several respects. The underlying genetic aberrations causing these syndromes are largely known, allowing novel treatments to be developed for some of these disorders. Early recognition and diagnosis allows for close follow-up and surveillance for associated malignancies.

The presence of pancreatic cancer (PC) in melanoma-prone families has been consistently associated with an increased frequency of CDKN2A mutations, the major high-risk susceptibility gene identified for melanoma. However, the precise relationship between CDKN2A, melanoma and PC remains unknown. We evaluated a recently identified PC susceptibility gene PALB2 using both sequencing and tagging to determine whether PALB2 might explain part of the relationship between CDKN2A, melanoma, and PC. No disease-related mutations were identified from sequencing PALB2 in multiple pancreatic cancer patients or other mutation carrier relatives of PC patients from the eight melanoma-prone families with CDKN2A mutations and PC. In addition, no significant associations were observed between 11 PALB2 tagging SNPs and melanoma risk in 23 melanoma-prone families with CDKN2A mutations or the subset of 11 families with PC or PC-related CDKN2A mutations. The results suggested that PALB2 does not explain the relationship between CDKN2A, melanoma, and pancreatic cancer in these melanoma-prone families.

Background

Germline CDKN2A mutations have been observed in 20-40% of high-risk melanoma-prone families, however little is known about their prevalence in population-based series of melanoma cases and controls.

Methods

We resequenced the CDKN2A gene, including the p14ARF variant and promoter regions, in approximately 703 registry-ascertained melanoma cases and 691 population-based controls from Iceland, a country in which the incidence of melanoma has increased rapidly.

Results

We identified a novel germline variant, G89D that was strongly associated with increased melanoma risk and appeared to be an Icelandic founder mutation. The G89D variant was present in about 2% of Icelandic invasive cutaneous malignant melanoma cases. Relatives of affected G89D carriers were at significantly increased risk of melanoma, head & neck cancers, and pancreatic carcinoma compared to relatives of other melanoma patients. Nineteen other germline variants were identified, but none conferred an unequivocal risk of melanoma.

Conclusions

This population-based study of Icelandic melanoma cases and controls showed a frequency of disease-related CDKN2A mutant alleles ranging from 0.7% to 1.0%, thus expanding our knowledge about the frequency of CDKN2A mutations in different populations. In contrast to North America and Australia where a broad spectrum of mutations was observed at a similar frequency, in Iceland, functional CDKN2A mutations consists of only one or two different variants. Additional genetic and/or environmental factors are likely critical for explaining the high incidence rates for melanoma in Iceland. This study adds to the geographic regions for which population-based estimates of CDKN2A mutation frequencies are available.

Epidemiological data on green/jasmine tea and esophageal as well as gastric cancer are limited and inconclusive. In order to study the effect of jasmine tea in upper gastrointestinal (UGI) cancers, we evaluated 600 esophageal squamous cell carcinoma (ESCC), 598 gastric cardia adenocarcinoma (GCA), and 316 gastric non-cardia adenocarcinoma (GNCA) cases and 1514 age-, gender-, and neighborhood-matched controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated from logistic regression adjusted for matching factors and potential confounders. Among controls, 35% of males and 8% of females reported consumption of jasmine tea; other tea consumption was rare. Consumption of jasmine tea (ever vs. never) was not associated with risk of ESCC (OR=1.15, 95% CI 0.92–1.44), GCA (OR=1.14, 95% CI 0.88–1.37), or GNCA (OR=0.85, 95% CI 0.64–1.15) in males and females combined. Among males, cumulative lifetime consumption showed a significant positive dose-response relation with ESCC risk, but not for GCA and GNCA. In exploratory analyses, occupation affected the relation between tea and ESCC such that consumption in males was associated with increased risk only in non-office workers. Overall, we found no evidence for a protective effect of tea in esophageal or gastric cancer. Further studies of the potential effects of thermal damage, tea quality, and water quality on UGI cancers are suggested.

Background

We determined the frequency of cancer, neurologic degeneration and mortality in xeroderma pigmentosum (XP) patients with defective DNA repair in a four decade natural history study.

Methods

All 106 XP patients admitted to the NIH from 1971 to 2009 were evaluated from clinical records and follow-up.

