The efficacy of on-site evaluation for identification of transplant pancreas.

OBJECTIVE: Pancreatic transplantation has been relatively uncommon until the last 2 decades, but is now an accepted therapeutic intervention for type I diabetes. Core biopsies of pancreas transplants are frequently performed to rule out rejection, which is a leading cause of graft failure. Often, it is difficult to localize the graft for biopsy by imaging. Our objective was to determine whether on-site assessment by cytopathology can assist in procedure protocols for biopsy of pancreas transplants. STUDY DESIGN: We reviewed cytology and surgical pathology reports from 68 instances of pancreas transplant biopsy at our institution and evaluated the correlation between cytology (fine needle aspiration, touch preparation or both) and the final core biopsy. RESULTS: Pancreatic tissue was identified by on-site assessment in 56/68 cases and was present on core biopsy in 53/68 cases. There were 3 cases where fine needle aspiration/touch preparation results were not concordant with final core biopsy results. The positive predictive value of a positive on-site assessment was 96.4%. More importantly, the negative predictive value of on-site assessment was 100%. CONCLUSIONS: On-site evaluation by cytopathology is highly effective for the identification of transplant pancreas. In cases where no pancreatic tissue is identified by on-site assessment, relocalization of the biopsy needle is recommended.

The cytologic criteria of malignancy.

Cytology and cell biology are two separate fields that share a focus on cancer. Cancer is still diagnosed based on morphology, and surprisingly little is known about the molecular basis of the defining structural features. Cytology uses the smallest possible biopsy for diagnosis by reducing morphologic "criteria of malignancy" to the smallest scale. To begin to develop common ground, members of the American Society of Cytopathology Cell Biology Liaison Working Group classify some of the "criteria of malignancy" and review their relation to current cell biology concepts. The criteria of malignancy are extremely varied, apparently reflecting many different pathophysiologies in specific microenvironments. Criteria in Group 1 comprise tissue-level alterations that appear to relate to resistance to anoikis, alterations in cell adhesion molecules, and loss of apical-basal polarity. Criteria in Group 2 reflect genetic instability, including chromosomal and possibly epigenetic instability. Criteria in Groups 3 are subcellular structural changes involving cytoplasmic components, nuclear lamina, chromatin and nucleoli that cannot be accounted for by genetic instability. Some distinct criteria in Group 3 are known to be induced by cancer genes, but their precise structural basis remains obscure. The criteria of malignancy are not closely related to the histogenetic classification of cancers, and they appear to provide an alternative, biologically relevant framework for establishing common ground between cytologists and cell biologists. To understand the criteria of malignancy at a molecular level would improve diagnosis, and likely point to novel cell physiologies that are not encompassed by current cell biology concepts.

C-fos assessment as a marker of anti-epidermal growth factor receptor effect.

Factors predicting sensitivity to epidermal growth factor receptor (EGFR) blockade are largely unknown and new strategies are being sought to individualize cancer therapy. This study evaluated the variation in the expression of the early response gene c-fos as a distal effect of EGFR inhibition and its relationship to antitumor effects. The growth-inhibitory and c-fos-modulating effects of gefitinib and erlotinib in human cancer cell lines (A431, CAL27, HN11, HuCCT1, and Hep2) were determined. Next, these cell lines were xenografted in mice and treated for 14 days with gefitinib (A431 and HuCCT1) or erlotinib (CAL27, HN11, and Hep2). Fine needle aspiration biopsy of tumors was done at baseline and after 14 days of therapy for c-fos assessment. In addition, we tested the feasibility of analyzing this marker in five paired tumor samples from a clinical trial of gefitinib in patients with solid tumors. In culture, gefitinib and erlotinib decreased c-fos mRNA levels in the susceptible cell lines A431, CAL27, and HN11; however, both drugs failed to achieve c-fos inhibition in resistant cells. Gefitinib or erlotinib abrogated the increase in c-fos expression in vivo in EGFR-sensitive A431, CAL27, and HN11 tumors but not in resistant strains. Ex vivo evaluation was feasible and predicted in vivo effects. The feasibility study in paired human tumor biopsies showed that this biomarker can be reliably measured in clinical materials. In summary, variations in c-fos expression reflect the pharmacologic actions of EGFR inhibitors in in vitro and in vivo models.

