Detection of metastases using circulating tumour DNA in uveal melanoma

Background: Approximately 50% of uveal melanoma (UM) patients will develop metastatic disease depending on the genetic features of the primary tumour. Patients need 3–12 monthly scans, depending on their prognosis, which is costly and often non-specific. Circulating tumour DNA (ctDNA) quantification could serve as a test to detect and monitor patients for early signs of metastasis and therapeutic response. Methods: We assessed ctDNA as a biomarker in three distinct UM cohorts using droplet-digital PCR: (A) a retrospective analysis of primary UM patients to predict metastases; (B) a prospective analysis of UM patients after resolution of their primary tumour for early detection of metastases; and (C) monitoring treatment response in metastatic UM patients. Results: Cohort A: ctDNA levels were not associated with the development of metastases. Cohort B: ctDNA was detected in 17/25 (68%) with radiological diagnosis of metastases. ctDNA was the strongest predictor of overall survival in a multivariate analysis (HR = 15.8, 95% CI 1.7–151.2, p = 0.017). Cohort C: ctDNA monitoring of patients undergoing immunotherapy revealed a reduction in the levels of ctDNA in patients with combination immunotherapy. Conclusions: Our proof-of-concept study shows the biomarker feasibility potential of ctDNA monitoring in for the clinical management of uveal melanoma patients

Early diagnosis of breast cancer by hair diffraction

A correlation between the incidence of breast cancer and an observed change in the X-ray diffraction pattern of hair from the afflicted individuals was first reported in 1999. Since that time, over 500 hair samples have been analyzed in double-blinded breast cancer studies with no false negatives being detected. To correlate this observed change with the presence of breast cancer, we examined whiskers removed from nude mice prior to and 8 weeks after subcutaneous implantation of a human breast cancer cell line. Here we show that the change observed in human hair was also evident in whiskers and that it appeared soon after cancer cell implantation

SLIRP KO males produce smaller litters and have fewer progressively motile sperm.

<p>(A) Litter sizes resulting from breeding wild type (WT) females with WT or SLIRP knockout (KO) males (*, significant difference by Maximum Likelihood analysis of repeated measures, p = 0.025). (B) Comparison of daily sperm production (B), motility (C) and progressive motility (D) between WT and SLIRP KO animals. **, ANOVA, p&lt;0.001.</p

Generation of the SLIRP KO mouse.

<p>(A) Wild type (WT), floxed SLIRP and SLIRP knockout (KO) mouse SLIRP locus configurations. Floxed mice contain a cDNA for SLIRP exons 2 to 4 (Ex 2–4) with polyadenylation signal (poly A) preceded by loxp and splice acceptor (SA) sites and followed by loxp, SA, Flag epitope, stop codon and c-<i>fos</i> sequences inserted within the first SLIRP intron (intron length in kb, nt, nucleotides). (B) Southern analysis to detect a 31.3 kb EcoRV fragment in WT (+/+) and heterozygous (+/−) mice and a 13.3 kb recombinant band in heterozygous and SLIRP homozygous KO (−/−) animals. (C) WT (334 nt) and recombinant (247 nt) RT-PCR products generated from WT and recombinant SLIRP mouse liver cDNAs. (D) Western analysis of WT and recombinant SLIRP mouse testis lysates for SLIRP and β–actin.</p

SLIRP is expressed in the mouse testis.

<p>(A &amp; B) Detection of SLIRP by immunohistochemistry in Leydig cells, spermatogonia (green arrow), early spermatocytes (yellow arrows), round spermatids (red arrows), elongate spermatids and sperm lining the lumen (white arrows). (C, F, I) Immunofluorescent staining for SLIRP (green), (D) Hsp60 (red), PCNA (G, red) and SP56 (J, red) overlayed in E, H &amp; K respectively. Yellow indicates colocalization in overlayed panels. Nuclear DAPI staining in blue E, H &amp; K. White bars: A, 80 µm, B, H &amp; K, 10 µm, E, 20 µm.</p

SLIRP localization in epididymal sperm.

<p>(A) Immunofluorescent staining for SLIRP (green) and Sp56 (B, red) in sperm heads (C, overlay). (D) SLIRP (green) and Septin 4 (E, red) staining of sperm tails (F, overlay). White bars: C, 10 µm; F, 1 µm.</p

Annulus and mitochondrial disruption in SLIRP KO sperm.

<p>Electron micrographs of the distal mid-piece and annulus region of (A) WT and (B) SLIRP KO sperm. White arrows, annulus; white bar, abnormal mid-piece/annulus junction in KO sperm; black arrows, electron light and dense areas in WT and KO mitochondria respectively. Black bars, 0.5 µm.</p