Michael B. Campion

A comparison of conventional cytology, DNA ploidy analysis, and fluorescence in situ hybridization for the detection of dysplasia and adenocarcinoma in patients with Barrett's esophagus ☆ ☆☆

New detection methods with prognostic power are needed for early identification of dysplasia and esophageal adenocarcinoma (EA) in patients with Barretts esophagus (BE). This study assessed the relative sensitivity and specificity of conventional cytology, DNA ploidy analysis with digital image analysis (DIA), and fluorescence in situ hybridization (FISH) for the detection of dysplasia and adenocarcinoma in endoscopic brushing specimens from 92 patients undergoing endoscopic surveillance for BE. FISH used probes to 8q24 (C-MYC), 9p21 (P16), 17q12 (HER2), and 20q13. Four-quadrant biopsies taken every centimeter throughout visible Barretts mucosa were used as the gold standard. The sensitivity of cytology, DIA, and FISH for low-grade dysplasia was 5%, 5%, and 50%, respectively; for high-grade dysplasia (HGD), 32%, 45%, and 82%, respectively; and for EA, 45%, 45%, and 100%, respectively. FISH was more sensitive (P < .05) than cytology and DIA for low-grade dysplasia, HGD, and EA. The specificity of cytology, DIA, and FISH among patients (n = 14) with tissue showing only benign squamous mucosa was 93%, 86%, and 100% (P = .22), respectively. All patients with a polysomic FISH result had HGD and/or EA within 6 months (n = 33). There was a significant difference between FISH categories (negative, 9p21 loss, gain of a single locus, and polysomy) for progression to HGD/EA (P < .001). These findings suggest that FISH has high sensitivity for the detection of dysplasia and EA in BE patients, with the power to stratify patients by FISH abnormality for progression to HGD/EA. Additional studies are needed to further evaluate the clinical use of FISH.

Prospective Cytological Assessment of Gastrointestinal Luminal Fluid Acquired During EUS: A Potential Source of False-Positive FNA and Needle Tract Seeding

OBJECTIVES:Endoscopic ultrasound (EUS) fine needle aspiration (FNA) can result in false-positive cytology and can also cause needle tract seeding. Our goal was to evaluate a potential cause, namely, the presence of malignant cells within gastrointestinal (GI) luminal fluid, either as a result of tumor sloughing from luminal cancers or secondary to FNA of extraluminal sites.METHODS:During EUS, luminal fluid that is usually aspirated through the echoendoscope suction channel and discarded was instead submitted for cytological analysis among patients with cancer and benign disease. Pre- and post-FNA luminal fluid samples were collected to discern the role of FNA in inducing a positive cytology. When not performing FNA, one sample was collected for the entire examination. The final diagnosis was based on strict clinicopathological criteria and ≥2-year follow-up. This study was conducted in a tertiary referral center.RESULTS:We assessed the prevalence of luminal fluid-positive cytology among patients with luminal (e.g., esophageal), extraluminal (e.g., pancreatic), and benign disease. Among the 140 patients prospectively enrolled with sufficient sampling and follow-up, an examination of luminal fluid cytology showed positive results for malignancy in luminal and extraluminal cancer patients, 48 and 10%, respectively. This included 8 out of 23 esophageal, 4 of 5 gastric, and 9 of 15 rectal cancers. The positive luminal fluid cytology rate with luminal cancers was not affected by performing FNA. Post-FNA luminal fluid cytology was positive in 3 out of 26 with pancreatic cancers. Cytological examination of luminal fluid aspirates did not demonstrate malignant cells in any patient with nonmalignant disease.CONCLUSIONS:Malignant cells are commonly present in the GI luminal fluid of patients with luminal cancers and can also be found in patients with pancreatic cancer after EUS FNA. Further study is needed to determine the impact of these findings on cytological interpretation, staging, risk of needle tract seeding, and patient care and outcomes.

International study on inter-reader variability for circulating tumor cells in breast cancer

IntroductionCirculating tumor cells (CTCs) have been studied in breast cancer with the CellSearch® system. Given the low CTC counts in non-metastatic breast cancer, it is important to evaluate the inter-reader agreement.MethodsCellSearch® images (N = 272) of either CTCs or white blood cells or artifacts from 109 non-metastatic (M0) and 22 metastatic (M1) breast cancer patients from reported studies were sent to 22 readers from 15 academic laboratories and 8 readers from two Veridex laboratories. Each image was scored as No CTC vs CTC HER2- vs CTC HER2+. The 8 Veridex readers were summarized to a Veridex Consensus (VC) to compare each academic reader using % agreement and kappa (κ) statistics. Agreement was compared according to disease stage and CTC counts using the Wilcoxon signed rank test.ResultsFor CTC definition (No CTC vs CTC), the median agreement between academic readers and VC was 92% (range 69 to 97%) with a median κ of 0.83 (range 0.37 to 0.93). Lower agreement was observed in images from M0 (median 91%, range 70 to 96%) compared to M1 (median 98%, range 64 to 100%) patients (P < 0.001) and from M0 and <3CTCs (median 87%, range 66 to 95%) compared to M0 and ≥3CTCs samples (median 95%, range 77 to 99%), (P < 0.001). For CTC HER2 expression (HER2- vs HER2+), the median agreement was 87% (range 51 to 95%) with a median κ of 0.74 (range 0.25 to 0.90).ConclusionsThe inter-reader agreement for CTC definition was high. Reduced agreement was observed in M0 patients with low CTC counts. Continuous training and independent image review are required.

