College Of American Pathologists Research Articles

A-377 The Selection and Implementation of POC Illicit Drug Toxicology Testing in Hamad Medical Corporation - Doha, Qatar

Abstract Background In 2013, Qatar launched the National Mental Health Strategy which is further supported by National Health Strategy 2018–2022 and Qatar National Vision 2030, where the mental health has been identified as a priority area of development. In doing so, the Ministry of Public Health worked closely with the Ministry of Interior in providing the appropriate treatment rehabilitation. With all considerable advantages of point-of-care testing (POCT) in rapid diagnosis, POC toxicological test has been implemented and used in screening of patients for drugs of abuse. In this study, we highlighted the challenges in selection and implementation process of POC toxicological test in Hamad Medical Corporation (HMC). Methods Due to unavailability of all required drug panels, it had to be customized and tested for the verification purpose before starting the evaluation process. Maintaining HMC procurements regulations and Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathologist (CAP) standards for POC toxicology tests, two POC illicit drug toxicology screening tests has been evaluated, comparison study has been performed against the HMC central laboratory. POCT team conducted training session ensuring the appropriate practice in place. The Extendable Laboratory Information System (LIS) functionality has been utilized for seamless data management and reporting for the automated analyzer. Results QUIDEL triage meter pro is an automated drug testing analyzer which detects Amphetamines, Barbiturates, Benzodiazepines, Cannabinoids, Methadone Metabolite, Methamphetamine, Opiates, Tricyclic and Cocaine Metabolites, while the customized manual testing kit FASTEP detects Tramadol (TRA), Methylenedioxymeth-amphetamine (MDMA), Phencyclidine (PCP), Buprenorphine (BUP), Oxycodone (OXY), Fentanyl (FYL), Ethyl Glucuronide (EtG), Cotinine (COT), Pregabalin (PGB) and Gamma Hydroxybutyrate (GHB). Both analytical performances were verified. Rapid bedside testing performed for more than 100 patients in last 8 months during admission, observation, and pre- discharge process. There is An Obvious improvement observed in testing turnaround time compared to the laboratory, easy results interpretation and reporting as well as cost-effective. Conclusion The implementation of POC illicit drug toxicology testing in HMC aligned with core pillars of the Qatar vision 2030 to deliver the right treatment at the right time and place. Its enhancing patient outcomes and overall Satellite health care sector in Qatar.

A-146 A Review of RDW-CV and RDW-SD Measurements in Patients With Iron Deficiency Anemia in an Acute Care Hospital in Singapore

Abstract Background Red Cell Distribution Width (RDW) is a routinely available parameter on automated haematology analysers. This parameter is often used to help in the diagnosis of iron deficiency anaemia. RDW quantitatively measures the degree of anisocytosis of the red blood cells. The more common way of reporting RDW is RDW-CV, which is based on the coefficient of variation of the red blood cell distribution volume and is calculated using mean red cell volume. RDW can also be presented as RDW-SD, which is a direct measurement of the width of the red blood cell distribution curve and is not influenced by mean red cell volume. Conventionally, laboratories report RDW-CV as part of the red blood cell indices in full blood count (FBC) reports. However, it has been proposed that RDW-SD is a better indicator of iron deficiency anaemia compared to RDW-CV. The authors seek to review and assess the clinical utility of both RDW measurements in patients with iron deficiency anaemia in an acute care hospital in Singapore. Methods RDW data of samples from 271 unique individuals from Sysmex XN-9000 (Sysmex, Japan) automated haematology analysers over a period of two months were reviewed. These individuals were selected as they had plasma iron measured in the same blood collection. The individuals were sorted into two groups based on the plasma iron measurement: with iron deficiency (80%) and without iron deficiency (20%). The correlation of RDW-CV and RDW-SD with the plasma iron concentration of the individuals was studied. The RDW-CV reference range used were established for the local population by an in-house reference range validation study while that of the RDW-SD is from a recent Participant Summary Report for a College of American Pathologists (CAP) Hematology Automated Differential survey (FH9-B 2022). The sensitivity, specificity, predictive values, and overall efficiency of RDW-CV and RDW-SD were calculated and compared using the data collected. Results The sensitivity and specificity of RDW-CV in detecting iron deficiency anaemia are 45.2% and 68.5%. The positive and negative predictive values for RDW-CV are 85.2% and 23.7% respectively. The sensitivity and specificity of RDW-SD in detecting iron deficiency are 33.2% and 64.8%. The positive and negative predictive value of RDW-SD are 79.1% and 19.4% respectively. The overall efficiency of RDW-CV is 49.8%, significantly higher than 39.4% of RDW-SD. Conclusion This retrospective study suggested that RDW-CV is still a better blood cell index than RDW-SD in the differential diagnosis of iron deficiency anaemia in a predominantly Asian population. However, the use of RDW-SD reference range that is appropriate for the local population may improve the clinical utility of the RDW-SD.