Endoscopic ultrasound-guided fine-needle aspiration: sampling, pitfalls, and quality management.

Endoscopic ultrasound-guided fine-needle aspiration (EUS FNA) is an accurate and versatile technique in the diagnosis of gastrointestinal tract lesions as well as other organ sites. EUS FNA is performed ideally with cytopathologic adequacy evaluation, and diagnosis at the time of tissue procurement. In this article, we review the most relevant issues in the process of EUS FNA-based diagnostics. Specifically, we describe the technical aspects of specimen collection, processing, and appropriate selection of ancillary studies. We also illustrate the most commonly encountered diagnostic pitfalls, and methods for their avoidance. Lastly, we discuss quality management, which emphasizes the communication between the endoscopist and the pathologist.

Epidermal growth factor receptor dynamics influences response to epidermal growth factor receptor targeted agents.

Analysis of gene expression of cancer cell lines exposed to erlotinib, a small molecule inhibitor of the epidermal growth factor receptor (EGFR), showed a marked increase in EGFR mRNA in resistant cell lines but not in susceptible ones. Because cetuximab induces EGFR down-regulation, we explored the hypothesis that treatment with cetuximab would interfere with erlotinib-induced EGFR up-regulation and result in antitumor effects. Exposure of the resistant biliary tract cancer cell line HuCCT1 but not the susceptible A431 epidermoid cell line to erlotinib induced EGFR mRNA and protein expression. Combined treatment with cetuximab blunted the erlotinib-induced EGFR up-regulation and resulted in inhibition of cell proliferation and apoptosis in the HuCCT1 cells. Blockage of erlotinib-induced EGFR synthesis in HuCCT1 cells by small interfering RNA resulted in identical antitumor effects as cetuximab, providing mechanistic specificity. In mice xenografted with A431, HuCCT1, and the pancreatic cancer cell line Panc430, maximal growth arrest and decrease in Ki67 proliferation index were documented with combined therapy, and EGFR down-regulation was observed in cetuximab-treated tumors. These results may indicate that resistance to EGFR kinase inhibition may be, at least in part, mediated by a highly dynamic feedback loop consisting of up-regulation of the EGFR upon exposure to EGFR kinase inhibitors. Abrogation of this response by small interfering RNA-mediated EGFR mRNA down-regulation and/or by cetuximab-mediated protein clearance induced tumor arrest across several cancer models with different EGFR expression levels, suggesting that resistance and sensitivity are dynamic events where proportional decrease in the target rather than absolute content dictates outcome.

Assessment of celecoxib pharmacodynamics in pancreatic cancer.

Cyclooxygenase-2 (COX-2) inhibitors are being developed as chemopreventive and anticancer agents. This study aimed to determine the biological effect of the COX-2 inhibitor celecoxib in pancreatic cancer as an early step to the further development of the agent in this disease. Eight patients scheduled for resection of an infiltrating adenocarcinoma of the pancreas were randomized to receive celecoxib at a dose of 400 mg twice daily or placebo for 5 to 15 days before the surgery. In addition, carcinomas from nine additional patients were xenografted in nude mice, expanded, and treated with vehicle or celecoxib for 28 days. Celecoxib markedly decreased the intra-tumor levels of prostaglandin E2 in patient carcinomas and in the heterotransplanted xenografts. However, this effect did not result in inhibition of cell proliferation or microvessel density (as assessed by Ki67 and CD31 staining). In addition, a panel of markers, including bcl-2, COX-1, COX-2, and VEGF, did not change with treatment in a significant manner. Furthermore, there was no evidence of antitumor effects in the xenografted carcinomas. In summary, celecoxib efficiently inhibited the synthesis of prostaglandin E2 both in pancreatic cancer surgical specimens and in xenografted carcinomas but did not exert evident antitumor, antiproliferative, or antiangiogenic effect as a single agent. The direct pancreatic cancer xenograft model proved to be a valuable tool for drug evaluation and biological studies and showed similar results to those observed in resected pancreatic cancer specimens.

