Kristin M. Post

Utilization of cell-transferred cytologic smears in detection of EGFR and KRAS mutation on adenocarcinoma of lung

Cell-transfer technique has been proven useful for performing immunocytochemistry on fine-needle aspiration smears. However, its utility for EGFR and KRAS molecular testing has not been validated. Molecular testing was performed using the cell-transfer technique on both Papanicolaou-stained ethanol-fixed and Hema 3-stained air-dried smears from 32 fine-needle aspiration samples that had diagnoses of adenocarcinoma of the lung, and then was compared to the results of the corresponding formalin-fixed paraffin-embedded tissues. The molecular testing was successfully performed on 32 of 32 ethanol-fixed and 31 of 32 air-dried samples. The molecular results on ethanol-fixed and air-dried smears showed 100% agreement. There is 100% (32/32) agreement for the EGFR and 97% (31/32) agreement for the KRAS between the cell-transfer technique and formalin-fixed paraffin-embedded tissues. One discrepant case was due to low percentage of tumor cells on the smears. Cell-transfer technique is a reliable alternative method for EGFR and KRAS testing if the cell blocks lack adequate cellularity.

Towards personalized therapy for patients with malignant melanoma: molecular insights into the biology of BRAF mutations

BRAF mutations have been identified as the most common oncogene mutation in melanomas, especially important in those originating on nonchronically sun-damaged skin. There is a large and continually growing body of evidence regarding the importance of this mutation in targeted therapy for melanoma. In this review, we outline these findings including: molecular pathways used by BRAF, the importance in nonmalignant neoplasms, histologic associations, the relationship of BRAF to KIT and NRAS mutations, and their impact on survival, as well as resistance mechanisms to BRAF inhibitors employed by melanoma. Understanding these topics and how they relate to one another may facilitate the development of new treatments and eventually improve the prognosis for those patients afflicted with this disease.

Detection of BRAF Mutations on Direct Smears of Thyroid Fine-Needle Aspirates Through Cell Transfer Technique

OBJECTIVES To determine the utility of the cell transfer technique (CTT) for BRAF molecular testing on thyroid fine-needle aspiration (FNA) specimens. METHODS Polymerase chain reaction (PCR)-based BRAF molecular testing was performed on tissues obtained through CTT from both air-dried and ethanol-fixed direct smears of thyroid FNA specimens and then compared with the corresponding thyroidectomy formalin-fixed, paraffin-embedded (FFPE) tissues on 30 cases. RESULTS BRAF testing was successfully performed on 29 of 30 air-dried CTT, 27 of 30 ethanol-fixed CTT, and 27 of 30 FFPE tissues. The results exhibited 11, 13, and 13 BRAF mutations and 18, 14, and 14 wild types for the air-dried CTT, the ethanol-fixed CTT, and the FFPE tissues, respectively. The concordance rate was 96% between air-dried and ethanol-fixed CTT tissues, 88% between air-dried CTT and FFPE tissues, and 92% between ethanol-fixed CTT and FFPE tissues. CONCLUSIONS PCR-based BRAF mutational testing can be reliably performed on the direct smears of the thyroid FNA specimens through the application of CTT.

EGFR alterations and EML4-ALK rearrangement in primary adenocarcinoma of the urinary bladder

The identification of mutations in epidermal growth factor receptor (EGFR) and translocations involving anaplastic lymphoma kinase (ALK) in lung adenocarcinoma has drastically changed understanding of the disease and led to the development of targeted therapies. Adenocarcinoma of the urinary bladder is rare and poorly understood at the molecular level. We undertook this study to determine whether EGFR mutations, increases in EGFR copy number, or ALK translocations are present in these tumors. Twenty-eight cases of primary bladder adenocarcinoma were analyzed. For EGFR mutational analysis, PCR-amplified products were analyzed on the Q24 Pyrosequencer with Qiagen EGFR Pyro Kits. All cases were analyzed via fluorescence in situ hybridization (FISH) using Vysis ALK Break Apart FISH Probes for detection of ALK chromosomal translocation and Vysis Dual Color Probes to assess for increased gene copy number of EGFR. None of the 28 cases examined showed mutational events in EGFR or ALK rearrangements. EGFR polysomy was seen in 10 out of 28 (36%) cases. No correlation with EGFR polysomy was seen in the tumors with respect to age, histologic subtypes, pathologic stage, or lymph node metastasis. In summary, EGFR mutations and ALK rearrangements do not appear to be involved in the development of primary adenocarcinoma of the urinary bladder. A subgroup of cases (36%), however, demonstrated increased gene copy number of EGFR by FISH.

KRAS mutation is present in a small subset of primary urinary bladder adenocarcinomas

Alexander R E, Lopez‐Beltran A, Montironi R, MacLennan G T, Post K M, Bilbo S A, Jones T D, Huang W, Rao Q, Sen J D, Meehan K, Cornwell A, Miravalle L & Cheng L 
(2012) Histopathology
KRAS mutation is present in a small subset of primary urinary bladder adenocarcinomas