Results

In the 65 percent (n=69) of patients with skin cancer, non-melanoma skin cancer (NMSC) was increased 10,000–fold and melanoma was increased 2,000-fold in patients under age 20. The 9 year median age at diagnosis of first non-melanoma skin cancer (NMSC) (n=64) was significantly younger than the 22 year median age at diagnosis of first melanoma (n= 38), a relative age reversal from the general population suggesting different mechanisms of carcinogenesis between NMSC and melanoma. XP patients with marked burning on minimal sun exposure (n=65) were less likely to develop skin cancer than those who did not. This may be related to the extreme sun protection they receive from an earlier age, decreasing their total UV exposure. Progressive neurologic degeneration was present in 24% (n=25) with 16/25 in complementation group XP-D. The most common causes of death were skin cancer (34%, n=10), neurologic degeneration (31%, n=9), and internal cancer (17%, n=5). The median age at death (29 years) in XP patients with neurodegeneration was significantly younger than those XP patients without neurodegeneration (37 years) (p=0.02).

Conclusion

This 39 year follow-up study of XP patients indicates a major role of DNA repair genes in the etiology of skin cancer and neurologic degeneration.

Chromosome 9p21 has been implicated in the pathogenesis of cutaneous malignant melanoma (CMM). In addition to CDKN2A, the major known high-risk susceptibility gene for CMM, recent studies suggest that other 9p21 genes may be involved in melanoma/nevi development. To identify 9p21 variants that influence susceptibility to CMM and number of nevi in CMM-prone families with and without CDKN2A mutations, we analyzed 562 individuals (183 CMM) from 53 families (23 CDKN2A+, 30 CDKN2A−) for 233 tagging SNPs in 21 genes at 9p21. Single SNP- and gene-based regression analyses were used to assess the risk of CMM, nevi count, skin complexion, and tanning ability associated with these SNPs and genes. We found that SNP rs7023329 in the MTAP gene was associated with number of nevi (Ptrend=0.003) confirming a recent finding by a genome-wide association study. In addition, three SNPs in the ACO1 gene, rs7855483 (Ptrend=0.002), rs17288067 (Ptrend=0.0009), and rs10813813 (Ptrend=0.005), showed the strongest associations with CMM risk. None of the examined 9p21 SNPs was associated with skin complexion, whereas two SNPs, rs10964862 in IFNW1 (Ptrend=0.003), and rs13290968 in TUSC1 (Ptrend=0.0006), were associated with tanning ability. Gene-based analyses suggested that the ACO1 gene was significantly associated with CMM (P=0.0004); genes IFNW1 (P=0.002) and ACO1 (P=0.0002) were significantly associated with tanning ability. Our findings are consistent with recent proposals that additional 9p21 genes may contribute to CMM susceptibility in CMM-prone families. These genetic variants may, at least partially, exert their effects through nevi and tanning ability.

Background

Melanoma rates are rising among young women, possibly due to increasing ultraviolet radiation to previously protected body sites. Therefore, we examined melanoma incidence trends by age, gender, and body site. Descriptive methods were complemented with the age-period-cohort parameters net drift and longitudinal age trend.

Methods

Case and population data were obtained from the Surveillance, Epidemiology, and End Results 9 Registries Database (1975-2006). Net drift summarized the average annual percentage change in log-linear rates per year of calendar-time (or year of diagnosis). Longitudinal age trend summarized the average annual percentage change by attained age at diagnosis. Early-and late-onset melanomas have low and high longitudinal age trends, respectively.

Results

There were 105,829 melanomas diagnosed in the SEER 9 Registries. The overall age-adjusted incidence rate (IR)for melanoma was 17.7/100,000 person-years. Age specific IRs were greater among women than men prior to age 40 years. Among women, Irs decreased for all anatomic sites relative to the trunk. The highest net drift occurred in truncal lesions among women (net drift = 3.8%/year of calendar time; 95% CI = 3.5%-4.0%). The lowest longitudinal age trends also were observed for truncal lesions among women (longitudinal age trend = 5.4%/year of attained age; 95% CI = 5.1-5.7).

Conclusions

Though melanoma Irs overall have risen for decades, the combination of high net drift and low longitudinal age trend demonstrate that melanomas are rising preferentially on the trunk among young women.

Impact

Future surveillance and analytic studies should consider melanoma effect modification by age, gender, and body site.

Purpose

The molecular drivers that determine histology in lung cancer are largely unknown. We investigated whether microRNA (miR) expression profiles can differentiate histological subtypes and predict survival for non-small cell lung cancer.