A proposed clinical test for monitoring fluoropyrimidine therapy: detection and stability of thymidylate synthase ternary complexes.

5-fluorouracil forms classic (covalent, ternary) complexes consisting of thymidylate synthase, fluoro-deoxyuridine monophosphate, and 5,10-methylene tetrahydrofolate. Despite a high pharmacologic interest in the classic complexes formed in cells treated with fluorouracil anticancer agents, the in vivo stability of the complexes and the possible interference in complex formation by other coadministered compounds have not been adequately described. We visualized classic complexes unaccompanied by unbound thymidylate synthase, inferring complete enzymatic inhibition, in 5-fluorouracil-treated S. cerevisiae and cancer cells in vitro and in murine tumors in vivo treated with 5-fluorouracil. Classic complexes persisted 13 days in cancer cells after a pulse of 5-fluorouracil. Classic complexes were reduced to absent in cancer cells in which the older antifolates methotrexate and aminopterin, or the modern antifolates pemetrexed and tomudex, were coadministered with 5-fluorouracil. Classic complexes were, however, detected when an alternate drug, 5-fluorodeoxyuridine, was administered with methotrexate. We visualized classic complexes at fifteen minutes to seven days after an acute single dose of 5-fluorouracil in mouse tumor models, in tumors and normal tissues. Using the same assay, we detected unbound thymidylate synthase in untreated human tissues, supporting the future use of this assay in evaluating the most appropriate dose of fluoropyrimidine and coadministered agents in clinical settings.

Myxopapillary ependymoma: cytomorphologic characteristics and differential diagnosis.

Myxopapillary ependymoma (ME) is a rare tumor with a predilection for sacrococcygeal area of adults. We present the cytomorphology of one such case in a 21-yr-old man, diagnosed by fine-needle aspiration (FNA). The tumor disclosed biphasic morphology with nests and aggregates of epithelioid malignant cells as well as branching cords of myxohyaline material. Most noticeable was the presence of distinct hyaline globules surrounded by neoplastic cells. The differential diagnosis of ME includes chordoma, adenoid cystic carcinoma, mucinous adenocarcinoma, and germ cell tumor. An accurate preoperative diagnosis of ME on FNA can be achieved based on its distinctive cytomorphologic features.

A phase I trial of aminolevulinic acid-photodynamic therapy for treatment of oral leukoplakia.

BACKGROUND: Photodynamic therapy with aminolevulinic acid (ALA PDT) for oral leukoplakia has shown promising effects in regression of oral leukoplakia. Although ALA has been extensively studied and is an ideal photosensitizer, the optimal light dose for treatment of oral leukoplakia has not been determined. We conducted a phase I study to determine MTD and DLT of PDT in patients treated with ALA for leukoplakia. METHODS: Patients with histologically confirmed oral leukoplakia received a single treatment of ALA PDT in cohorts with escalating doses of light (585nm). Clinical, histologic, and biologic markers were assessed. RESULTS: Analysis of 11 participants is reported. No significant toxicity from ALA PDT was observed in patients who received ALA with a light dose of up to 4J/cm(2). One participant experienced transient grade 3 transaminase elevation due to ALA. One participant had a partial clinical response 3months after treatment. Biologic mucosal risk markers showed no significant associations. Determination of MTD could not be accomplished within a feasible timeframe for completion of the study. CONCLUSIONS: ALA PDT could be safely administered with a light dose up to 4J/cm(2) and demonstrated activity. Larger studies are needed to fully elucidate the MTD and efficacy of ALA-PDT.

Optimal specimen processing of fine needle aspirates of non-Hodgkin lymphoma.

