Joel S. Bentz

Detection of epidermal growth factor receptor gene mutations in cytology specimens from patients with non-small cell lung cancer utilising high-resolution melting amplicon analysis

Background: Activating epidermal growth factor receptor (EGFR) mutations have been implicated in non-small cell lung cancer (NSCLC), and have also been clinically correlated with patient sensitivity to targeted EGFR inhibitors. Aim: To describe a technique for determining EGFR mutation status on archival fine needle aspirate (FNA) specimens from advanced NSCLC patients. Methods: Eleven archival FNA slides from patients with advanced NSCLC were examined for diagnostic material to identify tumour cell-enriched regions. EGFR mutation status was determined using a slide-scrape DNA extraction protocol of selected tumour cell regions on the smear slides, followed by real time PCR and high resolution melt analysis (HRMAA) of EGFR exons 18, 19, 20, and 21, followed by sequence analysis. Results: All DNA samples were successfully amplified by PCR. Three adenocarcinoma patient samples contained EGFR mutations in exon 19 (L747-P753insS). One of the three had an additional exon 19 mutation (A755D). Conclusions: Archival cytology slides from patients with NSCLC can be used to determine EGFR mutation status by PCR, HRMAA, and sequencing. The ability to use archival cytology slides greatly increases the potential material available for molecular analysis in diagnosis and selection of patients for targeted therapeutic agents.

Utility of BRAF V600E mutation detection in cytologically indeterminate thyroid nodules

Background Fine needle aspiration (FNA) is widely utilized for evaluation of patients with thyroid nodules. However, approximately 30% are indeterminate for malignancy. Recently, a mutation in the BRAF gene has been reported to be the most common genetic event in papillary thyroid carcinoma (PTC). In this retrospective study, we assessed the utility of BRAF V600E mutation detection for refining indeterminate preoperative cytologic diagnoses in patients with PTC. Methods Archival indeterminate thyroid FNAs and corresponding formalin-fixed, paraffin-embedded (FFPE) surgical samples with PTC were identified in our patient files. DNA extracted from slide scape lysates and 5 μm FFPE sections were evaluated for the BRAF V600E mutation using LightCycler PCR and fluorescent melting curve analysis (LCPCR). Amplification products that showed deviation from the wild-type genomic DNA melting peak, discordant FNA and FFPE matched pairs, and all benign control samples, underwent direct DNA sequencing. Results A total of 19 indeterminate thyroid FNAs demonstrating PTC on FFPE surgical samples were included in the study. Using BRAF mutation analysis, the preoperative diagnosis of PTC was confirmed in 3/19 (15.8%) FNA samples that could not be conclusively diagnosed on cytology alone. However, 9/19 (47.4%) FFPE tissue samples were positive for the V600E mutation. Of the discordant pairs, 5/6 FNAs contained less than 50% tumor cells. Conclusion When used with indeterminate FNA samples, BRAF mutation analysis may be a useful adjunct technique for confirming the diagnosis of malignancy in an otherwise equivocal case. However, overall tumor cell content of some archival FNA smear slides is a limiting factor for mutation detection.

Detection of B7-H4 and p53 in pancreatic cancer: potential role as a cytological diagnostic adjunct.

Objectives: This study compared p53 expression with B7-H4, a novel cancer biomarker, in pancreatic ductal adenocarcinoma (PDA) resection specimens and in a pilot series of endoscopic ultrasound-guided fine-needle aspirations (EUS-FNAs). Methods: B7-H4 and p53 expression were evaluated by immunoperoxidase methods in 36 PDA and 15 EUS-FNA specimens and were scored for intensity and proportion of positive cells; cases were then assigned a final sum score. Results: B7-H4 was detected in 33 (92%) of 36 PDA sections, 8 (89%) of 9 cytologically positive EUS-FNAs, and 1 (20%) of 5 cytologically negative EUS-FNAs. p53 was detected in 30 (83%) of 36 PDA sections, 4 (44%) of 9 cytologically positive EUS-FNAs, and 1 (20%) of 5 cytologically negative cases. One EUS-FNA case that was cytologically atypical but not diagnostic of malignancy expressed B7-H4 and p53. Some benign tissue components (intercalated cells/ducts, main pancreatic ducts, and acinar cells) were also positive for B7-H4 and/or p53. Overall expression of B7-H4 in benign tissues, however, was relatively low compared with that seen in most carcinoma cases. Conclusions: B7-H4 was expressed more often in PDA than was p53. Despite potentially problematic expression in benign/normal cells, the 2 markers target different cellular components and demonstrate potential diagnostic use for detection of PDA in resected and EUS-FNA specimens.