Experimental design

We analyzed miR expression in 165 adenocarcinoma (AD) and 125 squamous cell carcinoma (SQ) tissue samples from the Environmental And Genetics in Lung cancer Etiology (EAGLE) study using a custom oligo array with 440 human mature antisense miRs. We compared miR expression profiles using t-tests and F-tests and accounted for multiple testing using global permutation tests. We assessed the association of miR expression with tobacco smoking using Spearman correlation coefficients and linear regression models, and with clinical outcome using log-rank tests, Cox proportional hazards and survival risk prediction models, accounting for demographic and tumor characteristics.

Results

MiR expression profiles strongly differed between AD and SQ (global p<0.0001), particularly in the early stages, and included miRs located on chromosome loci most often altered in lung cancer (e.g., 3p21-22). Most miRs, including all members of the let-7 family, were down-regulated in SQ. Major findings were confirmed by QRT-PCR in EAGLE samples and in an independent set of lung cancer cases. In SQ, low expression of miRs down-regulated in the histology comparison was associated with 1.2 to 3.6-fold increased mortality risk. A 5-miR signature significantly predicted survival for SQ.

Conclusions

We identified a miR expression profile that strongly differentiated AD from SQ and had prognostic implications. These findings may lead to histology-based therapeutic approaches.

Previous studies have identified several dysregulated microRNAs in esophageal squamous cell carcinoma (ESCC); however, to date there are no ex vivo analyses comparing expression levels of these regulatory molecules in esophageal squamous cell tumors versus patient-matched normal epithelium. We describe here a technical strategy to evaluate microRNAs in normal esophageal basal cells (NB), normal esophageal differentiated cells (ND), and tumor cells (T). Laser capture microdissection was used to procure target populations from five cases and 18 ESCC-associated microRNAs were measured by RT-qPCR. Five microRNAs (miR-25, miR-106b, miR-21, miR-203, and miR-145) demonstrated consistent differential expression in at least one of the three comparisons: T vs. NB, T vs. ND, or NB vs. ND. The potential regulatory role of the microRNAs in ESCC was further evaluated by correlating their expression with a matched mRNA dataset, which included the same five cases and cell populations. In conclusion, the present work demonstrates the feasibility of studying microRNA levels in precisely dissected cell populations from clinical samples, and sheds light on the molecular mechanisms associated with ESCC.

Previous studies have identified several dysregulated microRNAs in esophageal squamous cell carcinoma (ESCC); however, to date there are no ex vivo analyses comparing expression levels of these regulatory molecules in esophageal squamous cell tumors versus patient-matched normal epithelium. We describe here a technical strategy to evaluate microRNAs in normal esophageal basal cells (NB), normal esophageal differentiated cells (ND), and tumor cells (T). Laser capture microdissection was used to procure target populations from five cases and 18 ESCC-associated microRNAs were measured by RT-qPCR. Five microRNAs (miR-25, miR-106b, miR-21, miR-203, and miR-145) demonstrated consistent differential expression in at least one of the three comparisons: T vs. NB, T vs. ND, or NB vs. ND. The potential regulatory role of the microRNAs in ESCC was further evaluated by correlating their expression with a matched mRNA dataset, which included the same five cases and cell populations. In conclusion, the present work demonstrates the feasibility of studying microRNA levels in precisely dissected cell populations from clinical samples, and sheds light on the molecular mechanisms associated with ESCC.

Objective

To quantify the risk of subsequent primary cancers among patients with primary cutaneous malignant melanoma.

Design

Population-based registry study.

Setting

We evaluated data from 9 cancer registries of the Surveillance, Epidemiology, and End Results program from 1973–2006.

Participants

We included 89 515 patients who survived at least 2 months after their initial melanoma diagnosis.

Results

Of the patients with melanoma, 10 857 (12.1%) developed 1 or more subsequent primary cancers. The overall risk of a subsequent primary cancer increased by 28% (observed to expected [O:E] ratio=1.28). One quarter of the cancers were subsequent primary melanomas (O:E=8.61). Women with head and neck melanoma and patients younger than 30 had markedly increased risks (O:E=13.22 and 13.40, respectively) of developing a subsequent melanoma. Second melanomas were more likely to be thin than were the first of multiple primary melanomas (thickness at diagnosis <1.00mm, 77.9% vs 70.3%, respectively; P<.001). Melanoma survivors had increased risk of developing several cancers; the most common cancers with elevated risks were breast, prostate, and non-Hodgkin lymphoma (O:E=1.10, 1.15, and 1.25, respectively).