UNLABELLED: Fine needle aspiration (FNA) in conjunction with flow cytometry (FC) is a useful technique for non-Hodgkin's lymphoma (NHL) diagnosis. We sought to investigate the effect of storage medium and time to processing on lymph node (LN) FNA viability. Benign LN FNAs were distributed among Roswell Park Memorial Institute (RPMI), Hanks' Balanced Salt Solution (HBSS) and Dulbecco's Modified Eagle's Medium (DMEM) storage media, and viability was compared at 0, 4.5 and 24 hours. FC survival analysis showed viable cells (%): at 0 hrs: HBSS 83.6%, RPMI 87.7%, DMEM 87.7%. At 4.5 hrs: HBSS 86.3%, RPMI 89.0%, DMEM 78.2%. At 24 hrs: HBSS 82.7%, RPMI 86.7%, DMEM 77.2%. FNA from a peri-pancreatic LN involved by grade 2 follicular lymphoma was stored in RPMI at 4° C and analyzed at 1, 3, 5 and 7 days. Over 90% of follicular lymphoma cells were suitable for FC analysis at 1, 3, and 5 days after collection, decreasing to 76% at 7 days. IN CONCLUSION: RPMI appears to be the optimal storage medium compared to DMEM and HBSS.An NHL FNA sample stored at 4° C remains suitable for FC analysis for up to 5 days.

Metastatic chromophobe renal cell carcinoma in pleural fluid cytology: review of literature and report of a case.

The majority of chromophobe renal cell carcinomas (CHRCC) are not aggressive, however, a subset are. The potential for metastasis makes recognition of CHRCC cells in cytologic preparations important for patient surveillance, management, and treatment. We report the detection of an aggressive form of CHRCC in pleural fluid cytology with histopathologic confirmation on subsequent pleural biopsy. Sarcomatoid portions of the original CHRCC, and the pleural metastasis, demonstrated loss of diffuse membranous staining with C-KIT.

Emerging concepts in the pathology and molecular biology of advanced non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is traditionally classified histologically, but until recently, the histologic subtype has had little impact on the selection of therapy. Drugs such as pemetrexed and bevacizumab are indicated for specific NSCLC subtypes, and this type of stratification represents the first step toward individualizing therapy in NSCLC. Beyond histologic features, the status of molecular targets, such as the epidermal growth factor receptor (EGFR) gene, has been shown to correlate with response to treatment with EGFR tyrosine kinase inhibitors in patients with relapsed or refractory disease and in the first-line therapy setting. New therapies targeting the EGFR and other molecular aberrations are under way to help define specific subsets of patients responsive to certain molecularly targeted treatments. The role of pathologists in guiding treatment decisions will increase because molecular profiling, together with pathologic and histologic analysis, represents the future of personalizing medicine for patients with NSCLC.

Epidermal growth factor receptor (EGFR) intron 1 polymorphism and clinical outcome in pancreatic adenocarcinoma.

BACKGROUND: Epidermal growth factor receptor (EGFR) intron 1 has a polymorphic region of CA repeats that is believed to be associated with increased EGFR expression, tumor aggressiveness, and worse survival in cancer patients. METHODS: A large population of pancreatic adenocarcinoma patients was investigated to evaluate this polymorphism as a potential prognostic marker of clinical outcome. Deoxyribonucleic acid obtained from 50 resected pancreatic adenocarcinomas and from 85 diagnostic endoscopic ultrasound-guided fine-needle aspiration procedures corresponding to patients with unresectable tumors was included. The correlation between CA repeat length and EGFR messenger ribonucleic acid levels was also examined. RESULTS: Analysis of the 135 patients revealed no correlation between EGFR intron 1 CA repeat length and tumor stage. There was no difference in overall patient survival when stratified by allele length. A correlation between EGFR intron 1 length and EGFR transcript and protein levels could not be established. CONCLUSIONS: The length of the EGFR intron 1 CA repeats does not correlate with levels of EGFR expression and cannot be used as marker of clinical prognosis in pancreatic cancer patients.