Detection of Cytomegalovirus Infection by Quantitative Polymerase Chain Reaction

Human cytomegalovirus (CMV), also known as human herpes virus-5 (HHV-5), is a common human pathogen acquired early in life in the majority of immunocompetent individuals. Primary infection establishes a state of latency and the virus can be reactivated during immunosuppression. CMV is a significant cause of morbidity and mortality in newborns and patients with impaired immune system. Prenatal infection can result in intrauterine growth retardation, hepatitis, myocarditis, pneumonitis, and neurologic abnormalities. Individuals with congenital or acquired immunosuppression can develop a primary CMV infection, infection with another CMV strain or experience reactivation of the latent virus. The hematopoietic stem cell and solid organ transplant recipients are at high risk of developing CMV infection, especially early in a post-transplant period. The definition of CMV disease includes the evidence of end-organ involvement in the presence of CMV detected by a validated laboratory assay. The selection of a laboratory method is highly dependent on the type of sample to be tested and the clinical presentation. In the clinical practice, the quantitative PCR-based assays are most helpful, since they can measure the level of CMV DNA in whole blood, plasma, cerebrospinal fluid, amniotic fluid, tissue, and urine, and follow the kinetics of infection. In this chapter we describe the PCR assay designed to quantify CMV DNA in human plasma by amplifying a 105 base-pair (bp) fragment of the CMV immediate-early DNA polymerase gene.

Distinct clinicopathological features in metanephric adenoma harboring BRAF mutation

BRAF mutation recently has been reported in metanephric adenoma. We sought to determine the clinical and morphologic features of BRAF-mutated metanephric adenoma and to correlate BRAF mutation with BRAF V600E immunohistochemical staining results. A series of 48 metanephric adenomas and 15 epithelial-predominant nephroblastomas were analyzed for the occurrence of BRAF mutation (BRAF V600E/V600E complex, BRAF V600D, BRAF V600K and BRAF V600R) using the BRAF RGQ PCR kit (Qiagen). Immunohistochemistry was performed using monoclonal mouse antibodies against p16INK4 and VE1 (Spring Bioscience), recognizing the BRAF V600E mutant protein. Forty-one of 48 cases (85%) showed BRAF V600E mutation; none of the other BRAF variants was detected. Of 41 BRAF-mutated metanephric adenomas, 33 showed positive VE1 immunostaining (sensitivity 80%, specificity 100%); in all cases we detected p16INK4 expression regardless of BRAF mutation status. All epithelial-predominant nephroblastomas were BRAF-wild-type and none expressed VE1. The following features were associated with BRAF V600E mutation: older patients (p=0.01), female predominance (p=0.005) and the presence of a predominantly acinar architecture (p=0.003). In summary, BRAF-mutated metanephric adenomas were associated with older age, female predominance, and the presence of a predominant acinar component. A subset (20%) of BRAF-mutated metanephric adenomas was not detected by VE1 immunostaining.

Detection of BRAF mutation in metastatic melanoma utilizing cell-transferred cytological smears.

Objectives: Fine-needle aspiration (FNA) is frequently used to diagnose metastatic melanoma. In this study, we validated the use of cell-transferred cytological smears for BRAF molecular testing. Study Design: We conducted a search of our laboratory information system for the period 2011-2013 in order to identify surgical pathology cases of either primary or metastatic melanomas in which BRAF mutation analyses had already been performed. Thirty FNA cases with diagnoses of metastatic melanoma from the same patients were identified. Direct smears from each FNA case were selected for mutation analyses using the cell transfer technique. Results: Mutation analyses were successfully performed on 28 of 30 FNA cases (93%) using the cell-transferred cytological smears. In 25 cases (8 BRAF mutations and 17 BRAF wild types), there was 100% agreement for the BRAF mutation between the cell-transferred cytological smears and the formalin-fixed paraffin-embedded tissues. Three FNA cases showed BRAF mutations that had not been detected in the correlated surgical specimens which were tested twice, and 2 cases failed to work. Conclusions: Cell-transferred cytological smears are a reliable and alternative resource for detecting BRAF mutations in metastatic melanoma.

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

In Memoriam - A Tribute to Alexander Meisels (1926-2014)

awards, among them the Goldblatt Award of the IAC (1975), the Papanicolaou Award of the ASC (1982) and the membership in the Order of Canada (2000). All members of the Executive Council and the entire membership of the IAC pay tribute to this great man and express their feelings of loss and sadness. Alex will be sadly missed. Volker Schneider, Freiburg i.B. Dr. Alexander Meisels died peacefully in September 2014 at the age of 88 years. His service to the International Academy of Cytology is legendary as he was a member of the Executive Council of the IAC for 39 years. He served as Secretary-Treasurer from 1971 to 1986 and continued subsequently as President, Treasurer and Member until 2010. He was responsible as Secretary or President for six International Congresses of Cytology organized by the IAC (Miami 1974, Tokyo 1977, Munich 1980, Montreal 1983, Brussels 1986 and Buenos Aires 1989). His contributions to the field of cytopathology were decisive and manifold. He considered his discovery of the relationship between the infection by human papillomavirus and the development of cervical carcinoma to be his most significant contribution, of which he was understandably proud. In 1976, he postulated that the koilocytotic changes in cervical epithelial cells represent an expression of viral infection and the initial step of carcinogenesis [1] , a hypothesis which was then controversially debated and is now common knowledge. Born in Berlin, he had to flee Germany with his parents and received his early schooling in Paris, France. He later attended the National University of Mexico, where he obtained his BSc and MD in 1951. In 1960 he moved to Quebec, Canada, where he worked until his retirement at the St. Sacrement Hospital and Laval University as Director of the Department of Pathology and the School of Cytotechnology. He educated numerous cytotechnologists, residents of pathology and foreign guests, who subsequently spread around the world. He was fluent in four languages, was an eloquent and gifted speaker, and travelled widely. He was particularly interested in the Spanish-speaking world and spread the cytologic gospel throughout Latin America. He received numerous Published online: October 29, 2014