Various detection techniques for mutations are available but
Various detection techniques for mutations are available, but screening of mutations for anti- therapy in CRC patients is done mostly by direct sequencing (DS) or real time PCR assays, which may be expensive and time consuming. In this issue of , Dr. Takayuki Yoshino and colleagues report on the utility of the MEBGEN RASKET KIT (RASKET), a multiplex assay using PCR-reverse sequence specific oligonucleotide with xMAP technology. This assay can detect 48 ( and ) mutations in exons 2, 3 and 4, with a short turnaround time of 4.5h for 96 specimens . Moreover, the RASKET results had high concordance with those obtained using DS and the TheraScreen RGQ Polymerase chain reaction kit. The RASKET can detect mutant fractions of around 1–5% which is similar to TheraScreen mutation kit and is higher than DS. There are other sensitive assays for detection such as the Allele Specific Competitive Blocker Polymerase Chain Reaction (ACB-PCR) that can detect mutations fractions as low as 0.001% (1 mutant out of 100,000 wild-type) . The sensitivity of an assay will impact whether a colorectal tumor is mutant or wild-type . With ACB-PCR, glucokinase inhibitor 12 mutations are detected in 100% of colon tumors and also in some normal colonic mucosa . The levels of different mutations that are “pathological” have not yet been rigorously established for CRC patients . Nevertheless, it has been demonstrated that the mutant subclones known to cause resistance to anti-EGFR therapy are quite prevalent and frequently occur at low levels (levels below the detection limit by DS ). There is evidence that a significant number of CRC patients characterized as wild-type fail to respond to EGFR therapy . This could be due in part to the insensitive techniques that clinicians use for mutation detection or due to the presence of other undetected mutations in different genes in the RAF–MEK–ERK kinase pathway. RASKET, similar to DS and the TheraScreen kit, does not provide quantitation of the detected mutations. Therefore, these methodologies may not be sufficient for establishing whether particular levels of mutation are associated with clinical response. mutations are frequently present as small subpopulations within colon tumors, which are remarkably heterogeneous and may often be polyclonal in origin . Therefore, it seems likely that both sensitive and quantitative methodologies to characterize mutations will be required to advance personalized treatment of colon cancer.
The RASKET kit has its limitations: it does not measure the mutations quantitatively and also there are other more sensitive assays available to measure mutations. Nonetheless, the RASKET multiplex assay detects exons 2, 3 and 4 and mutations from a small amount of DNA (50–100ng) that is extracted from formalin fixed paraffin embedded tissues in a short turnaround time of 4.5h.
Alopecia areata (AA) is an autoimmune disease due to T cell attack of the hair follicles and breakdown of their immune privilege resulting in transient non-scarring hair loss which may last from weeks to decades and which presents an enormous psychological burden. As so far no FDA approved successful treatments for AA are available, new hope and light arose on the horizon with the recently published genome wide association studies in AA patients revealing new molecular pathways disrupted in AA including autophagy/apoptosis, transforming growth factor beta/Tregs and JAK (Janus family of non-receptor protein tyrosine kinase) signaling (). Thus, today it is assumed that CD8+NKG2D+ T effector memory cells mediate alopecia areata in part through Janus kinase (JAK) signaling and that alopecia areata might be treated with JAK inhibitors. This assumption has been supported by clinical use of JAK inhibitors within a clinical study setting () or observed as a “side effect”, when JAK inhibitor treatment of another underlying disease resulted in regrowth of longstanding patchy or universal alopecia areata (). demonstrated that the interferon-γ (IFNγ)-signaling pathway is upregulated in AA-lesional skin, and that the use of JAK inhibitors was able to reverse symptoms of the disorder in mice and in three humans with AA. While different groups () reported successful use of ruxolitinib, a JAK1/2 inhibitor licensed for treating myelofibrosis, others reported successful use of the JAK 1/3 inhibitor tofacitinib in AA while using it for treating psoriasis ().