ErbB3 expression promotes tumorigenesis in pancreatic adenocarcinoma.

Historically, ErbB3 has been overlooked within the ErbB receptor family due to its perceived lack of tyrosine kinase activity. We have previously demonstrated that in pancreatic cancer ErbB3 is the preferred dimerization partner of EGFR, ErbB3 protein expression level directly correlates with the anti-proliferative effect of erlotinib (an EGFR-specific tyrosine kinase inhibitor), and transient knockdown of ErbB3 expression results in acquired resistance to EGFR-targeted therapy. In this study, we develop a stable isogenic model of ErbB3 expression in an attempt to decipher ErbB3's true contribution to pancreatic cancer tumorigenesis and to examine how this receptor affects cellular sensitivity to EGFR-targeted therapy. Analysis of the EGFR-ErbB3 heterodimer demonstrates that ligand-induced PI3K-AKT signaling is limited to ErbB3-expressing cells and that this signaling cascade can be partially abrogated by inhibiting EGFR function with erlotinib. Using our model of exogenous ErbB3 expression we showed a direct relationship between ErbB3 protein levels and increased pancreatic cancer cell proliferation in vitro. In vivo, ErbB3(+)PANC-1 xenografts had a significantly larger tumor volume than PANC-1 control xenografts (ErbB3-PANC-1) and displayed increased sensitivity to EGFR-targeted therapy. In pancreatic cancer, ErbB3 appears to be critically involved in EGFR signaling as evidenced by its profound effect on cellular proliferation and its ability to influence response to EGFR-targeted therapy.

Intrapancreatic schwannoma diagnosed by endoscopic ultrasound-guided fine-needle aspiration cytology.

Schwannoma is a tumor of neuro-ectodermal origin, usually occuring in the head and neck and extremities. A retroperitoneal, and particularly intra-pancreatic presentation is very rare, and poses a clinical and diagnostic challenge. We report a case of a male patient who underwent an Endoscopic Ultrasound-guided Fine Needle Aspiration (EUS-FNA) biopsy of a hypoechoic, intra-pancreatic mass. The onsite cytological evaluation was consistent with a spindle cell neoplasm. Further evaluation, aided by immunohistochemical stains, defined the mass as a Schwannoma. The patient then underwent a pancreaticoduodenectomy and the histopathological diagnosis of the surgical specimen confirmed the cytological diagnosis. To our knowledge, this is the first report of intra-pancreatic Schwannoma diagnosed preoperatively by EUS-FNA cytology.

Coordinated epidermal growth factor receptor pathway gene overexpression predicts epidermal growth factor receptor inhibitor sensitivity in pancreatic cancer.

The epidermal growth factor receptor (EGFR) inhibitor erlotinib is approved for treatment of pancreatic cancer but the overall activity is minimal, and known predictive factors for EGFR inhibitor efficacy are infrequent in this disease. We tested the hypothesis that global activation of the EGFR pathway is predictive of EGFR inhibitor efficacy. Pancreatic cancer tumors directly xenografted at surgery were treated with the EGFR inhibitors erlotinib and cetuximab and analyzed for biological features. Two of 10 tumors were sensitive, and by global gene expression profiling with gene set enrichment analysis, the EGFR pathway was highly expressed in sensitive compared with resistant tumors. The core gene components driving EGFR pathway overexpression were pathway ligands and positive effectors. In a prospective validation, the EGFR pathway-based signature correctly predicted anti-EGFR treatment response in eight additional tumors and was not predictive of response to gemcitabine and CI1040 (a MEK inhibitor). Analysis of EGFR, KRAS, and PIK3CA mutations and gene amplification by fluorescence in situ hybridization and multiplex ligation-dependent probe amplification showed that none of these genetic abnormalities were neither predictive nor responsible for the EGFR pathway activation. Coordinated overexpression of the EGFR pathway predicts susceptibility to EGFR inhibitors in pancreatic cancer. These results suggest a phenomenon of pathway addiction and support the value of unbiased system biology approaches in drug development.

