Microsatellite Instability Analysis and Its Prognostic Value in Invasive Nonampullary Duodenal Adenocarcinoma

Purpose: Nonampullary duodenal adenocarcinoma is a rare disease. Although several prognostic factors have been reported for this disease, they remain controversial due to their rarity. In this study, we retrospectively analyzed 54 cases of invasive nonampullary duodenal adenocarcinoma, focusing on the microsatellite instability phenotype, PD-L1 expression, and prognostic factors. Methods: Expression of the PD-L1 protein and cell differentiation markers in tumors was detected by immunohistochemistry. Microsatellite markers (NR-21, NR-22, NR-24, BAT-25 and BAT-26) were amplified for MSI assessment by PCR.Results: The incidence of microsatellite instability in invasive nonampullary duodenal adenocarcinoma was 35.2%. No significant correlation between the microsatellite instability phenotype and clinicopathological factors was observed. Positive expression of PD-L1 by immune cells was common in advanced-stage disease (P=0.054), and positive expression of PD-L1 in cancer cells correlated significantly with the histologically undifferentiated type (P=0.016). Kaplan-Meier survival analysis demonstrated a significantly better overall survival in patients with microsatellite instability (P=0.013) and at early-stage disease (P=0.000) than in those with microsatellite stability or at late tumor stages. Univariate and multivariate analyses showed that microsatellite instability (hazard ratio [HR]: 0.282, 95% confidence interval [CI]: 0.106-0.751, p=0.011) and early tumor stage (stage Ⅰ-Ⅱ) (hazard ratio [HR]: 8.81, 95% confidence interval [CI]: 2.545-30.500, p=0.001) were independent better prognostic factors of overall survival. Conclusions: Microsatellite instability and early tumor stage (stage Ⅰ-Ⅱ) were independent better prognostic factors of overall survival. A high proportion of microsatellite instability phenotypes and positive PD-L1 expression may be helpful for identifying immune checkpoint inhibitors as a novel therapeutic strategy.

Mitotic motor CENP-E cooperates with PRC1 in temporal control of central spindle assembly

Error-free cell division depends on the accurate assembly of the spindle midzone from dynamic spindle microtubules to ensure chromatid segregation during metaphase–anaphase transition. However, the mechanism underlying the key transition from the mitotic spindle to central spindle before anaphase onset remains elusive. Given the prevalence of chromosome instability phenotype in gastric tumorigenesis, we developed a strategy to model context-dependent cell division using a combination of light sheet microscope and 3D gastric organoids. Light sheet microscopic image analyses of 3D organoids showed that CENP-E inhibited cells undergoing aberrant metaphase–anaphase transition and exhibiting chromosome segregation errors during mitosis. High-resolution real-time imaging analyses of 2D cell culture revealed that CENP-E inhibited cells undergoing central spindle splitting and chromosome instability phenotype. Using biotinylated syntelin as an affinity matrix, we found that CENP-E forms a complex with PRC1 in mitotic cells. Chemical inhibition of CENP-E in metaphase by syntelin prevented accurate central spindle assembly by perturbing temporal assembly of PRC1 to the midzone. Thus, CENP-E-mediated PRC1 assembly to the central spindle constitutes a temporal switch to organize dynamic kinetochore microtubules into stable midzone arrays. These findings reveal a previously uncharacterized role of CENP-E in temporal control of central spindle assembly. Since CENP-E is absent from yeast, we reasoned that metazoans evolved an elaborate central spindle organization machinery to ensure accurate sister chromatid segregation during anaphase and cytokinesis.

