Germline Cancer Testing in Unselected Patients with Gastric and Esophageal Cancers: A Multi-center Prospective Study.

BACKGROUND AND AIMS: To determine prevalence and clinical utility of pathogenic germline variants (PGV) in gastric and esophageal cancer patients using universal genetic testing approach. METHODS: We undertook a prospective study of germline sequencing using an > 80 gene next-generation sequencing platform among patients with gastric and esophageal cancers receiving care at Mayo Clinic Cancer Center between April 1, 2018, and March 31, 2020. Patients were not selected based on cancer stage, family history of cancer, ethnicity, or age. Family cascade testing was offered at no cost. RESULTS: A total of 96 patients were evaluated. Median age was 66 years, 80.2% were male, 89.6% were white. Nearly 39% of the cohort had esophageal cancer, 35.4% gastric cancer and 26% gastroesophageal junction cancers. Approximately half (52%) of the patients had metastatic disease. Pathogenic germline variants (PGV) were detected in 15.6% (n = 15) patients. The prevalence of PGV was 10.8% in esophageal cancer, 17.6% in gastric cancer and 20% in gastroesophageal cancer. Eighty percent of patients with a positive result would not have been detected by screening with standard guidelines for genetic testing. Most PGV detected included genes with high and moderate penetrance related to DNA damage response including BRCA1, BRCA2, PALB2 and ATM. CONCLUSIONS: Universal multi-gene panel testing in gastric and esophageal cancers was associated with detection of heritable mutations in 15% of patients. The majority of PGV would not be detected with current screening guidelines and are related to DNA damage response.

DNA damage-induced ephrin-B2 reverse signaling promotes chemoresistance and drives EMT in colorectal carcinoma harboring mutant p53.

Mutation in the TP53 gene positively correlates with increased incidence of chemoresistance in different cancers. In this study, we investigated the mechanism of chemoresistance and epithelial-to-mesenchymal transition (EMT) in colorectal cancer involving the gain-of-function (GOF) mutant p53/ephrin-B2 signaling axis. Bioinformatic analysis of the NCI-60 data set and subsequent hub prediction identified EFNB2 as a possible GOF mutant p53 target gene, responsible for chemoresistance. We show that the mutant p53-NF-Y complex transcriptionally upregulates EFNB2 expression in response to DNA damage. Moreover, the acetylated form of mutant p53 protein is recruited on the EFNB2 promoter and positively regulates its expression in conjunction with coactivator p300. In vitro cell line and in vivo nude mice data show that EFNB2 silencing restores chemosensitivity in mutant p53-harboring tumors. In addition, we observed high expression of EFNB2 in patients having neoadjuvant non-responder colorectal carcinoma compared with those having responder version of the disease. In the course of deciphering the drug resistance mechanism, we also show that ephrin-B2 reverse signaling induces ABCG2 expression after drug treatment that involves JNK-c-Jun signaling in mutant p53 cells. Moreover, 5-fluorouracil-induced ephrin-B2 reverse signaling promotes tumorigenesis through the Src-ERK pathway, and drives EMT via the Src-FAK pathway. We thus conclude that targeting ephrin-B2 might enhance the therapeutic potential of DNA-damaging chemotherapeutic agents in mutant p53-bearing human tumors.

HDAC8 and STAT3 repress BMF gene activity in colon cancer cells.