Conclusions

Melanoma survivors have an approximately 9-fold increased risk of developing subsequent melanoma compared with the general population. The risk remains elevated more than 20 years after the initial melanoma diagnosis. This increased risk may be owing to behavioral factors, genetic susceptibility, or medical surveillance. Although the percentage of subsequent primary melanomas thicker than 1 mm is lower than for the first of multiple primary melanomas, it is still substantial. Melanoma survivors should remain under surveillance not only for recurrence but also for future primary melanomas and other cancers.

Epidemiological and mechanistic evidence on the association of quercetin-rich food intake with lung cancer risk and carcinogenesis are inconclusive. We investigated the role of dietary quercetin and the interaction between quercetin and P450 and glutathione S-transferase (GST) polymorphisms on lung cancer risk in 1822 incident lung cancer cases and 1991 frequency-matched controls from the Environment And Genetics in Lung cancer Etiology study. In non-tumor lung tissue from 38 adenocarcinoma patients, we assessed the correlation between quercetin intake and messenger RNA expression of the same P450 and GST metabolic genes. Multivariate odds ratios (ORs) and 95% confidence intervals (CIs) for sex-specific quintiles of intake were calculated using unconditional logistic regression adjusting for putative risk factors. Frequent intake of quercetin-rich foods was inversely associated with lung cancer risk (OR = 0.49; 95% CI: 0.37–0.67; P-trend < 0.001) and did not differ by P450 or GST genotypes, gender or histological subtypes. The association was stronger in subjects who smoked >20 cigarettes per day (OR = 0.35; 95% CI: 0.19–0.66; P-trend = 0.003). Based on a two-sample t-test, we compared gene expression and high versus low consumption of quercetin-rich foods and observed an overall upregulation of GSTM1, GSTM2, GSTT2, and GSTP1 as well as a downregulation of specific P450 genes (P-values < 0.05, adjusted for age and smoking status). In conclusion, we observed an inverse association of quercetin-rich food with lung cancer risk and identified a possible mechanism of quercetin-related changes in the expression of genes involved in the metabolism of tobacco carcinogens in humans. Our findings suggest an interplay between quercetin intake, tobacco smoking, and lung cancer risk. Further research on this relationship is warranted.

Background

Although pneumonia has been suggested as a risk factor for lung cancer, previous studies have not evaluated the influence of number of pneumonia diagnoses in relation to lung cancer risk.

Methods

The Environment And Genetics in Lung cancer Etiology (EAGLE) population-based study of 2,100 cases and 2,120 controls collected information on pneumonia more than one year before enrollment from 1,890 cases and 2,078 controls.

Results

After adjusting for study design variables, smoking, and chronic bronchitis, pneumonia was associated with decreased risk of lung cancer (odds ratio (OR), 0.79; 95% confidence interval (CI), 0.64–0.97), especially among individuals with ≥3 diagnoses versus none (OR, 0.35; 95% CI, 0.16–0.75). Adjustment for chronic bronchitis contributed to this inverse association. In comparison, pulmonary tuberculosis was not associated with lung cancer (OR, 0.96; 95% CI, 0.62–1.48).

Conclusions

The apparent protective effect of pneumonia among individuals with multiple pneumonia diagnoses may reflect an underlying difference in immune response and requires further investigation and confirmation.

Impact

Careful evaluation of number of pneumonia episodes may shed light on lung cancer etiology.

Background

The presence of recurrent high-risk mutations in CDKN2A and CDK4 among melanoma-prone kindreds suggests that a high-throughput, multiplex assay could serve as an effective initial screening tool. Moreover, with the emergence of new melanoma risk single nucleotide polymorphisms (SNPs) through genome-wide association studies, a flexible platform that can easily accommodate these new risk alleles is needed for more accurate genetic risk profiling. To this end, we have developed a novel melanoma-associated mutation detection method using a multiplex bead-based assay. This assay is suitable for high-throughput CDKN2A and CDK4 genotyping and can be eventually adapted to multiple loci across various constituent populations.

Methods

Genomic DNA from a 1603 subjects (1005 in training set, 598 in validation set) were amplified by multiplex PCR using five primer sets followed by multiplex allele-specific primer extension for 39 different known germline variants. The products were then sorted on an xMAP™ (formerly Tag-It™) array and detected by use of the Luminex xMAP™ system. Genotypes were compared to previously-determined sequence data.

