Optimization of RNA extraction from formalin-fixed, paraffin-embedded lymphoid tissues

Comprehensive analysis of gene expression using RNA extracted from frozen lymphoma specimens is becoming increasingly important for understanding disease pathogenesis, disease subclassification, and prognostication. As paraffin tissues are widely available whereas frozen specimens are not, development of gene expression analysis based on RNA extracted from paraffin-embedded tissues would facilitate application of the accumulated knowledge to a sample type that is typical of clinical practice. In the present study, we have developed and optimized methods of RNA extraction from paraffin-embedded lymphoid tissues. In contrast to previously suggested methods of RNA extraction from paraffin, our method uses sodium dodecyl sulfate that better preserves the extracted RNA and is optimized for more complete proteinase K digestion to release RNA from its complexes with protein. These modifications yield long RNA fragments up to 2000 bp enabling amplification of long amplicons. This allows usage of paraffin specimens for molecular rescue of RNA transcripted from rearranged clonal immunoglobulin genes-an advance that may increase the eligibility of lymphoma patients for immunotherapeutic approaches. Furthermore, real-time polymerase chain reaction analysis of expression of genes implicated in determination of prognosis of diffuse large B-cell lymphoma patients demonstrated an extremely high correlation (R>0.90) in normalized gene expression between paired frozen and formalin-fixed, paraffin-embedded specimens. Similarly, good correlation was also observed in gene array studies. These results suggest that the methods of RNA extraction we propose are suitable for giving accurate real-time quantitative reverse transcriptase-polymerase chain reaction results, array gene expression profiling, and molecular rescue of RNA transcripted from rearranged immunoglobulin genes for diagnostic and immunotherapeutic approaches

IRF-4 and c-Rel expression in antiviral-resistant adult T-cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATLL) is a generally fatal malignancy. Most ATLL patients fare poorly with conventional chemotherapy; however, antiviral therapy with zidovudine (AZT) and interferon alpha (IFN-alpha) has produced long-term clinical remissions. We studied primary ATLL tumors and identified molecular features linked to sensitivity and resistance to antiviral therapy. Enhanced expression of the proto-oncogene c-Rel was noted in 9 of 27 tumors. Resistant tumors exhibited c-Rel (6 of 10; 60%) more often than did sensitive variants (1 of 9; 11%). This finding was independent of the disease form. Elevated expression of the putative c-Rel target, interferon regulatory factor-4 (IRF-4), was observed in 10 (91%) of 11 nonresponders and in all tested patients with c-Rel+ tumors and occurred in the absence of the HTLV-1 oncoprotein Tax. In contrast, tumors in complete responders did not express c-Rel or IRF-4. Gene rearrangement studies demonstrated the persistence of circulating T-cell clones in long-term survivors maintained on antiviral therapy. The expression of nuclear c-Rel and IRF-4 occurs in the absence of Tax in primary ATLL and is associated with antiviral resistance. These molecular features may help guide treatment. AZT and IFN-alpha is a suppressive rather than a curative regimen, and patients in clinical remission should remain on maintenance therapy indefinitely

First lacustrine varve chronologies from Mexico: impact of droughts, ENSO and human activity since AD 1840 as recorded in maar sediments from Valle de Santiago

We present varve chronologies for sediments from two maar lakes in the Valle de Santiago region (Central Mexico): Hoya La Alberca (AD 1852–1973) and Hoya Rincón de Parangueo (AD 1839–1943). These are the first varve chronologies for Mexican lakes. The varved sections were anchored with tephras from Colima (1913) and Paricutín (1943/1944) and 210Pb ages. We compare the sequences using the thickness of seasonal laminae and element counts (Al, Si, S, Cl, K, Ti, Mn, Fe, and Sr) determined by micro X-ray fluorescence spectrometry. The formation of the varve sublaminae is attributed to the strongly seasonal climate regime. Limited rainfall and high evaporation rates in winter and spring induce precipitation of carbonates (high Ca, Sr) enriched in 13C and 18O, whereas rainfall in summer increases organic and clastic input (plagioclase, quartz) with high counts of lithogenic elements (K, Al, Ti, and Si). Eolian input of Ti occurs also in the dry season. Moving correlations (5-yr windows) of the Ca and Ti counts show similar development in both sequences until the 1930s. Positive correlations indicate mixing of allochthonous Ti and autochthonous Ca, while negative correlations indicate their separation in sublaminae. Negative excursions in the correlations correspond with historic and reconstructed droughts, El Niño events, and positive SST anomalies. Based on our data, droughts (3–7 year duration) were severe and centred around the following years: the early 1850s, 1865, 1880, 1895, 1905, 1915 and the late 1920s with continuation into the 1930s. The latter dry period brought both lake systems into a critical state making them susceptible to further drying. Groundwater overexploitation due to the expansion of irrigation agriculture in the region after 1940 induced the transition from calcite to aragonite precipitation in Alberca and halite infiltration in Rincón. The proxy data indicate a faster response to increased evaporation for Rincón, the lake with the larger maar dimensions, solar radiation receipt and higher conductivity, whereas the smaller, steeper Alberca maar responded rapidly to increased precipitation