Histone deacetylase (HDAC) inhibitors are undergoing clinical trials as anticancer agents, but some exhibit resistance mechanisms linked to anti-apoptotic Bcl-2 functions, such as BH3-only protein silencing. HDAC inhibitors that reactivate BH3-only family members might offer an improved therapeutic approach. We show here that a novel seleno-α-keto acid triggers global histone acetylation in human colon cancer cells and activates apoptosis in a p21-independent manner. Profiling of multiple survival factors identified a critical role for the BH3-only member Bcl-2-modifying factor (Bmf). On the corresponding BMF gene promoter, loss of HDAC8 was associated with signal transducer and activator of transcription 3 (STAT3)/specificity protein 3 (Sp3) transcription factor exchange and recruitment of p300. Treatment with a p300 inhibitor or transient overexpression of exogenous HDAC8 interfered with BMF induction, whereas RNAi-mediated silencing of STAT3 activated the target gene. This is the first report to identify a direct target gene of HDAC8 repression, namely, BMF. Interestingly, the repressive role of HDAC8 could be uncoupled from HDAC1 to trigger Bmf-mediated apoptosis. These findings have implications for the development of HDAC8-selective inhibitors as therapeutic agents, beyond the reported involvement of HDAC8 in childhood malignancy.

Effects of acclimation temperature on thermal tolerance, locomotion performance and respiratory metabolism in Acheta domesticus L. (Orthoptera: Gryllidae).

The effects of acclimation temperature on insect thermal performance curves are generally poorly understood but significant for understanding responses to future climate variation and the evolution of these reaction norms. Here, in Acheta domesticus, we examine the physiological effects of 7-9 days acclimation to temperatures 4 degrees C above and below optimum growth temperature of 29 degrees C (i.e. 25, 29, 33 degrees C) for traits of resistance to thermal extremes, temperature-dependence of locomotion performance (jumping distance and running speed) and temperature-dependence of respiratory metabolism. We also examine the effects of acclimation on mitochondrial cytochrome c oxidase (CCO) enzyme activity. Chill coma recovery time (CRRT) was significantly reduced from 38 to 13min with acclimation at 33-25 degrees C, respectively. Heat knockdown resistance was less responsive than CCRT to acclimation, with no significant effects of acclimation detected for heat knockdown times (25 degrees C: 18.25, 29 degrees C: 18.07, 33 degrees C: 25.5min). Thermal optima for running speed were higher (39.4-40.6 degrees C) than those for jumping performance (25.6-30.9 degrees C). Acclimation temperature affected jumping distance but not running speed (general linear model, p=0.0075) although maximum performance (U(MAX)) and optimum temperature (T(OPT)) of the performance curves showed small or insignificant effects of acclimation temperature. However, these effects were sensitive to the method of analysis since analyses of T(OPT), U(MAX) and the temperature breadth (T(BR)) derived from non-linear curve-fitting approaches produced high inter-individual variation within acclimation groups and reduced variation between acclimation groups. Standard metabolic rate (SMR) was positively related to body mass and test temperature. Acclimation temperature significantly influenced the slope of the SMR-temperature reaction norms, whereas no variation in the intercept was found. The CCO enzyme activity remained unaffected by thermal acclimation. Finally, high temperature acclimation resulted in significant increases in mortality (60-70% at 33 degrees C vs. 20-30% at 25 and 29 degrees C). These results suggest that although A. domesticus may be able to cope with low temperature extremes to some degree through phenotypic plasticity, population declines with warmer mean temperatures of only a few degrees are likely owing to the limited plasticity of their performance curves.

Referral and ascertainment bias in patients with synchronous and metachronous endometrial malignancy.

The purpose of this study was to evaluate the frequency in patients with endometrial cancer of other malignancies and the influence of referral and ascertainment biases on these associations. Analysis of 1,028 local and referred patients who had a hysterectomy for endometrial cancer was based on residence at the time of diagnosis. Altogether, 208 patients had a history of another malignancy, most frequently breast, colon, and ovary. At the time of surgery for endometrial cancer, the prevalence of lymphoma and breast and ovarian cancers was greater than expected although the higher prevalence of lymphoma was limited to referred patients. During follow-up after hysterectomy, the incidence of lung cancer was lower than expected, whereas the incidence of lymphoma was higher. Breast, colorectal, and bladder cancers were more common than expected although this finding was limited to local patients. We concluded that results of epidemiologic studies from tertiary care centers may be misleading if they do not account for referral and ascertainment biases.

STK11 status and intussusception risk in Peutz-Jeghers syndrome.

