Atkinson, Hunter

Introduction Establishment of reference intervals (RI) for quantitative assays requires a minimum of 120 qualified normal donor (ND) samples for each partition of an analyte. Based on CLSI EP28 A3C guideline [CLSI. Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline-Third Edition. CLSI document EP28-A3c. Wayne, PA: Clinical and Laboratory Standards Institute; 2008.], verification of previously established and/or published RI can be performed with a minimum of 20 NDs, especially given complexities of finding qualified NDs. Reagent manufacturers typically provide adult RI, thus facilitating RI verification as a viable option. NDs for the Pediatric (Peds) (<18 years of age) population are difficult to obtain, published RIs are performed with sample cohorts less than the suggested number or only performed with specific instrument/reagent combinations. Herein, we report our experiences with Peds RI. Methods After IRB approval, healthy pediatric outpatients ages 0-18yrs were recruited and consented. Plasma obtained from umbilical cord, finger/heel/ear stick, or venipuncture blood samples (no more than 50 mL or 3 mL per kg) and collected in 3.2% sodium citrate. Assays included Protein (P) C antigen (PCAG) and chromogenic activity (PCFX); P S total (PST), free (PSF) antigen, and functional (SFX); antithrombin antigen (ATAG) and chromogenic activity (ATFX); plasminogen (PLGN) and alpha-2-plasmin inhibitor (A2PI). Assay kits were PCFX - HemosIL® (Werfen, Beford, MA) Protein C, PST - LIATEST® (Diagnostica Stago S.A.S., Asnières sur Seine, France) Protein S, PSF - HemosIL® Free Protein S, SFX - HemosIL® Protein S Activity, ATAG - LIATEST® AT III , ATFX - HemosIL® Liquid Antithrombin, PLGN - HemosIL® Plasminogen, and A2PI - HemosIL® Plasmin Inhibitor performed on the ACL TOP 700 (Werfen) instrument. PCAG - REAADS® Protein C Antigen Test Kit (Corgenix, Inc. Broomfield, CO) was performed on the Janus G3 liquid handler (Revvity, Waltham, MA) and Synergy Neo2 microplate reader (Agilent, Santa Clara, CA). Assay mean and range were calculated. Results Insufficient numbers of patients <1yr were collected. Type of assay, age range in yr and number of samples (n), assay mean in % and range results respectively were as follows: PCAG 1-5 (n=15) 87(46-146); 5-10 (n=82) 81 (45-158); 10-15 (n=169) 84 (44-157); 15-18 (n=107) 91 (57-149). PCFX 1-5 (n=20) 97 (56-140); 5-10 (n=84) 89 (62-135); 10-15 (n=188) 91 (55-140); 15-18 (n=124) 96 (54-138). PST (males): 1-5 (n=10) 120 (96-145); 5-10 (n=46) 108 (75-150); 10-15 (n=91) 111 (80-168); 15-18 (n=63) 113 (72-184). PST (females): 1-5 (n=9) 104 (67-146); 5-10 (n=37) 111 (81-159); Ages 10-15 (n=94) 107 (78-153); 15-18 (n=63) 101 (65-148). PSF (males): 1-5 (n=10) 94 (74-109); 5-10 (n=47) 101 (66-141); 10-15 (n=93) 101 (69-151); 15-18 (n=62) 111 (74-187). PSF (females): 1-5 (n=9) 78 (9-124); 5-10 (n=37) 107 (79-137); 10-15 (n=95) 99 (63-138); 15-18 (n=63) 97 (60-132). SFX (males): 1-5 (n=9) 100 (85-128); 5-10 (n=48) 104 (66-144); 10-15 (n=93) 103 (66-168); 15-18 (n=59) 109 (72-157). SFX (females): 1-5 (n=8) 94 (18-121); 5-10 (n=37) 107 (81-143); 10-15 (n=90) 101 (65-147); 15-18 (n=60) 97 (53-135). ATAG 1-5 (n=19) 104 (80-121); 5-10 (n=83) 104 (78-121); 10-15 (n=188) 104 (66-126); 15-18 (n=126) 101 (67-126). ATFX 1-5 (n=20) 112 (94-135); 5-10 (n=84) 107 (87-123); 10-15 (n=188) 105 (84-138); 15-18 (n=126) 101 (84-129). PLGN 1-5 (n=17) 96 (76-124); 5-10 (n=85) 88 (65-125); 10-15 (n=183) 90 (49-151); 15-18 (n=119) 94 (64-151). A2PI: 1-5 (n=17) 134 (118-151); 5-10 (n=85) 126 (107-139); 10-15 (n=183) 121 (104-140); 15-18 (n=118) 116 (91-143). Conclusions Based on CLSI EP28 A3C criteria for verification, no more than 10% of results should fall outside of established/published RI, for example adult RI. Thus, compared to the SCL adult RI, PLGN assay meets criteria in all age ranges and no additional ND are needed. For the remaining assays, additional ND for ages 1-5 are needed. For ages 5-18 criteria for verification are satisfied for all analytes except PCAG. PCAG has 27, 61, and 19 patients falling below lower RI for ages 5-10, 10-15, and 15-18, respectively.