Detection of BRAF V600E activating mutation in papillary thyroid carcinoma using PCR with allele-specific fluorescent probe melting curve analysis

Background: A single hotspot mutation at nucleotide 1799 of the BRAF gene has been identified as the most common genetic event in papillary thyroid carcinoma (PTC), with a prevalence of 29–83%. Aims: To use a PCR assay to molecularly characterise the BRAF activating point mutation in a series of PTC and benign thyroid cases and correlate the mutation results with histological findings. Methods: Formalin-fixed paraffin-embedded (FFPE) sections were evaluated for the BRAF V600E mutation using LightCycler PCR with allele-specific fluorescent probe melting curve analysis (LCPCR). Results: 42 (37 PTC; 5 benign) surgical tissue samples were analysed for the BRAF V600E activating point mutation. Using LCPCR and direct DNA sequencing, the BRAF mutation was identified in 23/37 (62.2%) PTC FFPE samples. DNA sequencing results demonstrated confirmation of the mutation. Conclusions: Detection of BRAF-activating mutations in PTC suggests new approaches to management and treatment of this disease that may prove worthwhile. Identification of the BRAF V600E activating mutation in routine FFPE pathology samples by a rapid laboratory method such as LCPCR could have significant value.

Utility of ultrasound-guided fine-needle aspiration of parathyroid adenomas for localization before minimally invasive parathyroidectomy

OBJECTIVE To determine the sensitivity and specificity of ultrasound (US)-guided fine-needle aspiration (FNA) and measurement of parathyroid hormone (PTH) in the aspirate (FNA/PTH) as a preoperative localization procedure. METHODS The study group consisted of 34 consecutive patients with primary hyperparathyroidism. The FNA/PTH estimations in these patients were compared with those from 13 proven thyroid nodules. All patients underwent US study of the neck, which suggested the presence of a solitary adenoma in 30 patients and of hyperplasia in 2; no adenoma or hyperplasia could be visualized in 2 patients. Thirty-two patients underwent FNA/PTH, which yielded a mean PTH level of 22,060.0 +/- 6,653.0 pg/mL. This result was significantly different (P<0.001) from the mean PTH level in 13 thyroid nodules (9.0 +/- 1.0 pg/mL). RESULTS On the basis of the FNA/PTH results, 28 patients with suspected adenomas underwent minimally invasive parathyroidectomy (MIP), and 2 patients are awaiting a surgical procedure. Of these 28 patients, 27 had more than a 50% decline in intraoperative PTH level after removal of the suspected adenoma, confirming surgical success. In 1 patient, multigland hyperplasia was discovered during the operation. The 2 study subjects with US findings of suspected hyperplasia underwent 4-gland surgical procedures. All patients treated surgically continued to have normal serum calcium levels 6 to 18 months postoperatively. CONCLUSION Primary hyperparathyroidism is caused most commonly by a solitary adenoma and less commonly by multigland hyperplasia of the parathyroid glands. Surgical resection is the only curative therapy. MIP has become a frequently used strategy, but there are limitations to current preoperative localization techniques. We conclude that US-guided FNA is a useful technique that facilitates MIP, with a high degree of specificity (95%) and sensitivity (91%).

NQO1 expression in pancreatic cancer and its potential use as a biomarker.

Pancreatic ductal adenocarcinoma (PDA) is rarely curable due to regional/metastatic spread at diagnosis. Identification of molecular markers may enhance diagnosis and early detection of PDA. The 2-electron reductase, NAD(P)H:quinone oxidoreductase (NQO1) has been found to be overexpressed in many solid tumors including PDA, and may be a useful clinically relevant diagnostic marker of malignancy. For this study, we used 37 surgical resection cases: 24 PDAs and 13 benign pancreatic tissue specimens. An additional 16 specimens from pancreatic endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) were included as a pilot series. NQO1 was detected by avidin-biotin based immunohistochemical and immunocytochemical methods. Both staining intensity and proportion of NQO1 positive tumor cells were scored. Moderate to strong (2 to 3+) staining for NQO1 was detected in 22/24 (92%) surgically resected PDAs, 9/9 (100%) EUS-FNAs with malignant diagnoses, one cytologically atypical but not diagnostic for malignancy EUS-FNA, and 1/6 (17%) EUS-FNAs initially diagnosed as negative for malignancy. Subsequent histologic assessment confirmed malignancy in all 9 cytologically positive EUS-FNAs and in the atypical case. The NQO1 positive case initially diagnosed as negative for malignancy showed no evidence of carcinoma on subsequent tissue biopsy. NQO1 staining was also observed in some benign ducts/cells; however, correlation of NQO1 expression with cellular morphology assessment minimizes the risk of false positive diagnosis. NQO1 is consistently overexpressed in PDA. Although NQO1 is observed in some benign tissue components, this marker may be a clinically useful diagnostic adjunct for detection of PDA, independent of tumor grade/stage.