BACKGROUND: Peutz-Jeghers syndrome (PJS) is caused by germline STK11 mutations and characterised by gastrointestinal polyposis. Although small bowel intussusception is a recognised complication of PJS, risk varies between patients. OBJECTIVE: To analyse the time to onset of intussusception in a large series of PJS probands. METHODS: STK11 mutation status was evaluated in 225 PJS probands and medical histories of the patients reviewed. RESULTS: 135 (60%) of the probands possessed a germline STK11 mutation; 109 (48%) probands had a history of intussusception at a median age of 15.0 years but with wide variability (range 3.7 to 45.4 years). Median time to onset of intussusception was not significantly different between those with identified mutations and those with no mutation detected, at 14.7 years and 16.4 years, respectively (log-rank test of difference, chi(2) = 0.58, with 1df; p = 0.45). Similarly no differences were observed between patient groups on the basis of the type or site of STK11 mutation. CONCLUSIONS: The risk of intussusception in PJS is not influenced by STK11 mutation status.

The APC E1317Q variant in adenomatous polyps and colorectal cancers.

Genetic susceptibility may play a role in many colorectal cancers (CRCs). Known syndromes such as familial adenomatous polyposis and hereditary nonpolyposis CRC account for <5% of CRCs. The germ-line missense variant of the APC gene, E1317Q, has been proposed to confer a risk for colonic adenomatous polyps (adenomas), but not for CRCs in the general population. These findings are contradictory and controversial. In the present study, 608 cases (377 patients with CRC, 145 patients with 4-100 lifetime adenomas, and 86 with < or =3 lifetime adenomas), and 679 controls (362 spouses and 317 patients with normal colonoscopy) were screened for the APC E1317Q variant. The frequency of heterozygotes for E1317Q among patients with CRC (2.4%), patients with 4-100 adenomas (1.4%), and < or =3 adenomas (3.5%) did not differ from spouse controls (2.8%). When CRC patients were examined by DNA mismatch repair status, age at onset (< or =age 50 versus >50), or family history of CRC, no differences in the frequency of E1317Q were found. The APC variant E1317Q does not appear to be associated with increased risk for colorectal neoplasia in the general population. However, when we used normal colonoscopy controls (E1317Q carrier frequency = 0.3%), the prevalence of E1317Q was significantly increased in CRC patients, in patients with < or =3 adenomas, and in CRC patients with intact mismatch repair status, suggesting a possible role for E1317Q in colorectal tumorigenesis. These results underscore the importance of carefully defining the controls to be used in comparisons of allele frequencies.

Desmoid tumors in familial adenomatous polyposis: a pilot project evaluating efficacy of treatment with pirfenidone.

OBJECTIVES: Pirfenidone (Deskar, Marnac Inc., Dallas, TX), 5-methyl-1-phenyl-2-(1H)-pyridone, is a broad-spectrum, noncytotoxic, oral antifibrotic agent that is reported to inhibit or block the action of cytokine growth factors: transforming growth factor beta1, platelet-derived growth factor, epidermal growth factor, and fibroblast growth factor, and to prevent formation of new fibrotic lesions. METHODS: We enrolled 10 women and four men with extensive familial adenomatous polyposis (FAP)-associated desmoid disease in a 2-yr open-label treatment trial with oral pirfenidone. Imaging of desmoids was conducted at baseline and 6, 12, and 24 months. RESULTS: No drug toxicity or drug intolerance was encountered. Seven patients dropped out (three because of progressive disease), and seven continued for at least 18 months. Of those that continued, two had partial but significant reduction in the size of all desmoids beginning in the first 6 months of treatment, and two others experienced relief of symptoms without change in desmoid size. Three patients experienced no change in tumor size or symptoms. CONCLUSIONS: Pirfenidone is well tolerated by patients with FAP-associated desmoid tumors. Some patients with FAP/desmoid tumors treated with pirfenidone had regression of tumors, some had progression, and some had no response. Patients with rapidly growing tumors did not respond to pirfenidone. A placebo-controlled trial is needed to determine whether there is a subset of patients for whom pirfenidone may result in partial shrinkage of desmoid tumors, because the natural history of desmoid tumors is not predictable or understood.