Results

In the Toronto training cohort, variants were detected in 145 samples, giving complete concordance between the bead assay and direct sequencing results. Analysis of the 598 samples from the GenoMEL validation set led to identification of 150/155 expected variants (96.77% concordance). Overall, the bead assay correctly genotyped 1540/1603 (96.07%) of all individuals in the study and 1540/1545 (99.68%) of individuals whose mutations were represented in the probe set. Out of a total of 62,512 SNP calls, 62,517 (99.99%) were correctly assigned.

Conclusions

In this initial evaluation, the multiplex bead-based assay for familial melanoma appears to be a highly accurate method for genotyping CDKN2A and CDK4 variants.

Background

Lung cancer kills more than 1 million people worldwide each year. Whereas several human papillomavirus (HPV)–associated cancers have been identified, the role of HPV in lung carcinogenesis remains controversial.

Methods

We selected 450 lung cancer patients from an Italian population–based case–control study, the Environment and Genetics in Lung Cancer Etiology. These patients were selected from those with an adequate number of unstained tissue sections and included all those who had never smoked and a random sample of the remaining patients. We used real-time polymerase chain reaction (PCR) to test specimens from these patients for HPV DNA, specifically for E6 gene sequences from HPV16 and E7 gene sequences from HPV18. We also tested a subset of 92 specimens from all never-smokers and a random selection of smokers for additional HPV types by a PCR-based test for at least 54 mucosal HPV genotypes. DNA was extracted from ethanol- or formalin-fixed paraffin-embedded tumor tissue under strict PCR clean conditions. The prevalence of HPV in tumor tissue was investigated.

Results

Specimens from 399 of 450 patients had adequate DNA for analysis. Most patients were current (220 patients or 48.9%) smokers, and 92 patients (20.4%) were women. When HPV16 and HPV18 type–specific primers were used, two specimens were positive for HPV16 at low copy number but were negative on additional type-specific HPV16 testing. Neither these specimens nor the others examined for a broad range of HPV types were positive for any HPV type.

Conclusions

When DNA contamination was avoided and state-of-the-art highly sensitive HPV DNA detection assays were used, we found no evidence that HPV was associated with lung cancer in a representative Western population. Our results provide the strongest evidence to date to rule out a role for HPV in lung carcinogenesis in Western populations.

The authors investigated the relation between alcohol consumption and lung cancer risk in the Environment and Genetics in Lung Cancer Etiology (EAGLE) Study, a population-based case-control study. Between 2002 and 2005, 2,100 patients with primary lung cancer were recruited from 13 hospitals within the Lombardy region of Italy and were frequency-matched on sex, area of residence, and age to 2,120 randomly selected controls. Alcohol consumption during adulthood was assessed in 1,855 cases and 2,065 controls. Data on lifetime tobacco smoking, diet, education, and anthropometric measures were collected. Adjusted odds ratios and 95% confidence intervals for categories of mean daily ethanol intake were calculated using unconditional logistic regression. Overall, both nondrinkers (odds ratio = 1.42, 95% confidence interval: 1.03, 2.01) and very heavy drinkers (≥60 g/day; odds ratio = 1.44, 95% confidence interval: 1.01, 2.07) were at significantly greater risk than very light drinkers (0.1–4.9 g/day). The alcohol effect was modified by smoking behavior, with no excess risk being observed in never smokers. In summary, heavy alcohol consumption was a risk factor for lung cancer among smokers in this study. Although residual confounding by tobacco smoking cannot be ruled out, this finding may reflect interplay between alcohol and smoking, emphasizing the need for preventive measures.

Background

Genomic instability plays an important role in human cancers. We previously characterized genomic instability in esophageal squamous cell carcinomas (ESCC) in terms of loss of heterozygosity (LOH) and copy number (CN) changes in tumors using the Affymetrix GeneChip Human Mapping 500K array in 30 cases from a high-risk region of China. In the current study we focused on copy number neutral (CN = 2) LOH (CNNLOH) and its relation to gene expression in ESCC.

