In vitro and in vivo correlates of physiological and neoplastic human Fallopian tube stem cells.

High-grade serous cancer (HGSC) progresses to advanced stages without symptoms and the 5-year survival rate is a dismal 30%. Recent studies of ovaries and Fallopian tubes in patients with BRCA1 or BRCA2 mutations have documented a pre-metastatic intramucosal neoplasm that is found almost exclusively in the Fallopian tube, termed 'serous tubal intraepithelial carcinoma' or STIC. Moreover, other proliferations, termed p53 signatures, secretory cell outgrowths (SCOUTs), and lower-grade serous tubal intraepithelial neoplasms (STINs) fall short of STIC but share similar alterations in expression, in keeping with an underpinning of genomic disturbances involved in, or occurring in parallel with, serous carcinogenesis. To gain insight into the cellular origins of this unique tubal pathway to high-grade serous cancer, we cloned and both immortalized and transformed Fallopian tube stem cells (FTSCs). We demonstrated that pedigrees of FTSCs were capable of multipotent differentiation and that the tumours derived from transformed FTSCs shared the histological and molecular features of HGSC. We also demonstrated that altered expression of some biomarkers seen in transformed FTSCs and HGSCs (stathmin, EZH2, CXCR4, CXCL12, and FOXM1) could be seen as well in immortalized cells and their in vivo counterparts SCOUTs and STINs. Thus, a whole-genome transcriptome analysis comparing FTSCs, immortalized FTSCs, and transformed FTSCs showed a clear molecular progression sequence that is recapitulated by the spectrum of accumulated perturbations characterizing the range of proliferations seen in vivo. Biomarkers unique to STIC relative to normal tubal epithelium provide a basis for novel detection approaches to early HGSC, but must be viewed critically given their potential expression in lesser proliferations. Perturbations shared by both immortalized and transformed FTSCs may provide unique early targets for prevention strategies. Central to these efforts has been the ability to clone and perpetuate multipotent FTSCs.

Tumor profiling and the incidentalome: patient decisions and risks.

In recent years, the field of oncology has witnessed rapid advancements in genetic sequencing simultaneously with steeply declining costs of sequencing technology. As a result, genomics-driven cancer medicine and the use of tumor profiling are quickly becoming mainstays of cancer therapy. Oncology patients can benefit from tumor profiling by allowing the selection of targeted therapies tailored to their disease. However, it is increasingly recognized that the process of determining a tumor DNA sequence may lead to incidental discovery of underlying germline mutations which can impact other aspects of a patient's health, and that of their family. How to handle the 'incidentalome' has been the subject of recent public debate, yet patient education about the potential risks of tumor profiling remains sparse. Patient perspectives and clinical implications of the tumor incidentalome must be specifically addressed by the oncology community as tumor profiling expands to become a new standard of care.

The impact of tissue block sampling on the detection of p53 signatures in fallopian tubes from women with BRCA 1 or 2 mutations (BRCA+) and controls.

The tubal p53 signature is a putative precursor to pelvic serous carcinoma, but its frequencies in women with inherited mutations in the BRCA1 or BRCA2 genes (BRCA+) and controls has been controversial. An initial section and two levels (100-200 µm) from every block in BRCA+ (24) and control tubes (40) were stained for p53. The frequency of p53 signatures was computed between the populations and across the three levels from each block, and analyzed by Fisher exact test. A total of 17 (71%) BRCA+ and 20 (50%) control tubes were p53 signature positive (P=0.12); 21 and 16% of all tissue blocks sectioned harbored signatures (P=0.29), and 76 and 67% were found in the fimbria. In 49 and 32% of p53 signature positive cases in the two groups, the p53 signatures were not discovered until the second or third round of sectioning. In all, 38 and 40% of BRCA+ and control subjects harbored p53 signatures in more than one focus in a single block. In one case (BRCA+), a highly atypical proliferation was identified in one serial section. The p53 signatures are more common than previously reported and the frequency of detection increases as a function of sectioning through the tissue block, both in absolute frequency and in numbers of p53 signatures detected in a given block. There is a trend for a higher absolute frequency of p53 signatures (71 vs 50%; P=0.12) in BRCA+ subjects, but this is not reflected in a greater average number of p53 signatures or positive blocks per case. This study underscores the importance of systematic immunohistochemical examination of fallopian tubes when conducting epidemiological studies that compare the frequency of p53 signatures in different populations. Attention to this detail is critical when exploring risk factors germane to early serous carcinogenesis.