A comparison of endoscopic ultrasound, magnetic resonance imaging, and exam under anesthesia for evaluation of Crohn's perianal fistulas.

BACKGROUND & AIMS: To determine accuracy of endoscopic ultrasound (EUS) and magnetic resonance imaging (MRI) for evaluation of Crohn's disease perianal fistulas. METHODS: Thirty-four patients with suspected Crohn's disease perianal fistulas were prospectively enrolled in a blinded study comparing EUS, MRI, and examination under anesthesia (EUA). Fistulas were classified according to Parks' criteria, and a consensus gold standard was determined for each patient. Acceptable accuracy was defined as agreement with the consensus gold standard for > or =85% of patients. RESULTS: Three patients did not undergo MRI; 1 did not undergo EUS or EUA; and consensus could not be reached for 1. Thirty-two patients had 39 fistulas (20 trans-sphincteric, 5 extra-sphincteric, 6 recto-vaginal, 8 others) and 13 abscesses. The accuracy of all 3 modalities was > or =85%: EUS 29 of 32 (91%, confidence interval [CI] 75%-98%), MRI 26 of 30 (87%, CI 69%-96%), and EUA 29 of 32 (91%, CI 75%-98%). Accuracy was 100% when any 2 tests were combined. CONCLUSIONS: EUS, MRI, and EUA are accurate tests for determining fistula anatomy in patients with perianal Crohn's disease. The optimal approach may be combining any 2 of the 3 methods.

Risks and adverse outcomes of sexually transmitted diseases. Patients' attitudes and beliefs.

OBJECTIVE: To determine women's awareness and level of understanding of the risks and sequelae of sexually transmitted diseases (STDs). STUDY DESIGN: The study was a cross-sectional survey of 103 women seen at an urban center. We recruited women from several clinical sites: (1) patients with signs or symptoms of an STD seen in a busy women's urgent care unit, (2) women enrolled in clinical studies for STDs or bacterial vaginosis, (3) patients admitted to the hospital and found to have an STD, and (4) women at high risk based on age and sexual activity. A trained female interviewer administered the survey to women agreeing to participate. The questionnaire assessed the patient's awareness of various STDs and their association with adverse outcomes. The survey also evaluated women's knowledge regarding STD prevention. Descriptive analyses were performed using SAS (Cary, North Carolina) on the 103 patients surveyed. RESULTS: The median age of our survey population was 23, 66% were nonwhite, and 37% were on public assistance or uninsured. The population was at high risk for STDs based on the reported history of infection with Neisseria gonorrhoeae and Chlamydia trachomatis (6% and 38%, respectively). Of the 103 women surveyed, 33% had never heard of pelvic inflammatory disease (PID), and 79.6% could not identify any adverse sequelae of PID. Sixty-five percent were unaware that PID increases the risk of ectopic pregnancy, and 56.3% were unaware that it could result in chronic pelvic pain. When asked if they knew of any methods to prevent or reduce their risk of STDs, only 18% mentioned barrier contraception (condoms). Over 57% of respondents could not name a way to prevent STDs. CONCLUSION: Increased educational efforts targeted at high-risk individuals are necessary to improve knowledge of STDs and their adverse sequelae. With greater knowledge and awareness, individuals and couples may be more likely to practice protective behaviors, such as reducing high-risk exposure, and to increase preventive measures, such as barrier contraception use.

A search for germline APC mutations in early onset colorectal cancer or familial colorectal cancer with normal DNA mismatch repair.