Reporting and Establishment of Reference Intervals for Antiphospholipid Antibody Immunoassays: A Survey of Participants in the College of American Pathologists Proficiency Testing Program.

Misdiagnosis of antiphospholipid syndrome can occur owing to the wide diversity of antiphospholipid (aPL) assays and a lack of international calibrators and harmonized reference intervals. To assess laboratory practices regarding reporting and establishing reference intervals for immunoglobulin (Ig) G/IgM anti-cardiolipin (aCL) and anti-beta-2 glycoprotein I (anti-β2GPI) assays. Supplemental questions related to reporting and establishing reference ranges for aPL assays were sent as part of the Antiphospholipid Antibody (ACL)-B 2019 College of American Pathologists (CAP) proficiency testing survey. The response rate and methods assessment details were determined, as well as qualitative and quantitative results for 3 test samples. The number of participants reporting results for IgG aCL (n = 489), IgM aCL (n = 476), IgG anti-β2GPI (n = 354), and IgM anti-β2GPI (n = 331) varied by antibody type. The enzyme-linked immunosorbent assay (ELISA) (up to 58.6%, 260 of 444) was the most used method; others included multiplex (from 18.9% to 23.9%), fluorescence enzyme immunoassay (14.4%-17.6%), and chemiluminescence immunoassay (6.5%-9.0%). More respondents reported quantitative than qualitative results, and manufacturer cutoff ranges were used by 92.9% and 94.2% of respondents for aCL and anti-β2GPI, respectively. Despite variation in the use of semiquantitative ranges, qualitative negative/positive reporting of the test samples achieved almost 100% consensus. Qualitative consensus was met in contrast to the wide range of quantitative results obtained for each analyte across different kits. ELISA remains the most used method for detecting aPL antibodies, with most laboratories reporting quantitative results based on manufacturers' suggested reference ranges. The categorization of quantitative results as equivocal, weak positive, or positive for responders using kits from the same manufacturer was variable.

Pancreaticobiliary Cytology Practice in 2021: Results of a College of American Pathologists Survey.

The College of American Pathologists (CAP) surveys provide national benchmarks of pathology practice. To investigate pancreaticobiliary cytology practice in domestic and international laboratories in 2021. We analyzed data from the CAP Pancreaticobiliary Cytology Practice Supplemental Questionnaire that was distributed to laboratories participating in the 2021 CAP Nongynecologic Cytopathology Education Program. Ninety-three percent (567 of 612) of respondent laboratories routinely evaluated pancreaticobiliary cytology specimens. Biliary brushing (85%) was the most common pancreaticobiliary cytology specimen evaluated, followed by pancreatic fine-needle aspiration (79%). The most used sampling methods reported by 235 laboratories were 22-gauge needle for fine-needle aspiration (62%) and SharkCore needle for fine-needle biopsy (27%). Cell block was the most used slide preparation method (76%), followed by liquid-based cytology (59%) for pancreatic cystic lesions. Up to 95% (303 of 320) of laboratories performed rapid on-site evaluation (ROSE) on pancreatic solid lesions, while 56% (180 of 320) performed ROSE for cystic lesions. Thirty-six percent (193 of 530) of laboratories used the Papanicolaou Society of Cytopathology System for Reporting Pancreaticobiliary Cytology in 2021. Among all institution types, significant differences in specimen volume, specimen type, ROSE practice, and case sign-out were identified. Additionally, significant differences in specimen type, slide preparation, and ROSE practice were found. This is the first survey from the CAP to investigate pancreaticobiliary cytology practice. The findings reveal significant differences among institution types and between domestic and international laboratories. These data provide a baseline for future studies in a variety of practice settings.

