Loss of heterozygosity studies revisited: prior quantification of the amplifiable DNA content of archival samples improves efficiency and reliability

Polymerase chain reaction (PCR)-based loss of heterozygosity (LOH) studies of archival formalin-fixed, paraffin-embedded (FFPE) tumor tissues have become an important tool in the search for tumor suppressor genes and oncogenes and are also used increasingly in clinical practice. However, FFPE tissue samples may contain little amplifiable DNA, resulting in frequent reaction failures and unreliable LOH data. Using pairs of serial dilutions of reference DNA, we determined the minimum amplifiable DNA concentration necessary for reliable microsatellite-PCR LOH analysis. We then measured the amplifiable DNA content of a selection of frozen and FFPE-derived tumor specimens by real-time quantitative PCR. A minimum input of 600 pg of 100% amplifiable DNA per PCR was required for reliable LOH analysis. While the total DNA concentrations of all samples exceeded this figure, most FFPE-sample-derived DNA was non-amplifiable, with ratios of actually amplifiable DNA to total DNA as low as 1 to 3625. Many FFPE samples therefore contained substantially less than 600 pg/μl of actually amplifiable DNA, making them potentially unsuitable for LOH studies. Real-time quantitative PCR before LOH studies of FFPE tissues allows: identification of samples, which will fail microsatellite-PCR; exclusion of samples, which will yield unreliable results; and optimal adjustment of template input for the remainder. Amplification reactions undertaken without this precaution can result in unreliable LOH data.     

Is it HIV TTP or HIV‐associated thrombotic microangiopathy?

Thrombocytopenia is a common complication of Human Immunodeficiency Virus (HIV) infection. With advanced HIV disease, the presence of both thrombocytopenia and schistocytosis are frequently observed. In such cases, the diagnosis of HIV associated TTP is often considered. This article reviews emerging concepts of HIV associated microangiopathies. It concludes that the pathophysiology, in many cases seems to be distinct from idiopathic TTP (particularly with advanced HIV disease-<100 CD4/microliter). A sine que non for successful therapy of HIV-TMA appears to be the treatment of the underlying HIV infection.     

Mitogen-activated protein kinase kinase signaling promotes growth and vascularization of fibrosarcoma

We hypothesized that signaling through multiple mitogen-activated protein kinase (MAPK) kinase (MKK) pathways is essential for the growth and vascularization of soft-tissue sarcomas, which are malignant tumors derived from mesenchymal tissues. We tested this using HT-1080, NCI, and Shac fibrosarcoma-derived cell lines and anthrax lethal toxin (LeTx), a bacterial toxin that inactivates MKKs. Western blots confirmed that LeTx treatment reduced the levels of phosphorylated extracellular signal-regulated kinase and p38 MAPK in vitro. Although short treatments with LeTx only modestly affected cell proliferation, sustained treatment markedly reduced cell numbers. LeTx also substantially inhibited the extracellular release of angioproliferative factors including vascular endothelial growth factor, interleukin-8, and basic fibroblast growth factor. Similar results were obtained with cell lines derived from malignant fibrous histiocytomas, leiomyosarcomas, and liposarcomas. In vivo, LeTx decreased MAPK activity and blocked fibrosarcoma growth. Growth inhibition correlated with decreased cellular proliferation and extensive necrosis, and it was accompanied by a decrease in tumor mean vessel density as well as a reduction in serum expression of angioproliferative cytokines. Vital imaging using high-resolution ultrasound enhanced with contrast microbubbles revealed that the effects of LeTx on tumor perfusion were remarkably rapid (<24 h) and resulted in a marked reduction of perfusion within the tumor but not in nontumor tissues. These results are consistent with our initial hypothesis and lead us to propose that MKK inhibition by LeTx is a broadly effective strategy for targeting neovascularization in fibrosarcomas and other similar proliferative lesions.     

Cervical spine magnetic resonance imaging in primary care consulters with shoulder pain: a case control study

OBJECTIVES: To investigate the association between shoulder region pain presenting in primary care and cervical spine magnetic resonance imaging (MRI) abnormalities. METHODS: A matched case-control study of 48 pairs of participants. Patients had presented to primary care with a new episode of shoulder pain. Controls had no history of pain in the shoulder region and were individually matched with cases by age, gender and referring clinician. All participants underwent a structured clinical assessment and cervical spine MRI. Scans were scored by experienced musculoskeletal radiologists blinded to case-control status. RESULTS: Median age of participants was 51 years (range 19-79) and 21 (44%) were female. "Neck pain in the past week" was reported by 25 (52%) cases and seven (15%) controls (odds ratio, OR, 10.0; 95% confidence interval, CI, 2.4, 88.2). Cervical spine MRI from C3/4 to C6/7 revealed: 18 (38%) of both cases and controls had disc height loss >/=50% at any level; 10 (21%) cases and eight (17%) controls had disc disease with neural compromise; 11 (23%) cases and 16 (33%) controls had foraminal stenosis; nine (19%) of both cases and controls had canal narrowing. At least one of the above findings was present in 24 (50%) cases and 23 (48%) controls (OR 1.1; 95% CI 0.4, 3.4). CONCLUSIONS: Cervical spine MRI abnormalities were similar in both cases and controls, despite significantly more self-reported neck pain in cases with shoulder pain. Other possible mechanisms, such as muscular strain or postural problems, may explain the observed clinical association between shoulder region pain and neck associated symptoms.