RNA biomarkers discovered by RT-PCR-based gene expression profiling of archival formalin-fixed paraffin-embedded (FFPE) tissue form the basis for widely used clinical diagnostic tests; however, RT-PCR is practically constrained in the number of transcripts that can be interrogated. We have developed and optimized RNA-Seq library chemistry as well as bioinformatics and biostatistical methods for whole transcriptome profiling from FFPE tissue. The chemistry accommodates low RNA inputs and sample multiplexing. These methods both enable rediscovery of RNA biomarkers for disease recurrence risk that were previously identified by RT-PCR analysis of a cohort of 136 patients, and also identify a high percentage of recurrence risk markers that were previously discovered using DNA microarrays in a separate cohort of patients, evidence that this RNA-Seq technology has sufficient precision and sensitivity for biomarker discovery. More than two thousand RNAs are strongly associated with breast cancer recurrence risk in the 136 patient cohort (FDR <10%). Many of these are intronic RNAs for which corresponding exons are not also associated with disease recurrence. A number of the RNAs associated with recurrence risk belong to novel RNA networks. It will be important to test the validity of these novel associations in whole transcriptome RNA-Seq screens of other breast cancer cohorts.

Abstract Background: RNA biomarkers discovered by RT-PCR-based gene expression profiling of archival formalin-fixed paraffin-embedded (FFPE) tissue are the basis for very precise and sensitive clinical diagnostic tests, such as the 21 gene Oncotype DX® breast cancer assay. Both inherent limits of technical scalability and the small amounts of patient FFPE RNA available place practical constraints on the number of transcripts that can be interrogated by RT-PCR. We developed new methods for RNA profiling through massively parallel “next generation” sequencing (RNA-Seq) of archival FFPE specimens. We report here the technical performance of this methodology and compare the results to RT-PCR results obtained in one of the studies that were carried out to develop the 21 gene assay. Methods: RNA was extracted in 2002 from 136 invasive breast tumors that were formalin-fixed and paraffin-embedded between 1990 and 1997. RNA-Seq was carried out using minor modifications to methods we have reported previously (Sinicropi et al., Advances in Genome Biology and Technology Conference, p. 170, 2010 and p. 198, 2011). Briefly, 0.1 mg of total RNA was selectively depleted of ribosomal RNA and sequencing libraries were prepared using a modification of the ScriptSeq™ kit from Epicentre. The libraries were sequenced on an Illumina HiSeq 2000 instrument with multiplexing of two libraries per lane for 50 cycles in one direction. The resulting FASTQ sequences were mapped to version hg19 of the human genome using the Illumina CASAVA pipeline. The total number of sequences (reads) that uniquely mapped to all exons of each RefSeq entry was used for quantification of expression levels. Results: On average, there were 43 million reads per sample (range 31 - 58 million; SD=4.6 million) of which 69% uniquely mapped to the human genome. Ribosomal RNA was effectively removed and accounted for <0.3% of total counts. Significant coverage of a high proportion of the human genome was obtained, with 40% of RefSeq transcripts represented by a median of more than 100 reads. Using Cox proportional hazards analysis to evaluate the association of quantitative gene expression with breast cancer recurrence, the standardized hazard ratios and p-values for the 21 Oncotype DX genes determined by RNA-Seq were comparable to those originally obtained using RT-PCR. Moreover, whole transcriptome RNA-Seq identified more than 1800 new coding, intronic, and intergenic transcripts that strongly associated with breast cancer recurrence risk (at a false discovery rate <10%) and revealed heretofore unappreciated co-expressed gene networks. Summary: New methodology has been developed for application of next generation sequencing-based whole transcriptome profiling to small amounts of archival FFPE tissue. This technology has sensitivity and selectivity comparable to RT-PCR, can provide a vast increase in the number of interrogated transcripts, can reveal new biological relationships, and has excellent performance suitable for the discovery of RNA biomarkers. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD03-09.