Interpretable HER2 scoring by evaluating clinical guidelines through a weakly supervised, constrained deep learning approach.

The evaluation of the Human Epidermal growth factor Receptor-2 (HER2) expression is an important prognostic biomarker for breast cancer treatment selection. However, HER2 scoring has notoriously high interobserver variability due to stain variations between centers and the need to estimate visually the staining intensity in specific percentages of tumor area. In this paper, focusing on the interpretability of HER2 scoring by a pathologist, we propose a semi-automatic, two-stage deep learning approach that directly evaluates the clinical HER2 guidelines defined by the American Society of Clinical Oncology/ College of American Pathologists (ASCO/CAP). In the first stage, we segment the invasive tumor over the user-indicated Region of Interest (ROI). Then, in the second stage, we classify the tumor tissue into four HER2 classes. For the classification stage, we use weakly supervised, constrained optimization to find a model that classifies cancerous patches such that the tumor surface percentage meets the guidelines specification of each HER2 class. We end the second stage by freezing the model and refining its output logits in a supervised way to all slide labels in the training set. To ensure the quality of our dataset's labels, we conducted a multi-pathologist HER2 scoring consensus. For the assessment of doubtful cases where no consensus was found, our model can help by interpreting its HER2 class percentages output. We achieve a performance of 0.78 in F1-score on the test set while keeping our model interpretable for the pathologist, hopefully contributing to interpretable AI models in digital pathology.

Significant Variability in the Identification and Reporting of Band Neutrophils by Participants Enrolled in the College of American Pathologists Proficiency Testing Program: Time for a Change.

Increased band neutrophils in blood smear differential counts ("bandemia") are entrenched in medicine as a flag for sepsis. However, laboratory hematology experts have long advocated for discontinuation of reporting bands separately from segmented neutrophils because of poor sensitivity and specificity, poor interobserver agreement, and availability of alternative biomarkers for sepsis. To describe band neutrophil reporting practices and reproducibility of band classification among laboratories participating in the College of American Pathologists (CAP) proficiency testing (PT) program. A survey questionnaire was distributed to hematology PT participants. A subsequent morphologic challenge included 12 preselected cell identifications of segmented neutrophils, bands, and metamyelocytes, and a 100-cell manual differential count of a digitally scanned blood smear. Among laboratories that reported manual differentials, most respondents reported bands (4554 of 5268; 86.4%). Only 3222 of 4412 respondents (73.0%) provided band reference ranges. Though participants classified "easy" band neutrophils well (78.0%-98.3%), categorization of cell identifications for "moderate" and "difficult" bands was poor (3.1%-39.0% of laboratories), with classification instead as segmented neutrophils. This pattern was seen regardless of laboratory demographic characteristics. Marked variability in band counts was observed on the 100-cell differential count for both CAP PT participants and CAP Hematology and Clinical Microscopy Committee (HCMC) members (coefficients of variation, 55.8% and 32.9%, respectively). Variability was significantly improved when segmented and band neutrophils were grouped together (coefficients of variation, 6.2% and 5.0%, respectively). Most CAP PT-participating laboratories report band counts, many without reference ranges. The survey confirms significant interlaboratory variability of band enumeration when bands are separately identified from segmented neutrophils. This study reaffirms the CAP Hematology and Clinical Microscopy Committee's strong recommendation to group segmented and band neutrophils together in manual differential counts.

Relevance of shrinkage versus fragmented response patterns in rectal cancer.

