Linda Sercia

von Hippel-Lindau Gene Status and Response to Vascular Endothelial Growth Factor Targeted Therapy for Metastatic Clear Cell Renal Cell Carcinoma

PURPOSE The von Hippel-Lindau (VHL) gene is often inactivated (by mutation or promoter hypermethylation) in renal cell carcinoma but the relation to therapeutic outcome is unclear. MATERIALS AND METHODS Patients with metastatic clear cell renal cell carcinoma with available baseline tumor samples who received vascular endothelial growth factor targeted therapy were included in analysis. Patient characteristics, VHL gene status and clinical outcome were documented. Our primary end point was to test for response rate in relation to VHL inactivation. Progression-free survival and overall survival in relation to VHL status were investigated as secondary end points. RESULTS A total of 123 patients were evaluable. Response rate, median progression-free survival and median overall survival were 37% (95% CI 28-46), 10.8 (95% CI 7.7-14.8) and 29.8 (CI not estimable) months, respectively. Patients with VHL inactivation had a response rate of 41% vs 31% for those with wild-type VHL (p = 0.34). Patients with loss of function mutations (frameshift, nonsense, splice and in-frame deletions/insertions) had a 52% response rate vs 31% with wild-type VHL (p = 0.04). On multivariate analysis the presence of a loss of function mutation remained an independent prognostic factor associated with improved response. Progression-free survival and overall survival were not significantly different based on VHL status. CONCLUSIONS To our knowledge this is the largest analysis investigating the impact of VHL inactivation on the outcome of vascular endothelial growth factor targeted agents in metastatic renal cell carcinoma. We did not find a statistically significant increase in response to vascular endothelial growth factor targeted agents in patients with VHL inactivation. Loss of function mutations identified a population of patients with a greater response. Investigation of downstream markers is under way.

Renal angiomyolipoma: clinicopathologic study of 194 cases with emphasis on the epithelioid histology and tuberous sclerosis association.

The majority of renal angiomyolipoma (AML) is sporadic and occasionally it occurs as part of tuberous sclerosis complex (TSC). Epithelioid AML (EAML), an uncommon variant, is considered potentially malignant based on anecdotal case reports. The prognostic significance of epithelioid component in an otherwise typical AML is uncertain. We studied 194 AMLs for the clinicopathologic features of epithelioid and TSC-associated AMLs. Epithelioid component was present in 15 cases (7.7%) with an average amount of 51% (range: 10% to 100%). Histologically, the epithelioid tumor cells were categorized into small, intermediate, and large cell type based on the cell size. Worrisome histologic features were seen in many EAMLs, including coagulative tumor necrosis in 27% (4/15), nuclear atypia in 93% (14/15), mitosis in 47% (7/15), and atypical mitosis in 1 case. All 15 EAML patients had a mean follow-up time of 5.1 years and none had local recurrence or distant metastasis. Sixteen (8.2%) AMLs occurred in patients with definitive TSC. Three histologic features, namely microscopic AML foci, epithelioid component, and epithelial cysts, were present in 10 (62.5%), 4 (25%), and 44% (7/16), respectively, of TSC-associated AMLs, compared with 11 (6.2%), 11 (6.2%), and 6 (3.4%), respectively, in non–TSC-associated AMLs (P value all <0.01). In summary, all 15 cases of EAMLs in our study had benign clinical outcomes despite adverse pathologic features. Epithelioid component, epithelial cysts, and microscopic AML foci are strongly associated with TSC and the presence of all 3 features should raise strong suspicion for TSC.

Renal tubulocystic carcinoma is closely related to papillary renal cell carcinoma: implications for pathologic classification.

Tubulocystic carcinoma of the kidney (TC-RCC) is a rare renal tumor with unique gross and microscopic features unlike other types of renal cell carcinoma (RCC). Several recent studies recommend that it should be classified as a distinct RCC subtype. In this study, we provide pathologic and cytogenetic evidence supporting that TC-RCC is closely related to papillary RCC (PRCC). This study included 20 cases of renal tumors that partially or exclusively comprised a TC-RCC component. Pathologic examination documented the gross and microscopic features of TC-RCC, including multicentricity and the presence of concomitant PRCC and papillary adenoma. Formalin-fixed, paraffin-embedded sections from 12 TC-RCC and 20 PRCC were subjected to a multicolor fluorescence in situ hybridization assay containing probes for chromosomes 7, 17, and Y. One hundred nuclei were examined to enumerate the copy numbers of chromosomes in each tumor and its corresponding normal kidney tissue. A tumor with a percentage of cells harboring a chromosomal change ≥mean+3 SD of normal tissue was considered to harbor that chromosomal change, and a tumor with a percentage of cells with null Y chromosome count (loss of Y chromosome) ≥mean+3 SD of normal tissue was considered to harbor Y chromosome loss. Four of the 20 TC-RCCs were multicentric. Ten had associated PRCC or papillary adenoma within the same kidney as the TC-RCC. In 4 cases, the tubulocystic and papillary components were admixed together within the same lesion. The tumor cells lining both the tubulocystic and papillary components had similar cytologic features. Ten of 12 TC-RCCs had a chromosome 7 gain, 8 of 12 cases had a chromosome 17 gain, and 8 of 9 cases had a loss of Y chromosome. Six of 9 cases with all 3 chromosomes studied had a gain of chromosomes 7 and 17 and a loss of Y chromosome. Our study shows that TC-RCCs and PRCCs are closely related entities. With its distinctive gross and microscopic features, TC-RCC may be considered a unique “morphologic entity.” However, before it is accepted as a distinct renal cell carcinoma subtype, further studies are needed to document a characteristic molecular signature associated with this tumor.