Secretory cell outgrowth, PAX2 and serous carcinogenesis in the Fallopian tube.

The 'p53 signature' is a benign secretory cell outgrowth in the distal Fallopian tube that shares properties with ovarian serous cancer-including p53 mutations-and is a putative serous cancer precursor. We expanded the precursor definition to all secretory cell outgrowths (SCOUTs) of 30 or more cells and scored normal (N) and altered (A) expression of both p53 and PAX2, a gene down-regulated in ovarian and endometrial cancer. SCOUTs were identified by BCL2/p73 staining in tubes from women with serous carcinoma, inherited mutations in BRCA1 or BRCA2 and controls. SCOUTs were prevalent in both proximal and distal tube and significantly associated with serous carcinoma versus the others (p < 0.001); 89% were PAX2 (A) and 26% were PAX2 (A)/p53 (A) (p53 signatures). PAX2 (A)/p53 (N) SCOUTs were free of p53 mutations; however, 12 of 13 p53 signatures were PAX2 (A). A tubal carcinoma and contiguous SCOUT were p53 (A)/PAX2 (A) and shared the same p53 mutation. SCOUTs are discretely localized alterations commonly containing altered expression of multiple genes within histologically benign tubal epithelium. Geographic distribution in the tube varies by genotype and immunophenotype, from regionally unrestricted (PAX2) to greater likelihood specific area (fimbria) of shared prevalence (PAX2 and p53). This study reveals, for the first time, an entity (SCOUT) that is associated with serous cancer, expands the topography of altered PAX2 expression in the female genital tract mucosa and highlights another potential pathway disturbance involved in early serous carcinogenesis in the Fallopian tube.

The Li-Fraumeni syndrome (LFS): a model for the initiation of p53 signatures in the distal Fallopian tube.

A candidate early precursor to pelvic serous cancer, the 'p53 signature', is commonly found in the benign mucosa of the distal Fallopian tube and harbours p53 mutations and evidence of DNA damage. We examined tubes from women with pre-existing (germ-line) mutations in p53 [Li-Fraumeni syndrome (LFS)] for evidence of this precursor. Fallopian tubes from two cases of LFS were immunostained for p53, Ki-67 (proliferation) and H2AX (DNA damage response) and analysed for p53 mutations by laser capture microdissection (LCM) and p53 genomic sequencing (exons 2-11). A common single nucleotide repeat (snp) in exon 3 (rs1042522) and deletion sequencing chromatograms in exon 4 were examined in combination to estimate LOH in both LFS tubes and advanced serous carcinomas from the general population. LFS tubal epithelium contained abundant (10-20 per section) p53 signatures with evidence of DNA damage and low proliferative activity. Six of 11 LFS microdissected p53 signatures (55%) and 15 of 21 serous carcinomas (71%) revealed LOH at the p53 locus, relative to background epithelium. The LFS model confirms prior observations that the distal Fallopian tube is particularly prone to focal epithelial p53 gene inactivation-p53 mutation and LOH-in the absence of malignancy or increased epithelial proliferation. The fact that the LFS is not associated with ovarian cancers is consistent with the concept that loss of p53 function must be accompanied by at least one more genotoxic event (including BRCA1/2 functional inactivation) to produce the malignant phenotype. This is in keeping with a general model of carcinogenesis, in which different and often independent risk factors operate at multiple points in the serous carcinogenic spectrum.

Differentiated vulvar intraepithelial neoplasia contains Tp53 mutations and is genetically linked to vulvar squamous cell carcinoma.

Differentiated vulvar intraepithelial neoplasia is a unique precursor to vulvar squamous cell carcinoma that is typically HPV-negative and frequently associated with nuclear p53 staining. These features imply a mode of pathogenesis involving somatic mutations. However, the genetic relationship of differentiated vulvar intraepithelial neoplasm and vulvar squamous cell carcinoma and the role of Tp53 mutations in this process have not been resolved. We analyzed 11 differentiated vulvar intraepithelial neoplasms and 6 associated vulvar squamous cell carcinomas. Sections were stained for p53 and p63 and DNA from multiple epithelial sites, representing normal control tissues (n=10), differentiated vulvar intraepithelial neoplasias (n=18), and vulvar squamous cell carcinomas (n=6), were obtained by laser capture microdissection, and sequenced for exons 2-11 of Tp53. Six of 10 cases contained at least one Tp53 mutation-positive differentiated vulvar intraepithelial neoplasia focus; 4 strongly p53 immuno-positive and 2 negative. Staining for p53 and p63 co-localized, targeting the immature epithelium, but surface epithelium was Tp53 mutation-positive. Four of five vulvar squamous cell carcinomas were Tp53 mutation-positive; two shared identical Tp53 mutation with adjacent differentiated vulvar intraepithelial neoplasia. Disparate foci of differentiated vulvar intraepithelial neoplasia often showed different mutations consistent with multiple neoplastic clones. Differentiated vulvar intraepithelial neoplasia is, with few exceptions, associated with Tp53 mutations and will be p53 immunopositive when missense mutations (versus some nonsense and all deletion mutations) are present. Multiple Tp53 mutations in different sites supports the presence of multiple independent genetic events, but shared Tp53 mutations in both differentiated vulvar intraepithelial neoplasia and vulvar squamous cell carcinoma support a genetic relationship between the two. The confinement of p53 staining to immature cell nuclei is consistent with maturation-dependent degradation of mutant p53 protein.