Lung cancer adrenal gland metastasis: Optimal fine‐needle aspirate and touch preparation smear cellularity characteristics for successful theranostic next‐generation sequencing

Multigene molecular testing to guide personalized therapy for oncology patients is of increasing clinical relevance. Molecular testing of fine‐needle aspiration samples is underused, but when acquired with minimally invasive techniques could become the standard of care to obtain theranostic specimens. The aims of the current study were to identify key cytology specimen selection criteria suitable for next‐generation sequencing (NGS) and to determine the prevalence and spectrum of pathogenic alterations in a cohort of patients with AJCC stage IV lung cancer.

Direct uterine sampling with the Tao brush sampler using a liquid-based preparation method for the detection of endometrial cancer and atypical hyperplasia: A feasibility study

Endometrial cytology sampling devices for direct uterine sampling have been shown in previous studies to be a reliable and relatively painless method for detecting endometrial lesions. The purpose of the current study was to determine the performance characteristics of endometrial cytology for the detection of malignancy and atypical hyperplasia using liquid‐based cytology specimens collected with the Tao brush sampler.

Assessing the Value of Reflex Fluorescence In Situ Hybridization Testing in the Diagnosis of Bladder Cancer When Routine Urine Cytological Examination is Equivocal

PURPOSE We evaluated the usefulness of fluorescence in situ hybridization in the treatment of patients with equivocal cytology. MATERIALS AND METHODS Fluorescence in situ hybridization was performed in residual urine from 124 patients with a cytological diagnosis of cell clusters (22), atypical findings (46) and suspicious findings (56) who had a same day cystoscopy result and bladder biopsy within 6 months of the cytology diagnosis. Urologists and fluorescence in situ hybridization technologists were blinded to the matching fluorescence in situ hybridization and cystoscopy results, respectively. RESULTS In conjunction with cystoscopy fluorescence in situ hybridization was significantly more sensitive than cystoscopy alone for detecting cancer (87% vs 67%, p <0.001) and muscle invasive cancer (94% vs 56%, p = 0.031). Of the 124 equivocal cytology specimens 58 (47%) were positive by fluorescence in situ hybridization. Of these patients 53 (91%) had subsequent evidence of carcinoma, including Ta tumors in 17, Tis in 13, T1 in 8 and T2 or greater in 15, on the first followup biopsy. Three of the 5 remaining patients with a positive fluorescence in situ hybridization result and negative first followup biopsy had evidence of cancer at a later date, including TxN+ disease in 2 and Tis in 1. A total of 66 specimens were diagnosed as negative by fluorescence in situ hybridization. Of these patients 34 (52%) had negative biopsy results, whereas the remaining 32 (48%) demonstrated bladder cancer, including Ta disease in 20, Tis in 8, T1 in 2 and T2+ in 2. Cystoscopy detected 21 of the 32 tumors (66%) not detected by fluorescence in situ hybridization, while fluorescence in situ hybridization detected 17 of the 28 (61%) not detected by cystoscopy. CONCLUSIONS Our data suggest that fluorescence in situ hybridization with cystoscopy can aid clinicians in the diagnosis of bladder cancer in patients with equivocal cytology.

Comparison of Methods to Detect Neoplasia in Patients Undergoing Endoscopic Ultrasound-Guided Fine-Needle Aspiration