An Acquired High-Risk Chromosome Instability Phenotype in Multiple Myeloma: Jumping 1q Syndrome

Introduction Chromosome instability (CIN) is a driver of copy number aberrations (CNAs) in cancer, and is a major factor leading to tumor heterogeneity and resistance to therapy. By definition, CIN is an increased rate or ongoing acquisition and accumulation of CNAs and not simply the existence of structurally and numerically abnormal aneuploid clones. In multiple myeloma (MM), the most common whole-chromosome CNAs involve either hyperdiploid or non-hyperdiploid clones. Secondary segmental CNAs are associated with high-risk (HR) in MM and involve gains of 1q21 and deletions of 17p (del17p). These types of intra-chromosomal segmental CNAs are also found in the CIN phenotypes of the autosomal recessive (AR) chromosome instability syndromes. These syndromes include Fanconi anemia, Bloom's syndrome, and ICF syndrome (Immunodeficiency, Centromeric instability and Facial anomalies). These chromosome instability syndromes display a spectrum of aberrations characterized by higher rates of chromosomal breaks, chromatid exchanges, quadriradials, and pericentromeric aberrations. In particular, patients with ICF syndrome show a marked increase of 1q12 pericentromeric instability including 1q12 decondensation, triradials, multibranched chromosomes 1q, and 1q micronuclei. ICF patients also show transient 1q aberrations including isochromosome 1q (iso1q) and unbalanced translocations of 1q to 9q and 16q. In MM, we have previously reported increasing pericentromeric instability during tumor progression resulting in increasing CNAs of 1q21 by unbalanced jumping translocations of 1q12 (JT1q12). Strikingly, in a subset of MM patients with 1q21 CNAs of ≥ 5 a distinct cytogenetic phenotype emerges which demonstrates transient 1q12 aberrations including 1q12 decondensation, triradials, and multibranched chromosomes 1q morphologically identical to those seen in ICF patients. In MM this chromosome instability leads to a cascade of increasing clonal 1q21 duplications, iso 1qs, and unbalanced 1q translocations with 16q and 17p, resulting in losses in these receptor chromosomes (RC) and massive intra-clonal CNA heterogeneity. Methods To investigate the cytogenetic impact and progression of high CNAs of 1q21, we performed a comprehensive metaphase analysis of 50 patients showing segmental aneuploidies with 4 or more copies of 1q by G-banding. Locus specific FISH and spectral karyotyping were used to identify the key transient unstable and clonal structural aberrations of 1q12 resulting in segmental aneuploidies in the derivative RCs. Probe for 1q12 (Vysis) was used according to the manufacturer's protocol. Locus specific BAC clones for 1q21 (CKS1B) and 17p (TP53) were prepared and analyzed as previously described (Sawyer et al., Blood 123: 2014). IGH translocations were investigated with IGH break apart probes (Vysis). Results Data for 50 patients including CNAs of 1q21 of ≥ 4, IGH translocations, del(17p), derivative RCs, are presented. The t(4;14) was found in 15 patients, del(17p) in 23, and both aberrations were found in 8 patients. All patients showed unbalanced gains of 1q and deletions of RCs, the most frequent being 7 patients with der(1;16) and 6 with iso1q. In four of the 23 patients with del(17p), the deletion was due to a JT1q12 to 17p. Seven patients with 1q21 CNAs of ≥ 5 showed profound instability involving the 1q12 satellite DNA, demonstrating both transient and clonal aberrations driving the 1q21 CNAs. These aberrations included unstable 1q21 triplications, JT1q12s, iso1q formation with intra-arm 1q12 CNAs, and region specific breakage-fusion-bridge cycle amplifications. Conclusions Among patients with ≥ 5 CNAs of 1q21, a subset develop an acquired HR chromosome instability phenotype with an elevated rate of 1q12 pericentromeric instability characterized by concomitant deletions in 16q, iso1q, del(17p), and intra-arm segmental instability. These patients show pronounced instability in the 1q12 satellite DNA, morphologically identical to ICF syndrome, suggesting hypomethylation of this region as a driver of both 1q21 CNAs and deletions in RCs. We hypothesize that region specific hypomethylation of 1q12 provides the genomic background for the onset of an acquired 1q12 chromosome instability phenotype in MM similar to that found in ICF syndrome. For myeloma patients demonstrating this 1q12 chromosome instability phenotype we propose the term "jumping 1q syndrome." Disclosures Epstein: University of Arkansas for Medical Sciences: Employment. Davies:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; ASH: Honoraria; Abbvie: Consultancy; TRM Oncology: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; MMRF: Honoraria; Janssen: Consultancy, Honoraria. Morgan:Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria.