Evaluation of the Verigene Gram-positive blood culture nucleic acid test for rapid detection of bacteria and resistance determinants.

ABSTRACT Rapid identification of pathogens from blood cultures can decrease lengths of stay and improve patient outcomes. We evaluated the accuracy of the Verigene Gram-positive blood culture (BC-GP) nucleic acid test for investigational use only (Nanosphere, Inc., Northbrook, IL) for the identification of Gram-positive bacteria from blood cultures. The detection of resistance genes (mecA in Staphylococcus aureus and Staphylococcus epidermidis and vanA or vanB in Enterococcus faecium and Enterococcus faecalis) by the BC-GP assay also was assessed. A total of 186 positive blood cultures (in BacT/Alert FA bottles) with Gram-positive cocci observed with Gram staining were analyzed using the BC-GP assay. The BC-GP results were compared with the identification and susceptibility profiles obtained with routine methods in the clinical laboratory. Discordant results were arbitrated with additional biochemical, cefoxitin disk, and repeat BC-GP testing. The initial BC-GP organism identification was concordant with routine method results for 94.6% of the blood cultures. Only 40% of the Streptococcus pneumoniae identifications were correct. The detection of the mecA gene for 69 blood cultures with only S. aureus or S. epidermidis was concordant with susceptibility testing results. For 3 of 6 cultures with multiple Staphylococcus spp., mecA detection was reported but was correlated with oxacillin resistance in a species other than S. aureus or S. epidermidis. The detection of vanA agreed with susceptibility testing results for 45 of 46 cultures with E. faecalis or E. faecium. Comparison of the mean times to results for each organism group showed that BC-GP results were available 31 to 42 h earlier than phenotypic identifications and 41 to 50 h earlier than susceptibility results.

Multicenter Evaluation of the Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System for Identification of Gram-Positive Aerobic Bacteria

ABSTRACT Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF) is gaining momentum as a tool for bacterial identification in the clinical microbiology laboratory. Compared with conventional methods, this technology can more readily and conveniently identify a wide range of organisms. Here, we report the findings from a multicenter study to evaluate the Vitek MS v2.0 system (bioMérieux, Inc.) for the identification of aerobic Gram-positive bacteria. A total of 1,146 unique isolates, representing 13 genera and 42 species, were analyzed, and results were compared to those obtained by nucleic acid sequence-based identification as the reference method. For 1,063 of 1,146 isolates (92.8%), the Vitek MS provided a single identification that was accurate to the species level. For an additional 31 isolates (2.7%), multiple possible identifications were provided, all correct at the genus level. Mixed-genus or single-choice incorrect identifications were provided for 18 isolates (1.6%). Although no identification was obtained for 33 isolates (2.9%), there was no specific bacterial species for which the Vitek MS consistently failed to provide identification. In a subset of 463 isolates representing commonly encountered important pathogens, 95% were accurately identified to the species level and there were no misidentifications. Also, in all but one instance, the Vitek MS correctly differentiated Streptococcus pneumoniae from other viridans group streptococci. The findings demonstrate that the Vitek MS system is highly accurate for the identification of Gram-positive aerobic bacteria in the clinical laboratory setting.

Multicenter Study Evaluating the Vitek MS System for Identification of Medically Important Yeasts

ABSTRACT The optimal management of fungal infections is correlated with timely organism identification. Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) is revolutionizing the identification of yeasts isolated from clinical specimens. We present a multicenter study assessing the performance of the Vitek MS system (bioMérieux) in identifying medically important yeasts. A collection of 852 isolates was tested, including 20 Candida species (626 isolates, including 58 C. albicans, 62 C. glabrata, and 53 C. krusei isolates), 35 Cryptococcus neoformans isolates, and 191 other clinically relevant yeast isolates; in total, 31 different species were evaluated. Isolates were directly applied to a target plate, followed by a formic acid overlay. Mass spectra were acquired using the Vitek MS system and were analyzed using the Vitek MS v2.0 database. The gold standard for identification was sequence analysis of the D2 region of the 26S rRNA gene. In total, 823 isolates (96.6%) were identified to the genus level and 819 isolates (96.1%) were identified to the species level. Twenty-four isolates (2.8%) were not identified, and five isolates (0.6%) were misidentified. Misidentified isolates included one isolate of C. albicans (n = 58) identified as Candida dubliniensis, one isolate of Candida parapsilosis (n = 73) identified as Candida pelliculosa, and three isolates of Geotrichum klebahnii (n = 6) identified as Geotrichum candidum. The identification of clinically relevant yeasts using MS is superior to the phenotypic identification systems currently employed in clinical microbiology laboratories.

