Oscar Lin

Circulating Tumor Cell Analysis in Patients with Progressive Castration-Resistant Prostate Cancer

Purpose: To better direct targeted therapies to the patients with tumors that express the target, there is an urgent need for blood-based assays that provide expression information on a consistent basis in real time with minimal patient discomfort. We aimed to use immunomagnetic-capture technology to isolate and analyze circulating tumor cells (CTC) from small volumes of peripheral blood of patients with advanced prostate cancer. Experimental Design: Blood was collected from 63 patients with metastatic prostate cancer. CTCs were isolated by the Cell Search system, which uses antibodies to epithelial cell adhesion marker and immunomagnetic capture. CTCs were defined as nucleated cells positive for cytokeratins and negative for CD45. Captured cells were analyzed by immunofluorescence, Papanicolau staining, and fluorescence in situ hybridization. Results: Most patients (65%) had 5 or more CTCs per 7.5 mL blood sample. Cell counts were consistent between laboratories (c = 0.99) and did not change significantly over 72 or 96 h of storage before processing (c = 0.99). Their identity as prostate cancer cells was confirmed by conventional cytologic analysis. Molecular profiling, including analysis of epidermal growth factor receptor (EGFR) expression, chromosome ploidy, and androgen receptor (AR) gene amplification, was possible for all prostate cancer patients with ≥5 CTCs. Conclusions: The analysis of cancer-related alterations at the DNA and protein level from CTCs is feasible in a hospital-based clinical laboratory. The alterations observed in EGFR and AR suggest that the methodology may have a role in clinical decision making.

Prevalence and Co-Occurrence of Actionable Genomic Alterations in High-Grade Bladder Cancer

PURPOSE We sought to define the prevalence and co-occurrence of actionable genomic alterations in patients with high-grade bladder cancer to serve as a platform for therapeutic drug discovery. PATIENTS AND METHODS An integrative analysis of 97 high-grade bladder tumors was conducted to identify actionable drug targets, which are defined as genomic alterations that have been clinically validated in another cancer type (eg, BRAF mutation) or alterations for which a selective inhibitor of the target or pathway is under clinical investigation. DNA copy number alterations (CNAs) were defined by using array comparative genomic hybridization. Mutation profiling was performed by using both mass spectroscopy-based genotyping and Sanger sequencing. RESULTS Sixty-one percent of tumors harbored potentially actionable genomic alterations. A core pathway analysis of the integrated data set revealed a nonoverlapping pattern of mutations in the RTK-RAS-RAF and phosphoinositide 3-kinase/AKT/mammalian target of rapamycin pathways and regulators of G1-S cell cycle progression. Unsupervised clustering of CNAs defined two distinct classes of bladder tumors that differed in the degree of their CNA burden. Integration of mutation and copy number analyses revealed that mutations in TP53 and RB1 were significantly more common in tumors with a high CNA burden (P < .001 and P < .003, respectively). CONCLUSION High-grade bladder cancer possesses substantial genomic heterogeneity. The majority of tumors harbor potentially tractable genomic alterations that may predict for response to target-selective agents. Given the genomic diversity of bladder cancers, optimal development of target-specific agents will require pretreatment genomic characterization.

Fluorescence in situ Hybridization Analysis of Circulating Tumor Cells in Metastatic Prostate Cancer

Purpose: To assess the feasibility of characterizing gene copy number alteration by fluorescence in situ hybridization (FISH) of circulating tumor cells (CTC) isolated using the CellSearch system in patients with progressive castration-resistant metastatic prostate cancer. Experimental Design: We used probe combinations that included the androgen receptor (AR) and MYC genes for FISH analysis of CTC samples collected from 77 men with castration-resistant metastatic prostate cancer. Results: High-level chromosomal amplification of AR was detected in 38% and relative gain of MYC in 56% of samples analyzed. No such abnormalities were detected in samples with CTC counts of <10, reflecting ascertainment difficulty in these lower count samples. Conclusion: The CTC isolated from our patient cohort present a very similar molecular cytogenetic profile to that reported for late-stage tumors and show that FISH analysis of CTC can be a valuable, noninvasive surrogate for routine tumor profiling. That as many as 50% of these patients have substantial amplification of the AR locus indicates that androgen signaling continues to play an important role in late-stage prostate cancer.

