Identification and Characterization of Optimal Gene Expression Markers for Detection of Breast Cancer Metastasis

Sentinel lymph node (SLN) status is highly predictive of overall axillary lymph node involvement in breast cancer. Historically, SLN-positive patients have undergone axillary lymph node dissection in a second surgery. Intraoperative SLN analysis could reduce the cost and complications of a second surgery; however, existing histopathological methods lack standardization and exhibit poor sensitivity. Rapid molecular methods may lead to improved intraoperative diagnosis of SLN metastasis. In this study,we used a genome-wide gene expression analysis of breast and other tissues to identify seven putative markers for detecting breast cancer metastasis. We assessed the utility of these markers for identifying clinically actionable metastases in lymph nodes through reverse transcriptase-polymerase chain reaction analysis of SLNs from 254 breast cancer patients. Polymerase chain reaction signals were compared to pathology on a per-patient basis. The optimal two-gene combination, mammaglobin and cytokeratin 19, detected clinically actionable metastasis in breast SLNs with 90% sensitivity and 94% specificity. Application of stringent criteria for identifying presumptive hematoxylin- and eosin-positive samples increased sensitivity and specificity to 91 and 97%, respectively. This study represents the first comprehensive demonstration of the utility of gene expression markers for detecting clinically actionable breast metastases. An intraoperative molecular assay using these markers has the potential to significantly reduce second surgeries for patients undergoing SLN dissection. Originally published Journal of Molecular Diagnostics, Vol. 7, No. 3, Aug 200

Microarray analysis reveals genetic pathways modulated by tipifarnib in acute myeloid leukemia

Abstract Background Farnesyl protein transferase inhibitors (FTIs) were originally developed to inhibit oncogenic ras, however it is now clear that there are several other potential targets for this drug class. The FTI tipifarnib (ZARNESTRA™, R115777) has recently demonstrated clinical responses in adults with refractory and relapsed acute leukemias. This study was conducted to identify genetic markers and pathways that are regulated by tipifarnib in acute myeloid leukemia (AML). Methods Tipifarnib-mediated gene expression changes in 3 AML cell lines and bone marrow samples from two patients with AML were analyzed on a cDNA microarray containing approximately 7000 human genes. Pathways associated with these expression changes were identified using the Ingenuity Pathway Analysis tool. Results The expression analysis identified a common set of genes that were regulated by tipifarnib in three leukemic cell lines and in leukemic blast cells isolated from two patients who had been treated with tipifarnib. Association of modulated genes with biological functional groups identified several pathways affected by tipifarnib including cell signaling, cytoskeletal organization, immunity, and apoptosis. Gene expression changes were verified in a subset of genes using real time RT-PCR. Additionally, regulation of apoptotic genes was found to correlate with increased Annexin V staining in the THP-1 cell line but not in the HL-60 cell line. Conclusions The genetic networks derived from these studies illuminate some of the biological pathways affected by FTI treatment while providing a proof of principle for identifying candidate genes that might be used as surrogate biomarkers of drug activity.</p

Validation of the German Version of Functional Oral Intake Scale (FOIS-G) for Flexible Endoscopic Evaluation of Swallowing (FEES)

The Functional Oral Intake Scale (FOIS) is the most frequently used scale for the evaluation of functional oral intake by dysphagia patients. FOIS was validated using data from Videofluoroscopic Swallowing Study (VFSS). Until now, a validated German version of FOIS for Flexible Endoscopic Evaluation of Swallowing (FEES) is lacking. The aim of this study was a cross-cultural validation of the German version of FOIS (FOIS-G) for FEES. The translation of the original FOIS was carried out according to the Translation, Review, Adjudication, Pretesting, Documentation (TRAPD) translation methodology. For the validation process, six experienced language therapists (SLT) retrospectively analyzed charts of 93 stroke patients. Inclusion criteria were comprised of stroke, clinical examination by an SLT within 24 h of admission, and FEES within 72 h of admission. The validity was calculated by comparison with Modified Rankin Scale (MRS), Barthel Index (BI), the Penetration-Aspiration-Scale (PAS), and a water swallow test. Spearman rank correlation of all paired raters ranged from

Detection of Mutated K12- ras in Histologically Negative Lymph Nodes as an Indicator of Poor Prognosis in Stage II Colorectal Cancer

Stage II colorectal carcinoma is characterized by negative lymph node pathology as determined by conventional microscopic examination. These patients generally do not receive adjuvant therapy although 20%–30% will die from metastatic disease. To determine whether K- ras mutations at codon 12 could be used as a sensitive indicator of occult lymph node metastasis in stage II colon carcinoma, a retrospective study was performed using restriction endonuclease- mediated selective polymerase chain reaction (REMS-PCR) amplification. Of 106 colonic tumors analyzed, 46 were identified as positive for a K12- ras mutation in the primary tumor. Multiple lymph node samples from 38 of these 46 patients were examined by a sensitive nested PCR protocol for the presence of a K12- ras mutation. Of these 38 patients, 14 had 1 or more positive lymph nodes by PCR (37%) and 24 were negative for the mutation (63%). Of the 14 patients with a K12- ras mutation detected in lymph nodes, 8 died of the disease within 5 years (57%) compared to only 4 of the 24 patients with ras-negative lymph nodes (17%). The difference in time to death from disease, stratified using K12- ras status of lymph nodes, was statistically significant ( P = 0.036; log-rank test). These results suggest K- ras mutation status of lymph nodes in patients with stage II colon cancer might identify a subgroup of patients who are more likely to develop recurrent and/or metastatic disease and benefit from adjuvant therapy. Larger studies are indicated to determine whether detection of K- ras mutation positivity in histologically negative lymph nodes portends a poor prognosis and to determine whether more aggressive use of adjuvant therapy is warranted

Lymph node homeostasis and adaptation to immune challenge resolved by fibroblast network mechanics.