High-grade fimbrial-ovarian carcinomas are unified by altered p53, PTEN and PAX2 expression.

High-grade endometrioid and serous carcinomas of the ovary and fallopian tube are responsible for the majority of cancer deaths and comprise a spectrum that includes early or localized (tubal intraepithelial carcinoma) and advanced (invasive or metastatic) disease. We subdivided a series of these tumors into three groups, (1) classic serous, (2) mixed serous and endometrioid and (3) endometrioid carcinomas and determined: (1) the frequencies of coexisting tubal intraepithelial carcinoma, (2) frequency of a dominant ovarian mass suggesting an ovarian origin and (3) immuno-localization of WT-1, p53, PTEN, PAX2 and p16(ink4). All tumors were analyzed for p53 mutations. Thirty six, 25 and 8% of groups 1-3 were associated with tubal intraepithelial carcinoma (P=0.09) and 34, 45 and 62% predominated in one ovary (P=0.028), respectively. Differences in frequencies of diffuse p53 immunostaining (85-93%), WT-1 (70-98%) and p16(ink4) positivity (69-75%) were not significant for all groups. Greater than 95% reduction in PAX2 and PTEN occurred in 67-75 and 5-12%, respectively; however, PAX2 and PTEN staining intensity, when present, was often heterogeneous, highlighting different tumor populations. PAX2 and PTEN expression were markedly reduced or absent in 12 of 12 and 4 of 12 tubal intraepithelial carcinomas. In summary, high-grade müllerian carcinomas share identical frequencies of altered or reduced expression of p53, PTEN and PAX2, all of which can be appreciated in tubal intraepithelial carcinomas. Because only a subset of these tumors appears to arise in the fallopian tube, attention to expression of these biomarkers in the ovary and other müllerian sites might facilitate the identification of other carcinogenic pathways. PAX2 and PTEN, in addition to p53 and p16(ink4), comprise a potentially important gene combination in high-grade pelvic carcinogenesis.

A pathologist's road map to benign, precancerous, and malignant intraepithelial proliferations in the fallopian tube.

The fallopian tube has recently emerged as an important site of origin for not only early serous cancer in women with inherited mutations in BRCA1/BRCA2 but as a source of many pelvic serous carcinomas. With this increased attention has come the inevitable need to sort out what epithelial abnormalities are clinically important and how they should be reported by the practicing pathologist. This review addresses 4 categories of tubal epithelial change: (1) metaplasias; (2) nonmalignant atypias; (3) potential precursors, including secretory cell outgrowths and p53 signatures; and (4) tubal intraepithelial carcinomas. A modified protocol for sectioning the fallopian tube (SEE-FIM) is discussed and each of the above topics is covered in the context of its differential diagnosis and recommendations for reporting are included. Finally, the rationale for close inspection of the tube, both in presumed benign and malignant disease, is discussed, with reference to an ongoing multi-institutional web-based project (Pelvic-ovarian Cancer Interception project).

Diet, Physical Activity, and Body Weight in Cancer Survivorship.

Diet, physical activity, and body weight have been shown to play an important role in cancer survivorship. The impact of each of these lifestyle factors differs slightly among cancer types, and adherence to recommended diet and physical activity guidelines has been associated with positive outcomes, including decrease in the risk of cancer recurrence and improvement of quality of life. Although there are compelling data that appropriate diet, physical activity, and body weight have beneficial effects in cancer survivorship, additional trials are needed to understand the relationship.

STICS, SCOUTs and p53 signatures; a new language for pelvic serous carcinogenesis.