Role of KRAS and BRAF gene mutations in mucinous ovarian carcinoma

OBJECTIVES The goals of this study were to perform a comprehensive assessment of the prevalence of KRAS oncogene mutations in invasive epithelial ovarian carcinomas of various histologic subtypes, and for any subgroup(s) in which KRAS mutation was found to be common, to address the hypothesis that those tumors without KRAS mutation had sustained alternative activation of this signaling pathway through mutation of the BRAF oncogene. METHODS A total of 104 primary, invasive epithelial ovarian carcinomas from a 10-year period at this institution were selected for study based on histologic classification. The histologic cell type was serous in 21 cases, endometrioid in 30 cases, clear cell in 31 cases, and mucinous in 22 cases. Additional clinical and pathological information was abstracted from patient records, and pathology review was performed for all cases. Direct sequence analysis of exon 2 of the KRAS gene, containing codon 12, was performed using DNA isolated from all tumor specimens. Sequence analyses of exons 11 and 15 of the BRAF gene were performed for the 22 cases of mucinous ovarian carcinoma. RESULTS Activating KRAS mutations were more common in mucinous tumors (50%) than in all other histologic types combined (5%; P < 10(-7)). Mutation of KRAS was more common in stage I tumors than in advanced stage tumors (P = 0.0004). Of the 11 mucinous tumors with KRAS mutations, 6 were of Mullerian (endocervical) type and 5 were of gastrointestinal type. No mucinous tumor was found to harbor a BRAF mutation. CONCLUSIONS These data indicate that KRAS oncogene mutations exist in several histologic types of invasive epithelial ovarian carcinoma, especially stage I tumors, but are common only in tumors of mucinous histology. Mutations are equally prevalent in mucinous ovarian cancers of Müllerian and gastrointestinal types. In contrast to other solid tumor types frequently affected by KRAS mutation, mucinous ovarian cancers without a KRAS mutation have not sustained alternative activation of this signaling pathway through mutation of the BRAF oncogene.

IS THERE A DIFFERENCE IN OUTCOME BETWEEN STAGE I-II ENDOMETRIAL CANCER OF PAPILLARY SEROUS/CLEAR CELL AND ENDOMETRIOID FIGO GRADE 3 CANCER?