Partial response to neoadjuvant chemoradiotherapy (CRT) presents with one of two main response patterns: shrinkage or fragmentation. This study investigated the relevance of these response patterns in rectal cancer, correlation with other response indicators, and outcome. The study included a test (n = 197) and a validation cohort (n = 218) of post-CRT patients with rectal adenocarcinoma not otherwise specified and a partial response. Response patterns were scored by two independent observers using a previously developed three-step flowchart. Tumour regression grading (TRG) was established according to both the College of American Pathologists (CAP) and Dworak classifications. In both cohorts, the predominant response pattern was fragmentation (70% and 74%), and the scoring interobserver agreement was excellent (k = 0.85). Patients with a fragmented pattern presented with significantly higher pathological stage (ypTNM II-IV, 78% versus 35%; P < 0.001), less tumour regression with Dworak (P = 0.004), and CAP TRG (P = 0.005) compared to patients with a shrinkage pattern. As a predictor of prognosis, the shrinkage pattern outperformed the TRG classification and stratified patients better in overall (fragmented pattern, hazard ratio [HR] 2.04, 95% confidence interval [CI] 1.19-3.50, P = 0.008) and disease-free survival (DFS; fragmented pattern, HR 2.50, 95% CI 1.23-5.10, P = 0.011) in the combined cohorts. The multivariable regression analyses revealed pathological stage as the only independent predictor of DFS. The heterogeneous nature of tumour response following CRT is reflected in fragmentation and shrinkage. In rectal cancer there is a predominance of the fragmented pattern, which is associated with advanced stage and less tumour regression. While not independently associated with survival, these reproducible patterns give insights into the biology of tumour response.

Prognostic value of circumferential radial margin involvement in esophagectomy for esophageal cancer: a case series.

Residual tumor at the proximal or distal margin after esophagectomy is associated with worse survival outcomes; however, the significance of the circumferential resection margin (CRM) remains controversial. In this study, we sought to evaluate the prognostic significance of the CRM in patients with esophageal cancer undergoing resection. We identified patients who underwent esophagectomy for pathologic T3 esophageal cancer from 2000 to 2019. Patients were divided into three groups: CRM- (residual tumor >1mm from the CRM), CRM-close (residual tumor >0 to 1mm from the CRM), and CRM+ (residual tumor at the surgical CRM). CRM was also categorized and analyzed per the Royal College of Pathologists (RCP) and College of American Pathologists (CAP) classifications. Of the 519 patients included, 351 (68%) had CRM-, 132 (25%) had CRM-close, and 36 (7%) had CRM+. CRM+ was associated with shorter disease-free survival [DFS; CRM+ vs. CRM-: hazard ratio (HR), 1.53 [95% CI, 1.03-2.28]; P =0.034] and overall survival (OS; CRM+ vs. CRM-: HR, 1.97 [95% CI, 1.32-2.95]; P <0.001). Survival was not significantly different between CRM-close and CRM-. After adjustment for potential confounders, CAP+ was associated with poor oncologic outcomes (CAP+ vs. CAP-: DFS: HR, 1.47 [95% CI, 1.00-2.17]; P =0.050; OS: HR, 1.93 [95% CI, 1.30-2.86]; P =0.001); RCP+ was not (RCP+ vs. RCP-: DFS: HR, 1.21 [95% CI, 0.97-1.52]; P =0.10; OS: HR, 1.21 [95% CI, 0.96-1.54]; P =0.11). CRM status has critical prognostic significance for patients undergoing esophagectomy: CRM+ was associated with worse outcomes, and outcomes between CRM-close and CRM- were similar.

RNA Sequencing for Solid Tumor Fusion Gene Detection: Proficiency Testing Practice and Performance Comparison.