Twenty percent of colorectal cancers (CRCs) arise in people who have a family history of CRC in at least one other relative. Although a fraction of these CRCs are explained by two well-described autosomal dominant syndromes-5% by hereditary nonpolyposis colorectal cancer (HNPCC) and 1% by familial adenomatous polyposis (FAP)-the cause of the remaining 14% of familial aggregates of CRC is unknown. Many cases of HNPCC are due to germline mutations in DNA mismatch repair genes, leading to the tumor phenotype of microsatellite instability (MSI), and most cases of FAP are caused by germline APC mutations. To date, non-FAP familial CRC aggregates have not been evaluated for germline APC mutations. In this study, we examined the involvement of germline APC mutations in 79 individuals with CRC who had early-age onset of their cancer (age < 50 years) and/or a family history of CRC. Cases with FAP or HNPCC due to defective mismatch repair were excluded from the study. Using conformation-sensitive gel electrophoresis and the protein truncation test as the screening methods, no functionally significant germline mutations were detected for any of the cases. An apparently silent polymorphism resulting in a 1-bp alteration of A --> G (proline --> proline) in exon 4 was observed. Additionally, four intervening sequence (IVS) alterations were detected: IVS2-53t-->c in 3 cases; IVS4-17ins T in 3 cases; IVS5+32t-->c in 16 cases; and IVS5+33g-->a in 1 case. All appeared to be polymorphisms present in similar proportions in an average-risk population. We conclude that germline APC mutations do not account for familial MSS (stable microsatellite) CRC associated with few synchronous polyps.

Association of Peutz-Jeghers-like mucocutaneous pigmentation with breast and gynecologic carcinomas in women.

Most reports describe an increased risk of malignancy in Peutz-Jeghers syndrome (PJS). We identified individuals with PJS-like pigmentation but no polyposis, designated as isolated mucocutaneous melanotic pigmentation (IMMP), and 1) characterized their clinical features, 2) assessed them for cancer events, and 3) screened a sample of these subjects for mutations in LKB1, a gene responsible for a portion of PJS cases. Review of Mayo Clinic records from 1945 to 1996 identified 26 patients with IMMP. All were then interviewed or their medical records reviewed to determine if cancer had developed. Conformation-sensitive gel electrophoresis (CSGE) screening for LKB1 mutations was followed by direct sequencing. Ten of these 26 individuals (38%) developed 12 malignancies that arose in the cervix (n = 3), endometrium (n = 3), breast (n = 1), kidney (n = 1), lung (n = 2), colon (n = 1), and lymphatic tissue (n = 1). In females with IMMP, the relative risk for cancer was 3.2 (95% CI, 1.2-6.9), while that for males was not increased. The relative risk for breast and gynecologic cancers was 7.8 (95% CI, 2.5-18.1) in affected females. Of 9 individuals tested, no LKB1 mutations were detected. Classical PJS is associated with an increased cancer risk. Our results indicate that IMMP is another lentiginosis with cancer predisposition. In particular, the relative risk for cancer in females with IMMP was significantly increased, as is true in females with PJS. However, LKB1 mutations did not contribute to the development of IMMP in the patients tested.

Genetic heterogeneity in Peutz-Jeghers syndrome.

LKB1, the human gene encoding a serine threonine kinase, was recently identified as a susceptibility gene for Peutz-Jeghers syndrome (PJS), a disease characterized by the constellation of intestinal hamartomata, oral mucocutaneous hyperpigmentation, and an increased risk for gastrointestinal as well as extraintestinal malignancies. To date, the majority of individuals with PJS have been found to have genetic alterations in LKB1, most of which result in protein truncation. Additionally, linkage analyses have suggested a modicum of genetic heterogeneity, with the majority of PJS families showing linkage to the LKB1 locus. In this study, we evaluated five kindreds with greater than two affected family members, five PJS probands with only one other affected family member, as well as 23 individuals with sporadic PJS for mutations within the LKB1 gene. Conformation sensitive gel electrophoresis was utilized for the initial screen, followed by direct sequence analysis for characterization. Long-range PCR was used for the detection of larger genetic insertions or deletions. Mutation analysis revealed genetic alterations in LKB1 in two probands who had a family history of PJS. LKB1 mutations were detected in only four of the remaining 23 cases of sporadic PJS. These data suggest the presence of significant genetic heterogeneity for PJS and the involvement of other loci in this syndrome.