Pharmacodynamic-guided modified continuous reassessment method-based, dose-finding study of rapamycin in adult patients with solid tumors.

PURPOSE: Pharmacodynamic studies are frequently incorporated into phase I trials, but it is uncommon that they guide dose selection. We conducted a dose selection study with daily rapamycin (sirolimus) in patients with solid tumors employing a modified continuous reassessment method (mCRM) using real-time pharmacodynamic data as primary dose-estimation parameter. PATIENTS AND METHODS: We adapted the mCRM logit function from its classic toxicity-based input data to a pharmacodynamic-based input. The pharmacodynamic end point was skin phospho-P70 change after 28 days. Pharmacodynamic effect was defined as at least 80% inhibition from baseline. The first two dose levels (2 and 3 mg) were evaluated before implementing the mCRM, and the data used to estimate the next dose level based on statistical modeling. Toxicity-based boundaries limited the escalation steps. Other correlates analyzed were positron emission tomography (PET) and computed tomography, pharmacokinetics, phospho-P70 in peripheral-blood mononuclear cells, and tumor biopsies in patients at the maximum-tolerated dose (MTD). RESULTS: Twenty-one patients were enrolled at doses between 2 and 9 mg. Pharmacodynamic effect occurred across dose levels, and toxicity boundaries ultimately drove dose selection. The MTD of daily oral rapamycin was 6 mg. Toxicities in at least 20% were hyperglycemia, hyperlipidemia, elevated transaminases, anemia, leucopenia, neutropenia, and mucositis. Pharmacokinetics were consistent with prior data, and exposure increased with dose. No objective responses occurred, but five previously progressing patients received at least 12 cycles. PET showed generalized stable or decreased glucose uptake unrelated to antitumor effect. CONCLUSION: mCRM-based dose escalation using real-time pharmacodynamic assessment was feasible. However, the selected pharmacodynamic end point did not correlate with dose. Toxicity ultimately drove dose selection. Rapamycin is a well-tolerated and active oral anticancer agent.

Analysis of biologic surrogate markers from a Children's Oncology Group Phase I trial of gefitinib in pediatric patients with solid tumors.

This trial evaluated the effect of gefitinib on the plasma circulating levels of epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMP)-2 and -9 of patients treated on a pediatric Phase I trial. Complete plasma correlative studies were obtained from 16 of the 25 enrolled patients. There was a trend for lower MMP-2 baseline levels in patients with partial response or stable disease. The Ewing sarcoma from the only patient with partial response lacked egfr mutations. Gefitinib did not induce any significant variation in the levels of the assessed parameters, and none of these determinations showed significant predictive or prognostic value.

DNA-PKcs dependence of Artemis endonucleolytic activity, differences between hairpins and 5' or 3' overhangs.

During V(D)J recombination, the RAG proteins create DNA hairpins at the V, D, or J coding ends, and the structure-specific nuclease Artemis is essential to open these hairpins prior to joining. Artemis also is an endonuclease for 5' and 3' overhangs at many DNA double strand breaks caused by ionizing radiation, and Artemis functions as part of the nonhomologous DNA end joining pathway in repairing these. All of these activities require activation of the Artemis protein by interaction with and phosphorylation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). In this study, we have identified a region of the Artemis protein involved in the interaction with DNA-PKcs. Furthermore, the biochemical and functional analyses of C-terminally truncated Artemis variants indicate that the hair-pin opening and DNA overhang endonucleolytic features of Artemis are triggered by DNA-PKcs in two modes. First, autoinhibition mediated by the C-terminal tail of Artemis is relieved by phosphorylation of this tail by DNA-PKcs. Thus, C-terminally truncated Artemis derivatives imitate DNA-PKcs-activated wild type Artemis protein and exhibit intrinsic hairpin opening activity. Second, DNA-PKcs may optimally configure 5' and 3' overhang substrates for the endonucleolytic function of Artemis.