Abstract Background: Ovarian endometrioid carcinoma (OEC) is an uncommon, understudied histotype that is distinguished from other histotypes by unique genomic features and high hormone receptor expression. There is growing interest in the biological relevance of subtyping using immunohistochemistry (IHC). Methods: Women with primary invasive OEC diagnosed from 1989 to 2010 were retrospectively identified from the population-based Alberta Cancer Registry, Canada. Pathology slide review and IHC staining for Napsin A, TP53, and WT1 were used to reduce histologic misclassification, resulting in N=166 participants (low-stage N=138, high-stage N=28), excluding those who received neo-adjuvant chemotherapy. The following markers were stained and scored on tissue microarrays: TP53 (normal/abnormal), PMS2/MSH6 (mismatch repair proficient [MMRp]/deficient [MMRd]), PGR (loss/retained), and CTNNB1 (nuclear/cytoplasmic). Using a hierarchical decision tree by stage group (low/high), we categorized tumors as TP53 abnormal, MMRd, PGR loss, nuclear CTNNB1, and no specific immunohistochemical profile (NSIP). Distributions and five-year disease specific survival (DSS) was estimated for each group; hazard ratios (HRs) adjusted for age were estimated using Cox regression with NSIP as referent group. Results: Among low-stage cases, five-year survival was as follows: 80% for TP53 abnormal (N=10, 7%), 87% for MMRd (N=15, 11%), 80% for PGR loss (N=10, 7%), 98% for nuclear CTNNB1 (N=63, 46%), and 95% for NSIP group (N=40, 29%). Overall, IHC group was associated with DSS among low stage cases (LRT p-value=0.006), and the group with nuclear CTNNB1 had a small but statistically significant DSS advantage (HR=0.089, 95% CI 0.011-0.742, p=0.025). TP53 and PGR loss suggested shorter DSS than the referent NSIP group, but not statistically significantly so (HR=1.26 and 3.08, respectively). Among high-stage cases, five-year DSS was as follows: 57% for TP53 abnormal (N=7, 25%), 75% for MMRd (N=8, 29%), 33% for nuclear CTNNB1 (N=3, 11%), and 17% for NSIP (N=10, 36%). There were no high-stage cases in the PGR-loss group. Nuclear CTNNB1 group had a shorter DSS among high-stage cases, but this was not statistically significantly so (HR=1.16, p=0.85). Conclusion: Pending replication in additional datasets, results suggest that OEC cases in nuclear CTNNB1 or NSIP IHC groups may have a favorable prognosis in which de-escalation of therapy can be considered. In addition, abnormal TP53 and loss of PGR may identify a high-risk subset of low-stage OEC. Citation Format: Brooke Jorgensen, Stacey Winham, Julie Cunningham, Sebastian Armasu, Chen Wang, Hunter Atkinson, Bryan McCauley, Linda Kelemen, Martin Koebel, Ellen Goode. Towards a combined immunohistochemistry-based subtyping of ovarian endometrioid carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6499.