HRD Testing of Ovarian Cancer in Routine Practice: What Are We Dealing With?

Assessment of homologous recombination deficiency (HRD) status is now essential for ovarian cancer patient management. The aim of our study was to analyze the influence of ethnic variations, tumor purity, and neoadjuvant chemotherapy (CT) on the determination of HRD scores as well as to evaluate feasibility of HRD testing with the Amoy HRD Focus Assay in routine clinical practice. The HRD status, including the BRCA status and genomic scar score (GSS), was analyzed in 452 ovarian cancer specimens. The successful rate of HRD testing was 86% (388/452). The BRCA mutational rate was 29% (114/388); 252 samples (65%) were classified as HRD-positive. Our data demonstrate the feasibility of internal HRD testing by the AmoyDx HRD Focus Panel for high-grade serous ovarian cancer (HGSOC), showing results similar to other methods. The HRD rate in the Russian population is very similar to those of other European populations, as is the BRCA mutation frequency. The most substantial contribution to HRD level diversity is testing criteria depending on intrahospital arrangements. The analysis shows that biallelic BRCA alterations had higher GSS compared with those with monoallelic inactivation, consistent with positive HRD status. The study indicates that grades 1-2 of the pathological response caused by chemotherapy affect HRD scores and suggests controlling for tumor purity of 40% or more as a critical factor for GSS measurement.

Identification of a novel splice variant for mouse and human interleukin-5.

Expression of interleukins and their receptors is often regulated by alternative splicing. Alternative isoform of IL-5 receptor α-chain is well studied; however, no data on functional alternative splice variants of IL-5 has been reported up today. In the present study, we describe a novel splice variant for the mouse and human IL-5. The new form was found during analysis of PCR-products amplified from different mouse lymphoid tissues with a pair of primers designed to clone full-length mIL-5 ORF. A single short isoform of mIL-5 was detected along with the canonical full-length mRNA in ConA-stimulated lymphoid cells isolated from spleen, thymus, lymph nodes and blood. It was 30-40 nt shorter, and less abundant than classical form. The sequence analysis of an additional form of mIL-5 revealed that it lacks exon-2 (δ2). Using RT-PCR with the splice-specific primers we obtained an additional evidence for δ2 form expression. To verify whether mIL-5δ2 transcript is translated into protein, the coding sequences corresponding to full and δ2 forms of mIL-5 were cloned into an expression plasmid. After transfection into the human 293T cell line, we found that the short form of mIL-5 protein is expressed in cells and secreted into the supernatant, but at the reduced level than that detected for full isoform of mIL-5. Fluorescence microscopy examination revealed a partial translocation of mIL-5δ2 into cytoplasm, whereas mIL-5 resided mostly within endoplasmic reticulum. This can explain why the level of δ2 protein expression was reduced. Using a similar set of experimental approaches, we received the evidence that the human IL-5 mRNA has the δ2 splice form (hIL-5δ2) as well. It can be firmly detected by RT-PCR in PHA-activated mononuclear cells isolated from peripheral blood of healthy persons or patients with asthma. Altogether, our results showed that the human and mouse IL-5 have an alternative mRNA splice isoform, which loses exon-2, but nevertheless is expressed at protein level. However, more comprehensive studies will be required for evaluation of IL-5δ2 expression, regulation, biological function and clinical significance.

Elementary processes in cells after light absorption do not depend on the degree of polarization: implications for the mechanisms of laser phototherapy.

OBJECTIVE: The objective of this work was to evaluate the importance of the degree of light polarization in stimulation of cellular metabolism. BACKGROUND DATA: Although the possible role of polarization's effects on the mechanisms of laser phototherapy is sometimes discussed in the literature, there are still no clear answers. MATERIAL AND METHODS: A model system (HeLa cell suspension) was used in which the lengths of light scattering (l sc) and absorption (l a) were much larger than the thickness of the irradiated layer (L = 3 mm). The cell suspension (1 x 10(6) cells/cm3) was irradiated with a diode laser (lambda = 637 nm, D = 65.7 J/m2, tau = 10 sec, I = 6.57 W/m2). The polarization degree (99.4%, 60.9%, and 34.2%) of the beam was changed by means of optical fibers of different lengths. The irradiated suspension was incubated at 37 degrees C for 30 min, and the attached cells were counted afterwards. RESULTS: The cell fraction stimulated to adhere by red light at 637 nm was nearly the same in all three experimental groups (58.1% +/- 2.5%, 57.6% +/- 3.5%, and 62.5% +/- 3.2% for degree of beam polarization of 99.4%, 66.9%, and 34.2%, respectively). There was no statistically significant difference in these results (p <0.8, <0.6, and <0.7, respectively). At the same time, all three groups had statistically significant differences (p < 0.01) in adherence from the sham-irradiated control group (39.1% +/- 2.2%). CONCLUSION: The biological effect (stimulation of cell attachment) of light with lambda = 637 nm on cells in our model system was pronounced, but did not depend on the degree of light polarization. Elementary processes in cells (light absorption and photochemistry) do not appear to depend on the degree of light polarization.