Positive cone biopsy specimen margins in women infected with the human immunodeficiency virus.

OBJECTIVES: The purpose of this study was to compare the positive margin rate associated with cervical conization among women who are seropositive for human immunodeficiency virus with that among women who are seronegative. STUDY DESIGN: This was a cross-sectional study of 245 women who underwent cervical conization for the following indications: biopsy-proven cervical intraepithelial neoplasia grade 2 or 3, abnormal endocervical curettage specimen, cytologic-histologic examination discrepancy, persistent cervical intraepithelial neoplasia grade 1, or abnormal cytologic characteristics with inadequate colposcopic examination. RESULTS: Twenty-two (47.8%) of 46 women who were seropositive for human immunodeficiency virus and 65 (32.7%) of 199 women who were seronegative had positive cone biopsy specimen margins. In a multivariable logistic regression the human immunodeficiency virus-seropositive women had a 2-fold increased risk of having a positive cone biopsy margin (odds ratio, 2.25; 95% confidence interval, 1.07-4.76). CONCLUSION: If the presence of positive cone biopsy specimen margins represents the potential for disease progression, then our findings of a positive margin rate of nearly 50% in a human immunodeficiency virus-positive population may argue against the kind of conservative management of colposcopic follow-up that has been proposed for immunocompetent women.

Representative cDNA libraries and their utility in gene expression profiling.

An increasing interest in gene expression profiles in human diseases has led to the use of microdissected tumors and biopsies in gene discovery approaches. Since many of these clinical samples yield extremely small amounts of RNA, reproducible methods are needed to amplify this RNA while maintaining the original message profile. Using the SMART cDNA Synthesis Method, we show that high-, medium- and low-abundance transcripts can be amplified in a representative fashion and that the resulting cDNA can also be used as a complex probe to confirm gene expression differences identified by other techniques.

Erdheim-Chester disease: clinical, radiologic, and histopathologic findings in five patients with interstitial lung disease.

Erdheim-Chester disease is a clinicopathologic entity defined by a characteristic pattern of symmetric osteosclerosis caused by an infiltrate of mononuclear cells that include prominent numbers of foamy histiocytes. About half of patients have extraskeletal manifestations, including involvement of the hypothalamus/posterior pituitary, orbit, retroperitoneum, skin, lung, and heart. Pulmonary involvement is an uncommon but important manifestation of Erdheim-Chester disease because it causes significant morbidity and mortality. A review of the Mayo Clinic files produced four patients with confirmed Erdheim-Chester disease in whom lung biopsy had been performed. One additional patient was included from the University of Pittsburgh. Four patients were women. The mean age was 53.6 years (range 25-70 years). All patients had bilateral and symmetric sclerotic bone lesions characteristic of Erdheim-Chester disease, although in three the skeletal abnormalities were discovered only after lung biopsy. Four patients had dyspnea, and one also had a dry cough. One patient died 17 months after diagnosis. Chest radiographs showed diffuse interstitial infiltrates in all patients, with an upper zone predominance in three. Thoracic computed tomography (CT) scans showed thickening of the visceral pleura and interlobular septa with patchy associated fine reticular and centrilobular opacities and ground glass attenuation. Lung biopsy specimens showed an infiltrate of foamy histiocytes, lymphocytes, and scattered Touton giant cells with associated fibrosis in a striking lymphatic distribution. The infiltrate involved visceral pleura, interlobular septa, and bronchovascular bundles. Immunohistochemical stains were positive for CD68 in all cases and S-100 protein in four cases. Stains for CD1a were consistently negative. Ultrastructural studies in one case showed no Birbeck granules. Although in bone the histologic features of Erdheim-Chester disease may overlap with Langerhans' cell histiocytosis, its expression in the lung is distinct. Lung involvement in Erdheim-Chester disease has emerged as a unique radiographic and histologic entity.