The events leading to the most common and most lethal ovarian carcinoma - high grade serous carcinoma - have been poorly understood. However, the detailed pathologic study of asymptomatic women with germ-line BRCA 1 or BRCA2 (BCRA+) mutations has unearthed an early malignancy, serous tubal intraepithelial carcinomas (STIC), which has linked many peritoneal and ovarian serous carcinomas to the fimbria. The distinction between high-grade serous and endometrioid carcinomas continues to narrow, with shared alterations in expression of pTEN, PAX2 and p53. Moreover, the discovery of clonal alterations in p53 in benign tubal epithelium, - p53 signatures - has established a foundation for a serous cancer precursor in the fimbria. We have expanded this concept to include a generic secretory cell outgrowth (SCOUT) in the fallopian tube that is associated with altered PAX2 expression. As the repertoire of gene alterations is expanded and its link to serous carcinogenesis clarified, a cogent pathway to high-grade Mullerian carcinomas will emerge. This will challenge conventional thinking about ovarian carcinogenesis but will provide a new template for studies of ovarian cancer prevention.

Polymerase chain reaction detection of HPV in squamous carcinoma of the oropharynx.

Human papillomavirus (HPV) testing is routinely performed on oropharyngeal carcinomas. We compared the Access Genetics (Minneapolis, MN) polymerase chain reaction (PCR) assay (AGPCR), DNA-DNA in situ hybridization (ISH; Ventana, Tucson, AZ), and HPV-16 E7 PCR amplification in consecutively accessioned oropharyngeal cancers. We tested 126 cases by both PCR methods; 102 were positive by either for a maximum positive rate (MPR) of 81.0%. Relative to the MPR, the sensitivities of AGPCR and E7 PCR were 90.2% and 72.5%, respectively. Of 17 AGPCR+ cases tested by ISH, 14/14 unequivocally positive/negative were concordant. All cases (97/97) positive by either PCR assay were positive for p16. There was no relationship between level of histologic differentiation and HPV status. ISH and AGPCR have comparable performance for the detection of HPV in oropharyngeal carcinomas. PCR is a suitable and economical assay that is comparable to ISH in sensitivity and may provide logistical advantages relative to ISH for assessing HPV status in oropharyngeal malignancies. However, it is imperative that appropriate sensitivity controls be in place for such assays.

Hypersensitivity to intravenous ondansetron: a case report.

INTRODUCTION: Ondansetron, a 5-hydroxytryptamine3 receptor antagonist widely used in the prevention and treatment of chemotherapy-induced nausea and vomiting, is associated with various unusual adverse drug reactions. In this paper, we describe a hypersensitivity reaction to a single intravenous dose of ondansetron. CASE PRESENTATION: A 19-year-old woman presented to the emergency department of our institute with 3-4 episodes of nausea, vomiting and epigastric distress. She had a diagnosis of polycystic ovarian disease and had been on treatment with cyproterone acetate 2 mg, ethinyl estradiol 0.035 mg, finasteride 5 mg and metformin 500 mg for a month. She had been taking oral roxithromycin 500 mg per day for the past 3 days for treatment of a mild upper respiratory tract infection. She also occasionally took rabeprazole 10 mg for gastritis which had worsened after treatment with roxithromycin. She was treated with a single 4 mg dose of ondansetron intravenously. She immediately developed urticaria, which was treated with intravenous dexamethasone 4 mg and chlorpheniramine maleate 20 mg. The reaction abated within a few minutes and she was discharged within an hour. She was asymptomatic at 72 hours of follow-up.She had no history of ondansetron exposure, or drug or food allergies. On the Naranjo's causality assessment scale, the adverse event was 6 indicating a "probable" reaction to ondansetron. CONCLUSION: 5-hydroxytryptamine3 receptor antagonists have been associated with life-threatening adverse reactions such as hypotension, seizures and anaphylaxis. The wide availability of these drugs in India has promoted their off label use in the treatment of gastritis, migraine and so on. Our case represents an off label use in a patient who could have been treated with a safer drug.Some authors have suggested that anaphylaxis may be a class effect while others think it may be drug specific. In our case, the reaction could be either anaphylaxis or anaphylactoid, but the latter seems more likely given the history of absence of prior sensitization. Other components of the drug, such as solvent, also need to be considered as a cause of this reaction. Considering all of the existing evidence, we need to be more cautious while using ondansetron and also to be aware of the various unusual side effects, especially when used in an out-of-hospital set-up.Our case report underscores the importance of physicians judiciously using the drug, particularly in the outpatient setting so as to reduce the incidence of avoidable adverse drug reactions.