PURPOSE Several reports in the literature have shown that, compared with endometrioid adenocarcinoma, patients with papillary serous (PS) and clear cell (CC) histologic features do worse. However, it is unclear whether the outcome of PS/CC cancer is different from that of poorly differentiated endometrioid cancer. The purpose of this study was to compare the outcome between PS/CC and endometrioid International Federation of Gynecology and Obstetrics (FIGO) Grade 3 cancer and was limited to patients with Stage I-II uterine carcinoma. METHODS AND MATERIALS Between November 1987 and September 1999, 83 patients with Stage I endometrial cancer and Stage II occult endometrial cancer were treated with simple hysterectomy and high-dose-rate intravaginal brachytherapy. Forty-one patients (49%) had FIGO Grade 3 endometrioid tumors (Group 1) and 42 (51%) had PS/CC histologic features (Group 2). The mean age was 63 years (range 30-89). Comprehensive surgical staging was done in 23 (28%) of 83 patients. Capillary space-like invasion (CSLI) was seen in 24 (29%) of 83 patients. The median dose of intravaginal brachytherapy when used alone was 21 Gy in 3 fractions. Additional external beam radiotherapy was given to 42 (51%) of 83 patients to 45 Gy. The two groups were balanced with regard to age, race, comprehensive surgical staging, amount of myometrial involvement, CSLI, lower uterine segment involvement, cervical involvement, and use of external beam radiotherapy. The median follow-up was 46 months (range 4-147). RESULTS The pattern of relapse was as follows: vagina/pelvis in 5 of 14 patients, lungs in 8 of 15, intra-abdominal in 4 of 12, and supraclavicular lymph nodes in 1 of 14. One of the four intra-abdominal disseminations was in Group 1 and the other three in Group 2 (p = 0.6). The 5-year vaginal/pelvic control, disease-free survival (DFS), and overall survival (OS) rate was 93% (95% confidence interval [CI] 87-99%), 79% (95% CI 69-89%), and 74% (95% CI 64-85%), respectively. No significant difference in outcome was found between Groups 1 and 2. The 5-year vaginal/pelvic control rate was 97% (95% CI 91-100%) in Group 1 compared with 90% (95% CI 81-99%) in Group 2 (p = 0.2). The 5-year DFS rate was 79% (95% CI 64-95%) in Group 1 vs. 78% (95% CI 65-92%) in Group 2 (p = 0.6), and the 5-year OS rate was 71% (95% CI 55-87%) in Group 1 vs. 79% (95% CI 66-92%) in Group 2 (p = 0.3). The influence on outcome of age, race, comprehensive surgical staging, CSLI, amount of myometrial invasion, cervical involvement, lower uterine segment involvement, and presence of pure PS or CC histologic features was evaluated. On multivariate analysis, only CSLI correlated with poor DFS (p = 0.04; relative risk 3, 95% CI 1-9) and OS (p = 0.02; relative risk 3, 95% CI 1-6). CONCLUSION On the basis of the results of this study, no significant difference in outcome exists between patients with Stage I-II endometrial cancer with PS/CC histologic features and those with similar stage disease, but with FIGO Grade 3 endometrioid histologic features. CSLI was the only independent predictor of poor DFS and OS.

Genetic Analysis of the Early Natural History of Epithelial Ovarian Carcinoma

Background The high mortality rate associated with epithelial ovarian carcinoma (EOC) reflects diagnosis commonly at an advanced stage, but improved early detection is hindered by uncertainty as to the histologic origin and early natural history of this malignancy. Methodology/Principal Findings Here we report combined molecular genetic and morphologic analyses of normal human ovarian tissues and early stage cancers, from both BRCA mutation carriers and the general population, indicating that EOCs frequently arise from dysplastic precursor lesions within epithelial inclusion cysts. In pathologically normal ovaries, molecular evidence of oncogenic stress was observed specifically within epithelial inclusion cysts. To further explore potential very early events in ovarian tumorigenesis, ovarian tissues from women not known to be at high risk for ovarian cancer were subjected to laser catapult microdissection and gene expression profiling. These studies revealed a quasi-neoplastic expression signature in benign ovarian cystic inclusion epithelium compared to surface epithelium, specifically with respect to genes affecting signal transduction, cell cycle control, and mitotic spindle formation. Consistent with this gene expression profile, a significantly higher cell proliferation index (increased cell proliferation and decreased apoptosis) was observed in histopathologically normal ovarian cystic compared to surface epithelium. Furthermore, aneuploidy was frequently identified in normal ovarian cystic epithelium but not in surface epithelium. Conclusions/Significance Together, these data indicate that EOC frequently arises in ovarian cystic inclusions, is preceded by an identifiable dysplastic precursor lesion, and that increased cell proliferation, decreased apoptosis, and aneuploidy are likely to represent very early aberrations in ovarian tumorigenesis.

Anorectal cytology as a screening tool for anal squamous lesions: cytologic, anoscopic, and histologic correlation.

Anorectal cytology has been increasingly used as a screening method for anal squamous lesions, particularly in high‐risk, homosexual, patients with human immunodeficiency virus infection. The diagnostic cytologic, anoscopic, and histologic criteria bear some resemblance to the criteria used in cervicovaginal samples with few differences. It is important to recognize these differences because they can lead to an erroneous diagnosis of dysplasia and unnecessary procedures.

