Exploring the intratumoral heterogeneity of DNA ploidy in prostate cancer.

BACKGROUND: Prostate cancer is morphologically and molecularly heterogeneous. Genomic heterogeneity might be mirrored by variability in DNA ploidy. Aneuploidy is a hallmark of genomic instability and associated with tumor aggressiveness. Little attention has been paid to the biological significance of the diploid tumor cell population that often coexists with aneuploid populations. Here, we investigated the role of DNA ploidy in tumor heterogeneity and clonal evolution. METHODS: Three radical prostatectomy specimens with intratumoral heterogeneity based on nuclear features on H&E were selected. DNA content of each subpopulation was determined by DNA image cytometry and silver in situ hybridization (SISH). Genomic evolution was inferred from array comparative genomic hybridization (aCGH). Additionally, immunohistochemistry was used to examine the stemness-associated marker ALDH1A1. RESULTS: Nuclear morphology reliably predicted DNA ploidy status in all three cases. In one case, aCGH analysis revealed several shared deletions and one amplification in both the diploid and the aneuploid population, suggesting that these populations could be related. In the other two cases, a statement about relatedness was not possible. Furthermore, ALDH1A1 was expressed in 2/3 cases and exclusively observed in their diploid populations. CONCLUSIONS: In this proof-of-concept study, we demonstrate the feasibility to predict the DNA ploidy status of distinct populations within one tumor by H&E morphology. Future studies are needed to further investigate the clonal relationship between the diploid and the aneuploid subpopulation and test the hypothesis that the aneuploid population is derived from the diploid one. Finally, our analyses pointed to an enrichment of the stemness-associated marker ALDH1A1 in diploid populations, which warrants further investigation in future studies.

Pure intraductal carcinoma of the parotid gland: Cytologic findings on FNA sample. Report of one case.

One case of intraductal carcinoma of the parotid gland in a 67-year-old male patient is here introduced. The patient, who had a one-year history of a parotid mass, had undergone ultrasound and MRI examination that disclosed a 13x4x3 mm well delimited nodular mass of the accessory lobe of his left parotid gland. Ultrasound-guided Fine Needle Aspiration (FNA) had been performed by the clinician. The obtained smears showed widespread cellular necrosis in which cellular clusters with moderate and focally severe atypias displayed papillary and cribriform architecture and were admixed with sheets of epithelial cells with less striking nuclear atypias, squamous, or apocrine metaplasia. Histopathological examination disclosed a pure intraductal carcinoma of the parotid gland with classical morphology, which was radically excised. The differential cytological diagnosis of pure intraductal carcinoma of salivary glands may be difficult and comprises mucoepidermoid carcinoma as well as "in situ" carcinomas developping in the context of sclerosing polycystic adenosis, mammary analogue secretory carcinoma (MASC) of the salivary glands and cystic variants of salivary adenocarcinoma NOS (formerly called cystadenocarcinomas).

Brush cytology for the detection of high-risk HPV infection in oropharyngeal squamous cell carcinoma.

BACKGROUND: High-risk human papillomavirus (HR-HPV) infection is associated with improved prognosis and a better response to treatment in patients with oropharyngeal squamous cell carcinoma (OPSCC). Brush cytology is a noninvasive method with which to collect cells from the surface of mucosal lesions. The objective of the current study was to assess the performance of OPSCC brush cytology for the detection of HR-HPV. METHODS: Liquid-based brush cytology specimens were prospectively collected during panendoscopy from 51 patients with OPSCC. Cell suspensions were analyzed with Papanicolaou staining, polymerase chain reaction-based HPV DNA testing, and p16 immunostaining. HPV testing and p16 staining were also performed on paired OPSCC biopsy or surgical resection specimens. The detection of HR-HPV DNA alone and the combined positivity for HR-HPV DNA and p16 protein in dysplastic squamous cells were used to calculate accuracy, sensitivity, specificity, and positive and negative predictive values for HR-HPV detection using brush cytology samples. RESULTS: Approximately 96% of OPSCC brush cytology samples (49 of 51 samples) were classified as satisfactory for evaluation. Dysplastic squamous cells were found in 88% of samples (43 of 49 samples). HPV DNA testing was conclusive in 95% of samples (41 of 43 samples) and revealed HR-HPV DNA in approximately 54% of patients (22 of 41 patients) (HPV type 16 in 19 patients and HPV type 33 in 3 patients). Approximately 49% of brush cytology samples (20 of 41 samples) were positive for HR-HPV DNA and p16 expression. The accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of brush cytology to identify HR-HPV DNA-positive and p16-positive OPSCC samples were 88%, 83%, 94%, 95%, and 81%, respectively. CONCLUSIONS: Brush cytology appears to be a valid approach with which to determine the HR-HPV status of patients with OPSCC.