Osteoclastic and pleomorphic giant cell tumors of the pancreas diagnosed via EUS-guided FNA: unique clinical, endoscopic, and pathologic findings in a series of 5 patients

INTRODUCTION Osteoclastic and pleomorphic giant cell tumors of the pancreas are rare entities that have been typically described only in single case reports. We report on our experience with a series of 5 patients with pancreatic giant cell tumors seen at our institution. METHODS This was a retrospective study involving a search of the study institutions medical records from 2001 to 2007 for patients diagnosed with giant cell-containing neoplasms of the pancreas. RESULTS Five patients (2 women, 3 men) were identified. Age range was 59 to 81 years, with a mean of 70.2 years. None were current or former smokers. None had a history of alcohol abuse or preexisting pancreatitis of any kind. On EUS, tumors tended to be large, with a mean diameter of 47 mm (range 20-70 mm). All tumors had a heterogeneous echotexture and a distinct appearance when compared with the typical appearance of adenocarcinoma when viewed via EUS. The diagnosis of giant cell tumor of the pancreas, as well as the subtype, was made via EUS-guided FNA of the pancreatic lesion. Patients with pleomorphic giant cell tumors of the pancreas had a poor clinical course with a rapid decline, whereas those with mixed or osteoclastic giant cell tumors tended to have a better outcome, with a greater long-term survival. One patient is still alive more than 18 months after diagnosis. LIMITATION Retrospective study. CONCLUSIONS Giant cell tumors of the pancreas have unique clinical, endoscopic, and cytologic features. The risk factors for these lesions may be different from those associated with pancreatic adenocarcinoma. Some giant cell tumor subtypes may carry a more favorable prognosis than pancreatic adenocarcinoma, and awareness and recognition of these differences can affect patient care.

Liquid-based cytology for cervical cancer screening.

In multiple studies during the last decade, liquid-based cytology for cervical cancer screening has been shown to increase the detection rate for preneoplastic squamous intraepithelial lesions equal to or greater than the conventional Papanicolaou (Pap) smear method. Liquid-based collection and processing provide more representative cervical sampling than conventional smearing of the specimen on a glass slide. Currently, there are two test methodologies that are widely marketed and available to clinical laboratories, health systems and clinicians that undertake cervical cytology. The purpose of this article is to provide an overview of the methodology and performance of SurePath™ Liquid-Based Pap Test in cervical cytology screening. The SurePath liquid-based Pap test significantly reduces the unsatisfactory rate of Pap test slides, and detects a significantly higher number of low- and high-grade squamous lesions when compared with the conventional Pap smear technique. Biopsy confirmation shows that this increased detection does not come at a cost of decreasing specificity, and sensitivity for histologic dysplasia is equal to or greater than the best available data for the conventional Pap method. The SurePath collection vial provides residual cellular material for adjunctive out-of-the-vial molecular testing, including sexually transmitted diseases and oncologic biomarkers associated with cervical carcinoma. Finally, SurePath slides can be placed on an automated cervical cytology screening device (FocalPoint™), thus providing improved disease detection and enhanced laboratory productivity.

A phase II study of erlotinib as initial treatment for patients with stage IIIB-IV non-small cell lung cancer.

Introduction: Erlotinib improves survival in patients with advanced non-small cell lung cancer who have been previously treated with systemic chemotherapy. The current trial was designed to evaluate erlotinib as a primary therapy before chemotherapy in patients with minimally restricted eligibility criteria. Methods: Eligibility criteria included stage IIIB/IV or recurrent non-small cell lung cancer, no prior chemotherapy for systemic disease, performance status = 0 to 1, no history of brain metastases, and weight loss less than 10%. Patients received erlotinib 150 mg/d until objective or symptomatic progression when they were offered conventional chemotherapy. The primary end point was progression-free survival. Results: Forty patients were accrued. The median age was 65 years, 35 had performance status = 1, 8 were never-smoker, and 23 were former smokers. Histologies were adenocarcinoma in 22 and squamous cell in six. The major toxicity was rash (grade 1, 12; grade 2, 16; grade 3, 3). Partial responses were observed in six (15%), stable in 11 (28%), and progressive disease in 23 (58%). The median time on erlotinib was 8 weeks. The median survival was 50 weeks with 1, 2, and 3 years survivals of 44%, 18%, and 16%. Retrospective epidermal growth factor receptor mutational analysis was performed in 18 subjects and four mutations (22%) were identified. Only 25 patients have received subsequent chemotherapy (too early, 4; refused, 9; and unable because of performance status, 2), and, of these, 9 (36%) achieved unconfirmed responses. Conclusions: Despite a modest response rate, lack of enrichment for never-smokers and absence of conventional chemotherapy in many patients, the median and long-term survivals were comparable with those expected after conventional sequencing of chemotherapy. Erlotinib as initial therapy was well tolerated and warrants randomized evaluation as first-line treatment for advanced lung cancer.

Osteoclastic and pleomorphic giant cell tumors of the pancreas: A review of clinical, endoscopic, and pathologic features

Giant cell tumors of the pancreas come in three varieties-osteoclastic, pleomorphic, and mixed histology. These tumors have distinctive endoscopic, clinical, and cytological features. Giant cell tumors have a controversial histogenesis, with some authors favoring an epithelial origin and others favoring a mesenchymal origin. The true origin of these lesions remains unclear at this time. These are also very rare tumors but proper identification and differentiation from more common pancreatic adenocarcinoma is important. The risk factors of these tumors and the prognosis may be different from those associated with standard pancreatic adenocarcinoma. Recognition of these differences can significantly affect patient care. These lesions have a unique appearance when imaged with endoscopic ultrasound (EUS), and these lesions can be diagnosed via EUS guided Fine Needle Aspiration (FNA). This manuscript will review the endoscopic, clinical, and pathologic features of these tumors.