Emergent physical properties of tissues are not readily understood by reductionist studies of their constituent cells. Here, we show molecular signals controlling cellular, physical, and structural properties and collectively determine tissue mechanics of lymph nodes, an immunologically relevant adult tissue. Lymph nodes paradoxically maintain robust tissue architecture in homeostasis yet are continually poised for extensive expansion upon immune challenge. We find that in murine models of immune challenge, cytoskeletal mechanics of a cellular meshwork of fibroblasts determine tissue tension independently of extracellular matrix scaffolds. We determine that C-type lectin-like receptor 2 (CLEC-2)-podoplanin signaling regulates the cell surface mechanics of fibroblasts, providing a mechanically sensitive pathway to regulate lymph node remodeling. Perturbation of fibroblast mechanics through genetic deletion of podoplanin attenuates T cell activation. We find that increased tissue tension through the fibroblastic stromal meshwork is required to trigger the initiation of fibroblast proliferation and restore homeostatic cellular ratios and tissue structure through lymph node expansion

Clinical and biologic activity of the farnesyltransferase inhibitor R115777 in adults with refractory and relapsed acute leukemias: a phase 1 clinical-laboratory correlative trial

R115777 is a nonpeptidomimetic enzyme-specific inhibitor of farnesyl protein transferase (FT) that was developed as a potential inhibitor of Ras protein signaling, with antitumor activity in preclinical models. This study was a phase 1 trial of orally administered R115777 in 35 adults with poor-risk acute leukemias. Cohorts of patients received R115777 at doses ranging from 100 mg twice daily (bid) to 1200 mg bid for up to 21 days. Dose-limiting toxicity occurred at 1200 mg bid, with central neurotoxicity evidenced by ataxia, confusion, and dysarthria. Non–dose-limiting toxicities included reversible nausea, renal insufficiency, polydipsia, paresthesias, and myelosuppression. R115777 inhibited FT activity at 300 mg bid and farnesylation of FT substrates lamin A and HDJ-2 at 600 mg bid. Extracellular signal-regulated kinase (ERK), an effector enzyme of Ras-mediated signaling, was detected in its phosphorylated (activated) form in 8 (36.4%) of 22 pretreatment marrows and became undetectable in 4 of those 8 after one cycle of treatment. Pharmacokinetics revealed a linear relationship between dose and maximum plasma concentration or area under the curve over 12 hours at all dose levels. Weekly marrow samples demonstrated that R115777 accumulated in bone marrow in a dose-dependent fashion, with large increases in marrow drug levels beginning at 600 mg bid and with sustained levels throughout drug administration. Clinical responses occurred in 10 (29%) of the 34 evaluable patients, including 2 complete remissions. Genomic analyses failed to detect N-ras gene mutations in any of the 35 leukemias. The results of this first clinical trial of a signal transduction inhibitor in patients with acute leukemias suggest that inhibitors of FT may have important clinical antileukemic activity

The spectrum and severity of FUS-immunoreactive inclusions in the frontal and temporal lobes of ten cases of neuronal intermediate filament inclusion disease

Neuronal intermediate filament inclusion disease (NIFID), a rare form of frontotemporal lobar degeneration (FTLD), is characterized neuropathologically by focal atrophy of the frontal and temporal lobes, neuronal loss, gliosis, and neuronal cytoplasmic inclusions (NCI) containing epitopes of ubiquitin and neuronal intermediate filament proteins. Recently, the 'fused in sarcoma' (FUS) protein (encoded by the FUS gene) has been shown to be a component of the inclusions of familial amyotrophic lateral sclerosis with FUS mutation, NIFID, basophilic inclusion body disease, and atypical FTLD with ubiquitin-immunoreactive inclusions (aFTLD-U). To further characterize FUS proteinopathy in NIFID, and to determine whether the pathology revealed by FUS immunohistochemistry (IHC) is more extensive than a-internexin, we have undertaken a quantitative assessment of ten clinically and neuropathologically well-characterized cases using FUS IHC. The densities of NCI were greatest in the dentate gyrus (DG) and in sectors CA1/2 of the hippocampus. Anti-FUS antibodies also labeled glial inclusions (GI), neuronal intranuclear inclusions (NII), and dystrophic neurites (DN). Vacuolation was extensive across upper and lower cortical layers. Significantly greater densities of abnormally enlarged neurons and glial cell nuclei were present in the lower compared with the upper cortical laminae. FUS IHC revealed significantly greater numbers of NCI in all brain regions especially the DG. Our data suggest: (1) significant densities of FUS-immunoreactive NCI in NIFID especially in the DG and CA1/2; (2) infrequent FUS-immunoreactive GI, NII, and DN; (3) widely distributed vacuolation across the cortex, and (4) significantly more NCI revealed by FUS than a-internexin IHC