Evaluation of chromosomal aberrations in patients with benign conditions and reactive changes in urinary cytology.

BACKGROUND: Fluorescence in situ hybridization (FISH) is routinely used to help clarify atypical urinary cytology. However, to the authors' knowledge, little is known regarding the frequency of chromosomal aberrations in non-neoplastic conditions that could potentially lead to false-positive FISH results. The objective of the current study was to evaluate the frequency of chromosomal aberrations in benign cells of the urinary tract using the UroVysion FISH test. METHODS: The authors analyzed 77 Papanicolaou-stained benign urine cytology specimens with reactive epithelial atypia using a FISH assay detecting the chromosomes 3, 7, and 17 and the gene locus 9p21. A positive test result was defined as an increased copy number of at least 2 chromosomes in ≥ 4 of 25 cells, or > 10 cells with a tetraploid or octaploid pattern, or homozygous or heterozygous deletion of 9p21 (≥ 12cells). RESULTS: FISH was positive in 27 of 77 bladder washings (35.1%) including 25 of 65 bladder washings (40.5%) and 2 of 15 voided urines (13.5%) from patients with irritative bladder (15 of 36 patients), a history of radiotherapy (7 of 12 patients), nonspecific cystitis (3 of 11 patients), hematuria (3 of 8 patients), and lithiasis (1 of 4 patients) . In 7 of 27 FISH-positive urothelial specimens, the positivity was solely due a polyploid pattern (tetraploid/octaploid pattern) in > 10 of the cells. CONCLUSIONS: Chromosomal aberrations can occur in reactive urothelial cells, with a tetraploid pattern being the most common. Even an aneuploid pattern of FISH signals does not always prove malignancy because it rarely occurs in reactive urothelial cells. Correlation of FISH results with cytomorphology and patient history is crucial to avoid false-positive diagnoses.

Fluorescence in situ hybridization in the definitive diagnosis of malignant mesothelioma in effusion cytology.

BACKGROUND: Distinction of malignant mesothelioma (MM) from reactive mesothelial cells (RM) in effusions is notoriously difficult. The aim of our study was to test chromosomal aberrations detected by fluorescence in situ hybridization (FISH) in the diagnosis of MM in effusion cytology and to explore the potential role of p16, p14, and p15 gene methylation as an alternative mechanism of tumor suppressor gene inactivation. METHODS: Fifty-two effusions of biopsy-proven MM and 28 benign effusions were retrospectively analyzed by multitarget FISH assay for aberrations of chromosomes 3, 7, 17, and 9p21. In case of a negative result, the corresponding MM biopsy specimen was analyzed. Methylation-specific polymerase chain reaction (MSP) for p16, p14, and p15 was performed on FISH-negative MM biopsy specimens. RESULTS: Seventy-nine percent of effusions with biopsy-proven MM had chromosomal aberrations, with loss of 9p21 as the most common finding. All benign effusions were FISH negative. Sensitivity, specificity, and positive and negative predictive values for detection of MM by FISH were 79%, 100%, 100%, and 72%, respectively. Six of nine FISH-negative effusions with biopsy-proven MM were also FISH negative in the MM biopsy specimens. Four of five FISH-negative biopsy specimens showed promoter methylation in p16 and p14 as compared with one of 12 benign controls. CONCLUSIONS: FISH is a sensitive and highly specific method for the definitive diagnosis of MM in effusion cytology. In the subset of FISH-negative MM, tumor suppressor genes on the chromosomal region 9p21 are often inactivated by promoter methylation.