Results

Overall we found that 70% of all LOH observed was CNNLOH. Ninety percent of ESCCs showed CNNLOH (median frequency in cases = 60%) and this was the most common type of LOH in two-thirds of cases. CNNLOH occurred on all 39 autosomal chromosome arms, with highest frequencies on 19p (100%), 5p (96%), 2p (95%), and 20q (95%). In contrast, LOH with CN loss represented 19% of all LOH, occurred in just half of ESCCs (median frequency in cases = 0%), and was most frequent on 3p (56%), 5q (47%), and 21q (41%). LOH with CN gain was 11% of all LOH, occurred in 93% of ESCCs (median frequency in cases = 13%), and was most common on 20p (82%), 8q (74%), and 3q (42%). To examine the effect of genomic instability on gene expression, we evaluated RNA profiles from 17 pairs of matched normal and tumor samples (a subset of the 30 ESCCs) using Affymetrix U133A 2.0 arrays. In CN neutral regions, expression of 168 genes (containing 1976 SNPs) differed significantly in tumors with LOH versus tumors without LOH, including 101 genes that were up-regulated and 67 that were down-regulated.

Conclusion

Our results indicate that CNNLOH has a profound impact on gene expression in ESCC, which in turn may affect tumor development.

Background

Esophageal squamous cell carcinomas (ESCC) are usually asymptomatic and go undetected until they are incurable. Cytological screening is one strategy to detect ESCC at an early stage and has shown promise in previous studies, although improvement in sensitivity and specificity are needed. Proteases modulate cancer progression by facilitating tumor invasion and metastasis. In the current study, matrix metalloproteinases (MMPs) were studied in a search for new early detection markers for ESCC.

Methods

Protein expression levels of MMPs were measured using zymography in 24 cases of paired normal esophagus and ESCC, and in the tumor-associated stroma and tumor epithelium in one sample after laser capture microdissection (LCM). MMP-3 and MMP-10 transcripts in both the epithelium and stroma in five cases were further analyzed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).

Results

Gelatin zymography showed bands corresponding in size to MMP-2, MMP-3, MMP-9, and MMP-10 enzymes in each of the 24 cancer cases. MMP levels tended to be higher in tumors than paired normal tissue; however, only the 45 kDa band that corresponds to the activated form of MMP-3 and MMP-10 was strongly expressed in all 24 tumors with little or no expression in the paired normal foci. LCM-based analysis showed the 45 kDA band to be present in both the stromal and epithelial components of the tumor microenvironment, and that MMP-3 and MMP-10 mRNA levels were higher in tumors than paired normal tissues for each compartment.

Conclusions

Increased levels of MMPs occur in ESCC suggesting their up-regulation is important in esophageal tumorigenesis. The up-regulated gene products have the potential to serve as early detection markers in the clinic.

Objective

To investigate the risk of uterine fibroids and other reproductive risk factors in women with hereditary leiomyomatosis and renal cell cancer (HLRCC).

Design

Case-control study.

Setting

National Institutes of Health, Rockville, Maryland.

Patients

A family-based case-control study was conducted between July 1, 2004, and June 30, 2006, including 105 women from families with HLRCC ascertained throughout North America. A telephone interview was conducted with all participants using a standardized questionnaire that elicited information about their menstrual, pregnancy, uterine fibroid, and hormonal contraceptive use history. Diagnosis of uterine fibroids was confirmed by pathologic diagnosis and by medical record review. DNA was extracted from blood samples and was screened for germline mutations in the fumarate hydratase (FH) gene.

Main Outcome Measures

FH germline mutation status, presence of uterine fibroids, age at diagnosis, and symptoms and treatment of uterine fibroids.

Results

Of 105 women, 77 reported a history of uterine fibroids. Regardless of uterine fibroid status, 75 of 105 women had a germline mutation in FH (FHmut positive). The risk of uterine fibroids in FHmut-positive women was statistically significantly increased compared with that in FHmut-negative women (odds ratio [OR], 7.6; 95% confidence interval [CI], 2.9–20.0), as it was among women clinically affected with HLRCC compared with those clinically unaffected with HLRCC (8.6; 3.1–24.0). The median age at uterine fibroid diagnosis for FHmut-positive women (28 years) was significantly younger than that for FHmut-negative women (38 years) (P=.03). Women with a germline mutation in FH or clinically affected with HLRCC reported younger age at menarche (P < .004) compared with FHmut-negative women (P =.02) or women who were clinically unaffected with HLRCC. Women with HLRCC were more likely to have had treatment for uterine fibroids (OR, 4.6; 95% CI, 1.4–15.8), including hysterectomy (P=.02) at an earlier age compared with women who were clinically unaffected with HLRCC.

Conclusions

This study provides the first evidence (to our knowledge) that women with germline mutations in FH and with clinical HLRCC have an increased risk of developing uterine fibroids. These women also have a younger age at uterine fibroid diagnosis and are more likely to have treatment for uterine fibroids at a younger age than women without HLRCC in their families.