The aim of our study was to evaluate the use of a 1-molar gadolinium chelate (gadobutrol) as an alternative contrast medium for computed tomography angiography (CTA) exams of the aorta. CTA exams of the thoracic and/or abdominal aorta were performed on 15 patients with contraindications for the use of iodine who were not suitable for magnetic resonance examinations. The exams were performed with a 16-detector row scanner, injecting a mean dose of 0.37 mmol Gd/kg of body weight at a flow rate of 4 ml/s. Creatinine levels were obtained prior to the exam in patients with impaired renal function, and 24 and 48 h afterwards. The mean attenuation values obtained in the middle ascending and middle descending thoracic aorta were 202.3 and 216.8, respectively. The mean HU values of the abdominal aorta were 210.4 at the level of the renal arteries and 186.8 in the aortic bifurcation. All the exams were considered diagnostically adequate. No significant increase in serum creatinine was observed 24 and 48 h after the exam. We believe that gadobutrol could be an alternative contrast medium for CTA exams with 16-detector row scanners in patients with contraindications for iodinated contrast medium.

Antiphospholipid syndrome (APS) is a cause of infertility and fetal loss. Ovarian stimulation can induce previously unknown APS. Ovarian hyperstimulation syndrome (OHS) is uncommon but potentially life-threatening, as well as catastrophic APS. A woman that simultaneously developed a severe OHS and a catastrophic APS is described in this paper. Both entities produced thrombotic cardiac and brain thrombosis. A peculiar mechanism of cardiac ischemia is also described. In spite of the life-threatening risk of this situation, the indication for preventive anti-aggregation and/or anticoagulation is not clear.

Virtual endoscopy of the aorta is a new three-dimensional reconstruction method from multislice computed tomography or magnetic resonance that offers a virtual navigation through the aorta, and the possibility of having a new preoperative endoluminal vision. We present a case of subacute aortic dissection with a preoperative virtual endoscopy of the aorta. (c) 2005 by The Society of Thoracic Surgeons.

In order to evaluate the clinical utility of non-enhanced CT with perfusion and angio CT in the assessment of acute ischaemic stroke, 42 patients with symptoms of acute stroke were examined within the first 6 h from onset of symptoms with non-enhanced CT (NECT), perfusion CT (PCT) and CT angiography (CTA). Maps of cerebral blood flow (CBF), cerebral blood volume (CBV) and mean transit time (MTT) were analysed visually, and after drawing regions of interest (ROIs) in the territory of anterior, middle and posterior cerebral arteries, maximum-intensity projection and volume-rendering images of the cervical and cerebral vessels were created. All patients underwent a control CT or MR examination 24-48 h after the initial examination. Twenty-nine patients developed an area of infarction at control examinations. Significant perfusion abnormalities were found in 27 cases, whilst in two patients the perfusion studies were considered to be normal. All the cases with perfusion abnormalities showed arterial stenoses or occlusions on angio CT. Small infarctions at levels other than the ones selected for perfusion CT, and arteriosclerotic changes, were observed in the two cases with no perfusion abnormalities. In conclusion, combining non-enhanced CT with PCT and CTA is a simple and a very valuable tool in the initial assessment of acute stroke.

Throughout the List decade many laboratories have shown that mRNA levels in formalin-fixed and paraffin-embedded (FPE) tissue specimens can be quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) techniques despite the extensive RNA fragmentation that occurs in tissues so preserved. We have developed RT-PCR methods that are sensitive, precise, and that have multianalyte capability for potential wide use in clinical research and diagnostic assays. Here it is shown that the extent of fragmentation of extracted FPE tissue RNA significantly increases with archive storage time. Probe and primer sets for RT-PCR assays based on amplicons that are both short and homogeneous hi length enable effective reference gene-based data normalization for cross comparison of specimens that differ substantially in age. A 48-gene assay used to compare gene expression profiles from the same breast cancer tissue that had been either frozen or FPE showed very similar profiles after reference gene-based normalization. A 92-gene assay, using RNA extracted from three 10-mum FPE sections of archival breast cancer specimens (dating from 1985 to 2001) yielded analyzable data for these genes in all 62 tested specimens. The results were substantially concordant when estrogen receptor, progesterone receptor, and HER2 receptor status determined by RT-PCR was compared with immunohistochemistry assays for these receptors. Furthermore, the results highlight the advantages of RTPCR over immunohistochemistry with respect to quantitation and dynamic range. These findings support the development of RT-PCR analysis of FPE tissue RNA as a platform for multianalyte clinical diagnostic tests.