Benefit of baseline cytometry for surveillance of patients with Barrett's esophagus.

BACKGROUND: The current gold standard for the surveillance of Barrett's esophagus is the Seattle four-quadrant biopsies protocol (4-QB). Using endoscopic brush cytology, this study prospectively investigated whether digital image cytometry (DICM) is of additional benefit over regular histology as a predictor for progression to high-grade dysplasia or cancer during a surveillance of at least 3 years. METHODS: The prospective cohort in this study included 93 patients (72% male) with Barrett's esophagus, baseline endoscopies, and at least one DICM in addition to 4-QB who had been followed up a minimum of 3 years at the time of analysis. High-grade dysplasia (HGD) and adenocarcinoma were defined as primary end points. The DICM was performed on Feulgen-restained cytology smears with a continuous collision detection (CCD) three-chip color video camera (Sony) and an AutoCyte QUIC DNA workstation. RESULTS: Of the 93 patients, 11 presented with the diagnosis of HGD and adenocarcinoma at baseline endoscopy. The remaining 82 patients were analyzed after a median follow-up time of 44 months (range, 36-65 months). Of these 82 patients, 9 (11%) had low-grade dysplasia (LGD) at baseline histology: One of two patients with LGD and aneuploid DICM showed HGD at follow-up assessment, whereas none of seven patients with LGD and diploid DICM had development of HGD. Of the 82 patients, 73 (89%) had either specialized intestinal metaplasia (SIM) without dyplasia or indefinite findings for dysplasia at baseline histology. Of the eight patients with SIM and intermediate/aneuploid DICM, two had development of HGD. None of those with negative or indefinite findings for dysplasia and diploid DICM had HGD at the follow-up evaluation. In summary, the three patients who had development of HGD showed a pathologic DICM at baseline, and no patient with diploid DICM had HGD. CONCLUSIONS: Cytometry from brush cytology as an add-on to histology appears to be of additional benefit during surveillance of Barrett's esophagus. Whereas an aneuploid/intermediate DICM warrants an early re-endoscopy, a diploid DICM underscores the low-risk status especially of patients with low-grade dysplasia.

HER2 gene status in primary breast cancers and matched distant metastases.

INTRODUCTION: The status of the gene encoding human EGF-like receptor 2 (HER2) is an important prognostic and predictive marker in breast cancer. Only breast cancers with HER2 amplification respond to the targeted therapy with trastuzumab. It is controversial to what degree the primary tumour is representative of distant metastases in terms of HER2 status. Discrepancies in HER2 status between primary tumours and distant metastases have been described, but their reasons remain unclear. Here, we compared HER2 status on cytological specimens of distant metastases with the result from the primary carcinomas, and explored the prevalence of and the reasons for discrepant results. METHODS: HER2 status was determined by fluorescence in situ hybridisation. HER2 gene amplification was defined as a HER2/chromosome 17 signal ratio of 2 or more. HER2 results from cytological specimens of matched distant metastases were compared with the results from the corresponding primary tumours (n = 105 patients). In addition, lymph node metastases were analysed in 31 of these patients. RESULTS: HER2 amplification was found in 20% of distant metastases. HER2 status was discordant between the primary tumour and distant metastasis in 7.6% of the 105 patients. Re-evaluation revealed that in five patients (4.7%), discrepancies were due to interpretational difficulties. In two of these patients, focal amplification had initially been overlooked as a result of heterogeneity in the primary tumours or in the metastases, respectively. A further three patients had borderline amplification with a ratio close to 2. Discrepancy remained unexplained in three patients (2.9%). CONCLUSION: HER2 gene status remains highly conserved as breast cancers metastasise. However, discrepant results do occur because of interpretational difficulties and heterogeneity of HER2 amplification. Cytological specimens from distant metastases are well suited for HER2 fluorescence in situ hybridisation analysis.