Phosphorylation by CK2 Regulates MUS81/EME1 in Mitosis and After Replication Stress

ABSTRACTThe MUS81 complex is crucial for preserving genome stability through the resolution of branched DNA intermediates in mitosis. However, untimely activation of the MUS81 complex in S-phase is dangerous. Little is known about the regulation of the human MUS81 complex and how deregulated activation affects chromosome integrity. Here, we show that the CK2 kinase phosphorylates MUS81 at Serine 87 in late-G2/mitosis, and upon mild replication stress. Phosphorylated MUS81 interacts with SLX4, and this association promotes the function of the MUS81 complex. In line with a role in mitosis, phosphorylation at Serine 87 is suppressed in S-phase and is mainly detected in the MUS81 molecules associated with EME1. Loss of CK2-dependent MUS81 phosphorylation contributes modestly to chromosome integrity, however, expression of the phosphomimic form induces DSBs accumulation in S-phase, because of unscheduled targeting of HJ-like DNA intermediates, and generates a wide chromosome instability phenotype. Collectively, our findings describe a novel regulatory mechanism controlling the MUS81 complex function in human cells. Furthermore, they indicate that, genome stability depends mainly on the ability of cells to counteract targeting of branched intermediates by the MUS81/EME1 complex in S-phase, rather than on a correct MUS81 function in mitosis.

Correlation of benefit from immune checkpoint inhibitors with next gen sequencing (NGS) profiles in esophagogastric cancer (EGC) patients.

4025 Background: Immuno-oncology (IO) with anti-PD-1 and –PD-L1 antibodies (Abs) is active in EGC but only benefits a minority of Pts. Biomarkers are needed to identify responders. Methods: We reviewed our experience of Pts treated with anti-PD-1/PD-L1 Abs and correlated their outcomes with PD-L1 and mismatch repair protein (MMR) status by immunohistochemistry (IHC), as well as MSK-IMPACT (≥340-gene) NGS profile. MSIsensor from IMPACT assesses microsatellite instability phenotype, while ≥20 mutations (or 17 mutations/Mb) strongly correlates with MMR-deficiency (dMMR) by IHC (J Clin Oncol 2016;34:2141). Progression-free (PFS) and overall survival (OS) were analyzed from the start of IO. Results: 71 Pts were identified, with 3 Pts receiving 2 IO regimens. 66 had adenoCAs and 5 had squamous CAs. Median age 58, 77% male, 96% had received ≥2 prior chemo regimens. 39 (55%), 18 (25%) and 17 Pts (24%) respectively received anti-PD-1, anti-PD-L1 and anti-CTLA-4 plus anti-PD-1/PD-L1 Abs. 6 Pts (8%) had objective response (2 complete responses or CRs) and the median PFS and OS are 1.6 and 4.7 mos; 2-yr OS is 17%. PD-L1 IHC was performed in 16 Pts (23%; 7 +ve), MMR was tested in 20 Pts (28%; 4 dMMR) and IMPACT was obtained in 44 Pts (62%). All 4 dMMR tumors were also MSI by MSIsensor and had a median of 46 mutations (range, 29-63) or, equivalently, 33 mutations/Mb (range, 21-46); 2 of 2 dMMR tumors tested PD-L1 +ve. 3 of the 4 Pts with dMMR/MSI tumors had a response (including 1 CR) and the median OS of these 4 Pts is not reached with 23+ months of follow-up. Finally, a patient whose tumor is MMR-proficient, not MSI but has 15 mutations (including in POLD1), achieved an ongoing CR at 37+ mos. For the 44 Pts with IMPACT testing, there appeared to be improved OS for tumors with ≥10 vs. <10 mutations/Mb (2-yr OS 80% vs. 12%, p=0.03). Conclusions: Pts with tumors that are MSI or have ≥10 mutations/Mb on MSK-IMPACT appear to derive significant benefit from IO. MSK-IMPACT can offer novel information, identify novel mutations (e.g. POLD1) and may be used to help select Pts for IO. We are seeking to define a mutation no. cut-off that can serve as a biomarker and updated data will be presented.