Throughout the last decade many laboratories have shown that mRNA levels in formalin-fixed and paraffin-embedded (FPE) tissue specimens can be quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) techniques despite the extensive RNA fragmentation that occurs in tissues so preserved. We have developed RT-PCR methods that are sensitive, precise, and that have multianalyte capability for potential wide use in clinical research and diagnostic assays. Here it is shown that the extent of fragmentation of extracted FPE tissue RNA significantly increases with archive storage time. Probe and primer sets for RT-PCR assays based on amplicons that are both short and homogeneous in length enable effective reference gene-based data normalization for cross comparison of specimens that differ substantially in age. A 48-gene assay used to compare gene expression profiles from the same breast cancer tissue that had been either frozen or FPE showed very similar profiles after reference gene-based normalization. A 92-gene assay, using RNA extracted from three 10- micro m FPE sections of archival breast cancer specimens (dating from 1985 to 2001) yielded analyzable data for these genes in all 62 tested specimens. The results were substantially concordant when estrogen receptor, progesterone receptor, and HER2 receptor status determined by RT-PCR was compared with immunohistochemistry assays for these receptors. Furthermore, the results highlight the advantages of RT-PCR over immunohistochemistry with respect to quantitation and dynamic range. These findings support the development of RT-PCR analysis of FPE tissue RNA as a platform for multianalyte clinical diagnostic tests.

Reversible posterior leukoencephalopathy syndrome (RPLS) is an uncommon entity related to multiple and different pathologies, the most common being hypertensive crisis. It is believed to be secondary to the breakdown on the blood-brain barrier. At the beginning, it is undistinguishable from other leukoencephalopathies. However, the disappearance of brain lesions after removal of the potential cause, establish the differential diagnosis with other leukoencephalopathies. We present the case of an HIV-infected patient with a RPLS related to a hypertensive crisis short after the initiation of indinavir-containing highly active antiretroviral therapy. Once blood pressure was controlled and indinavir replaced by nelfinavir, white matter lesions at magnetic resonance imaging disappeared. The clinical and radiologic evolution excludes other diagnosis as progressive multifocal leukoencephalopathy and points indinavir as a potential hypertension-inducing agent in HIV-infected predisposed subjects.

This study was undertaken to investigate the usefulness of contrast-enhanced power Doppler ultrasound (PDU) and contrast-enhanced Harmonic imaging (CHI) for the detection of residual hepatocellular carcinoma (HCC) after chemoembolization therapy (CHT).

Benign uterine leiomyoma metastasizing to the lung is a recognized entity that has been reported infrequently in the medical literature. There is persisting controversy regarding the pathogenesis and biology of these lesions. We report a well-studied and well-characterized case of benign leiomyoma metastasizing to the lung. The patient was a 72-year-old woman with an enlarged uterus that contained several leiomyomas with usual histology. Areas of fibrosis, hyalinization, edema, and focal infarction together with small foci with mildly increased cellularity and minimal nuclear pleomorphism were seen. Careful and repeated mitotic counts ranged from 0 to 2 mitoses per 10 high-power fields. In summary, based on histopathologic criteria, the neoplasm was determined to be a focally cellular benign leiomyoma. Four years later, the patient underwent surgical resection of a single nodule in the lung, which had been detected on routine radiographs. Histopathologic evaluation showed a low-grade leiomyosarcoma with moderate nuclear pleomorphism, necrosis, and brisk mitotic activity. Immunohistochemical studies performed on both neoplasms showed them to be of mesenchymal derivation with smooth muscle differentiation. Both neoplasms expressed estrogen receptors with moderate to strong intensity. The patient received no further treatment and, to date, shows no evidence of recurrent disease. The diagnosis of benign metastasizing leiomyoma can only be made with certainty after careful and extensive sampling of the primary tumor to exclude small foci of sarcoma and of the pulmonary tumor to rule out a primary neoplasm. Although it is biologically peculiar, benign metastasizing leiomyoma should continue to be recognized as a distinct entity because current morphologic criteria do not allow primary myometrial tumors to be reclassified as leiomyomas of uncertain malignant potential even if they have metastasized to the lung.