Identification of Enterobacteriaceae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using the VITEK MS system.

This multicenter study evaluated the accuracy of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry identifications from the VITEK MS system (bioMérieux, Marcy l’Etoile, France) for Enterobacteriaceae typically encountered in the clinical laboratory. Enterobacteriaceae isolates (n = 965) representing 17 genera and 40 species were analyzed on the VITEK MS system (database v2.0), in accordance with the manufacturer’s instructions. Colony growth (≤72 h) was applied directly to the target slide. Matrix solution (α-cyano-4-hydroxycinnamic acid) was added and allowed to dry before mass spectrometry analysis. On the basis of the confidence level, the VITEK MS system provided a species, genus only, or no identification for each isolate. The accuracy of the mass spectrometric identification was compared to 16S rRNA gene sequencing performed at MIDI Labs (Newark, DE). Supplemental phenotypic testing was performed at bioMérieux when necessary. The VITEK MS result agreed with the reference method identification for 96.7 % of the 965 isolates tested, with 83.8 % correct to the species level and 12.8 % limited to a genus-level identification. There was no identification for 1.7 % of the isolates. The VITEK MS system misidentified 7 isolates (0.7 %) as different genera. Three Pantoea agglomerans isolates were misidentified as Enterobacter spp. and single isolates of Enterobacter cancerogenus, Escherichia hermannii, Hafnia alvei, and Raoultella ornithinolytica were misidentified as Klebsiella oxytoca, Citrobacter koseri, Obesumbacterium proteus, and Enterobacter aerogenes, respectively. Eight isolates (0.8 %) were misidentified as a different species in the correct genus. The VITEK MS system provides reliable mass spectrometric identifications for Enterobacteriaceae.

Multi-centre evaluation of mass spectrometric identification of anaerobic bacteria using the VITEK® MS system

Accurate and timely identification of anaerobic bacteria is critical to successful treatment. Classic phenotypic methods for identification require long turnaround times and can exhibit poor species level identification. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an identification method that can provide rapid identification of anaerobes. We present a multi-centre study assessing the clinical performance of the VITEK(®) MS in the identification of anaerobic bacteria. Five different test sites analysed a collection of 651 unique anaerobic isolates comprising 11 different genera. Multiple species were included for several of the genera. Briefly, anaerobic isolates were applied directly to a well of a target plate. Matrix solution (α-cyano-4-hydroxycinnamic acid) was added and allowed to dry. Mass spectra results were generated with the VITEK(®) MS, and the comparative spectral analysis and organism identification were determined using the VITEK(®) MS database 2.0. Results were confirmed by 16S rRNA gene sequencing. Of the 651 isolates analysed, 91.2% (594/651) exhibited the correct species identification. An additional eight isolates were correctly identified to genus level, raising the rate of identification to 92.5%. Genus-level identification consisted of Actinomyces, Bacteroides and Prevotella species. Fusobacterium nucleatum, Actinomyces neuii and Bacteroides uniformis were notable for an increased percentage of no-identification results compared with the other anaerobes tested. VITEK(®) MS identification of clinically relevant anaerobes is highly accurate and represents a dramatic improvement over other phenotypic methods in accuracy and turnaround time.

Detection of a novel point mutation of the prothrombin gene at position 20209.

Detection of the prothrombin G20210A mutation was performed on the LightCycler Instrument (Roche Molecular Biochemicals, Mannheim, Germany) using commercially available primers and hybridization probes. Herein we report four cases from unrelated African American individuals where LightCycler analysis showed atypical melting curves. Sequence analysis of the four samples showed heterozygosity for a C to T mutation 1 bp upstream from the known 20210 mutation at position 20209. The clinical significance of this mutation is not known, but three patients in whom it was detected had a history of venous thrombosis or stroke. The fourth patient had severe liver disease, which may have masked thrombotic predisposition. Since most assays used to detect the G20210A mutation use a PCR/restriction digestion assay, which would not detect the C20209T mutation, this new mutation may be underrecognized when prothrombin gene mutation testing is performed by PCR/restriction digestion assay.

Renal neuroendocrine tumours: a clinicopathological study.

To report cases of primary neuroendocrine tumours (NETs) of the kidney, including carcinoid tumour, large cell neuroendocrine carcinoma (LCNEC) and small cell carcinoma (SCC), which show a wide range of NE differentiation and biological behaviour, and are exceedingly rare.