BACKGROUND & AIMS Digital image analysis (DIA) and fluorescence in situ hybridization (FISH) can be used to evaluate biliary strictures with greater accuracy than conventional cytology (CC). We performed a prospective evaluation of the accuracy of CC, compared with that of DIA and FISH, in detection of malignancy in patients undergoing endoscopic ultrasonography (EUS) fine-needle aspiration (FNA). METHODS We collected a minimum of 6 FNA samples from each of 250 patients during EUS. CC or DIA and FISH analyses were performed on every other specimen (from every other FNA pass); patients were randomly assigned to the first test performed. CC slides were reviewed by gastrointestinal cytopathologists who were blinded to all data. Findings from cytohistologic analysis, after a minimum 24-month follow-up period, were used as the standard (n = 202; median age, 65 years). RESULTS Aspirates were collected from lymph nodes (n = 111), pancreas (n = 61), gastrointestinal lumen wall (n = 9), periluminal mass (n = 4), liver (n = 8), and miscellaneous sites (n = 9). Matched samples provided a mean of 3.2 passes for CC and 1.6 passes for DIA and FISH. The data indicate a potential lack of utility for DIA. The combination of CC and FISH detected malignancy with 11% greater sensitivity than CC alone (P = .0002), but specificity was reduced from 100% to 96%. CONCLUSIONS FISH analysis identifies neoplastic lesions with significantly greater sensitivity than CC in patients with diverse pathologies who underwent EUS with FNA, despite limited tissue sampling for FISH analysis.

Kinase Genotype Analysis of Gastric Gastrointestinal Stromal Tumor Cytology Samples Using Targeted Next-Generation Sequencing

Gastric gastrointestinal stromal tumors (GISTs) usually contain the mast/stem cell growth factor receptor Kit gene (KIT) or platelet-derived growth factor receptor A (PDGFRA) mutations that can be targeted by, or mediate resistance to, imatinib. Diagnostic material often is obtained by endoscopic ultrasound-guided fine-needle aspiration, which often is unsuitable for molecular analysis. We investigated whether targeted next-generation sequencing (NGS) can be used in multiplex genotype analysis of cytology samples collected by endoscopic ultrasound-guided fine-needle aspiration. We used the Ion AmpliSeq V2 Cancer Hotspot NGS Panel (Life Technologies, Carlsbad, CA) to identify mutations in more than 2800 exons from 50 cancer-associated genes in GIST samples from 20 patients. We identified KIT mutations in 58% of samples (91% in exon 11 and 9% in exon 17) and PDGFRA mutations in 26% (60% in exon 18 and 40% in exon 12); 16% of samples had no mutations in KIT or PDGFRA. No pathogenic alterations were found in PIK3CA, BRAF, KRAS, NRAS, or FGFR3. We predicted that 32% of patients would have primary resistance to imatinib, based on mutations in exon 17 of KIT, exon 18 of PDGFRA (D842V), or no mutation in either gene. Targeted NGS of cytology samples from GISTs is feasible and provides clinically relevant data about kinase genotypes that can help guide individualized therapy.

Endoscopic ultrasound fine-needle aspiration cytology mutation profiling using targeted next-generation sequencing: personalized care for rectal cancer

OBJECTIVES In an era of precision medicine, our aim was to determine the frequency and theranostic potential of mutations identified in malignant lymph nodes (LNs) sampled by endoscopic ultrasound fine-needle aspiration (EUS FNA) of patients with rectal cancer by targeted next-generation sequencing (NGS). METHODS The NGS Ion AmpliSeq Cancer Hotspot Panel v2 (Life Technologies, Carlsbad, CA) and MiSeq (Illumina, San Diego, CA) sequencers were used to sequence and assess for 2,800 or more possible mutations in 50 established cancer-associated genes. RESULTS Among 102 patients, 89% had 194 pathogenic alterations identified in 19 genes. The identification of KRAS, NRAS, or BRAF mutations suggests that 42% are likely nonresponders to anti-epidermal growth factor receptor therapy. Among KRAS, NRAS, or BRAF wild-type patients, alterations in eight genes linked to alternative therapies were identified in 44%. CONCLUSIONS Our data demonstrate the successful ability to apply a single multiplex test to allow multigene mutation detection from malignant LN cytology specimen DNA collected by EUS FNA.

Chromosomal Alterations Detected by Fluorescence In Situ Hybridization in Urothelial Carcinoma and Rarer Histologic Variants of Bladder Cancer

Fluorescence in situ hybridization (FISH) with the UroVysion probe set (Abbott Molecular, Des Plaines, IL) was used to assess 31 bladder cancers for chromosomal abnormalities, including 4 adenocarcinomas, 5 urachal adenocarcinomas, 6 small cell carcinomas, 7 squamous cell carcinomas, and 9 typical urothelial carcinomas. FISH was also used to assess the benign urothelium in 4 cases. There was a significant increase (P < .001) in the mean number of chromosome 3 (2.64 vs 1.51), chromosome 7 (2.61 vs 1.48), and chromosome 17 (2.41 vs 1.41) centromeric signals observed in cells from patients with cancer compared with patients without cancer. Of the 31 tumors, 29 (94%) demonstrated polysomic signal patterns in more than 10% of cells. In the 2 remaining tumor specimens, there was a high percentage of cells (>75%) demonstrating homozygous 9p21 deletion. The data from this study suggest that chromosomal abnormalities detectable by FISH in urothelial carcinoma are also common in rarer histologic variants of bladder cancer.