A digital method to interpret the C-MYC stain in diffuse large B cell lymphoma.

Diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS) is a heterogenous group of aggressive lymphomas. C-MYC expression by immunohistochemical stain (IHC) is shown to be an independent prognostic factor in DLBCL. In the clinical setting, MYC stain is currently evaluated by manual quantification with a minimum positivity cut-off 40%. Manual quantification methods can be subjective and may show intra- and interobserver variability and variability between centers. Thus, stains which require definitive quantification such as MYC needs better standardized and precise methods. Here we present a simple digital algorithm for quantitative evaluation of MYC stain in DLBCL, NOS. For this, slides immunostained for C-MYC were scanned at 40X with a high-resolution, Philips Ultra Fast scanner (Koninklijke Philips N.V. Cambridge, MA). The images were manually assessed and appropriate areas with neoplastic cells were selected. For quantification, positive and negative C-MYC staining nuclei were scored using a modified Visiopharm APP Nuclei Detection, AI (Brightfield) using Visiopharm Image Analysis software (Visiopharm, Hørsholm, Denmark version 2018.09). The percentage positivity resulted by the digital method was concordant with the pathologist's interpretation with statistical significance (rs: 0.85968; p (2-tailed) = 0). Minor disadvantages were observed including failure to detect very weak staining and inability to separate neoplastic and non-neoplastic nuclei when admixed in the same area. If combined with a quick manual evaluation, a digital method like this with precision and reproducibility will be of great use in quantitative evaluation of MYC and other similar stains in clinical setting and will reduce intra- and interobserver variability.

KMT2D loss drives aggressive tumor phenotypes in cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the second most lethal skin cancer. Due to ultraviolet light-induced damage, cSCCs have a high mutation rate, but some genes are more frequently mutated in aggressive cSCCs. Lysine-specific histone methyltransferase 2D (KMT2D) has a two-fold higher mutation frequency in metastatic cSCCs relative to primary non-metastatic associated cSCCs. The role of KMT2D in more aggressive phenotypes in cSCC is uncharacterized. Studies of other tumor types suggest that KMT2D acts to suppress tumor development. To determine whether KMT2D loss has an impact on tumor characteristics, we disrupted KMT2D in a cSCC cell line using CRISPR-cas9 and performed phenotypic analyses. KMT2D loss modestly increased cell proliferation and colony formation (1.4- and 1.6-fold respectively). Cells lacking KMT2D showed increased rates of migration and faster cell cycle progression. In xenograft models, tumors with KMT2D loss showed slight increases in mitotic indices. Collectively, these findings suggest that KMT2D loss-of-function mutations may promote more aggressive and invasive behaviors in cSCC, suggesting that KMT2D-related pathways could be targets for cancer therapies. Future studies to determine the downstream genes and mechanism of phenotypic effect are needed.