ICUD-EAU international consultation on bladder cancer 2012: Pathology

CONTEXT To present a summary of the 2nd International Consultation on Bladder Cancer recommendations on the pathology of bladder cancer using an evidence-based strategy. OBJECTIVE To standardize descriptions of the diagnosis and reporting of urothelial carcinoma of the bladder and help optimize uniformity between individual pathology practices and institutions. EVIDENCE ACQUISITION A detailed Medline analysis was performed for original articles addressing bladder cancer with regard to pathology. Proceedings from the last 5 yr of major conferences were also searched. EVIDENCE SYNTHESIS The major findings are presented in an evidence-based fashion. Large retrospective and prospective data were analyzed. CONCLUSIONS Providing the best management for patients with bladder neoplasia relies on close cooperation and teamwork among urologists, oncologists, radiologists, and pathologists.

Immunohistochemical Staining of Cytologic Smears With MIB-1 Helps Distinguish Low-Grade From High-Grade Neuroendocrine Neoplasms

Neuroendocrine neoplasms (NENs) of the lung and gastrointestinal tract constitute a pathologic and biologic spectrum of tumors. Accurate cytologic diagnosis of a neuroendocrine neoplasm is important since definitive treatment frequently is based on low- and high-grade categories without histologic sampling. In many instances, however, low- and high-grade NENs share cytologic features, hindering a precise classification. Since the histologic diagnostic criteria for separation of low- from high-grade categories can be based on the proliferation rate, we proposed to evaluate the usefulness of the immunocytochemical stain for the proliferation marker MIB-1 in the grading of NENs. Cytologic preparations of 63 NENs were retrieved from the files of Memorial Sloan-Kettering Cancer Center, New York, NY. One representative alcohol-fixed slide from each case was destained and restained immunocytochemically for MIB-1. When MIB-1 immunoreactivity was considered, all low-grade NENs showed immunoreactivity in fewer than 25% of the neoplastic cells, and all high-grade NENs demonstrated immunoreactivity in more than 50% of neoplastic cells. Our study demonstrates that MIB-1 dramatically stratifies NENs as low-grade or high-grade. Therefore, the proliferation index also correlates with grade of NEN in cytology specimens.

Somatic mutation of fibroblast growth factor receptor-3 (FGFR3) defines a distinct morphological subtype of high-grade urothelial carcinoma.

FGFR3 mutations are common in low‐grade urothelial carcinoma and represent a potential therapeutic target in this disease. Their incidence and functional role in high‐grade urothelial carcinoma (HGUC), which displays an increased propensity for recurrence and muscularis propria invasion, is less well defined. We developed a mass spectrometry‐based genotyping assay to define the incidence of FGFR3 mutations in a large clinically annotated set of urothelial carcinomas. FGFR3 mutations were found in 17% of HGUC versus 84% of low‐grade lesions. Retrospective pathological review of the class of FGFR3 mutant HGUC revealed unique histological features, characterized by a bulky, exophytic component with branching papillary architecture as well as irregular nuclei with a koilocytoid appearance. The predictive value of this histological appearance was confirmed using a prospective set of 49 additional HGUCs. Prospective histological review was able to correctly predict for the presence of an FGFR3 mutation in 13/24 HGUC specimens that exhibited the distinct morphology (54%). All 25 specimens lacking the defined histological features were FGFR3 wild‐type for a negative predictive value of 100%. Macrodissection of individual tumours confirmed the presence of the FGFR3 mutant allele in non‐invasive and invasive, low and high‐grade regions of individual tumours and in the lymph node metastases of patients whose tumours possessed the characteristic morphological signature, suggesting that FGFR3 mutations are not restricted to the more clinically indolent regions of HGUCs. These data suggest that histological screening of HGUCs followed by confirmatory genotyping can be used to enrich for the population of HGUCs most likely to harbour activating mutations in the FGFR‐3 receptor tyrosine kinase. Histological review could thus aid in the development of targeted inhibitors of FGFR‐3 by facilitating the identification of the subset of patients most likely to harbour activating mutations in the FGFR3 gene. Copyright