Next-generation sequencing-based approaches using RNA have increasingly been used by clinical laboratories for the detection of fusion genes, intragenic rearrangements, and exon-skipping events. Correspondingly, the College of American Pathologists (CAP) has advanced RNA sequencing proficiency testing (PT) to ensure optimal performance of these assays. To report on laboratory performance and practices of RNA sequencing for the detection of fusion genes, intragenic rearrangements, and exon-skipping events using CAP PT data from 8 mailings (2018-A through 2021-B). CAP PT RNA sequencing program results from 153 laboratories across 24 proficiency test specimens, interrogating 22 distinct engineered fusion transcripts, were analyzed for correct identification of the fusion event, associated performance variables, and laboratory practices. Overall, the 4-year program detection rate (sensitivity) was 95.5% (1486 of 1556 results). False-negative rates were 3.6% (53 of 1463) and 18.3% (17 of 93) for fusion gene and intragenic rearrangement/exon-skipping events, respectively. Only 19 false-positive results were reported among the 8 PT mailings, and most were likely the result of preanalytical or postanalytical errors. There were no practice characteristics (eg, instrumentation, sequencing method) significantly associated with the fusion detection results. These data reveal a high overall sensitivity and specificity for fusion gene detection by participating laboratories using clinical RNA sequencing. Performance was comparable across all laboratories, regardless of methodology. The fraction of false-negative results for intragenic rearrangement/exon-skipping events was greater than that for the chimeric fusion genes. False-negative results could not be attributed to any specific practice characteristics.

HER-2 overexpression in female genital tract clear cell carcinomas: Evaluation of different scoring guidelines, clinicopathological features and prognostic impact

BackgroundClear cell carcinoma (CCC) is a rare high-grade adenocarcinoma associated with poor response to platinum-based chemotherapy agents in the female genital tract. Human epidermal growth factor receptor 2 (HER2) overexpression is routinely used as a biomarker for targeted therapy in breast and gastric carcinomas, but its role in CCC remains unclear. MethodsIn this study, HER2 overexpression was evaluated by immunohistochemistry (IHC) using College of American Pathologists (CAP) HER2 scoring guidelines for breast and endometrial serous carcinoma (ESC) on tissue microarray blocks. In equivocal and positive cases, fluorescence in situ hybridization (FISH) was performed. IHC score 3, and all amplified cases on FISH test were considered positive. ResultsThirty-six cases of ovarian (OCCC), 36 endometrial (ECCC), and 2 cervical CCC were included. According to ESC and breast scoring guidelines, 20 % and 15.1 % of ECCC and 14.7 % and 6 % of OCCC were HER2 positive, respectively. Both cases of cervical CCC were negative. Scoring based on breast carcinoma guideline showed higher concordance (100 %) with gene amplification results, in comparison with ESC guideline (82.7 %). On multivariate survival analysis, HER2 positive ECCC and OCCC (based on ESC scoring methods) had significantly lower overall and disease-free survivals (OS, DFS) (P < 0.05). ConclusionHER2 immunoscoring based on ESC guideline can yield a higher sensitivity with relevant clinical and prognostic features in OCCC and ECCC. HER2 can be considered a potential biomarker for targeted therapy and future clinical trials.

Prognostication and optimal criteria of circumferential margin involvement for esophageal cancer after chemoradiation and esophagectomy.

Circumferential radial margin (CRM) involvement by tumor after resection for esophageal cancer has been suggested as a significant prognostic factor. However, the prognostic value of CRM involvement after surgery with neoadjuvant concurrent chemoradiotherapy (CCRT) is unclear. This study aimed to evaluate the prognostic value of and survival outcomes in CRM involvement as defined by the Royal College of Pathologists (RCP) and the College of American Pathologists (CAP) for patients with esophageal cancer undergoing neoadjuvant CCRT and esophagectomy. A total of 299 patients with esophageal cancer who underwent neoadjuvant CCRT followed by esophagectomy between 2006 and 2016 were enrolled in our study. The CRM status of the specimens obtained was determined pathologically according to both the CAP and RCP criteria. Survival analyses were performed and compared according to the two criteria. Positive CRM was found in 102 (34.1%) and 40 (13.3%) patients according to RCP and CAP criteria, respectively. The overall and progression-free survival rates were significantly lower in the CRM-positive group than in the CRM-negative group according to both the RCP and CAP criteria. However, under multivariate analysis, in addition to pathological T and N staging of the tumor, only CAP-defined CRM positivity was a significant prognostic factor with adjusted hazard ratios of 2.64 (1.56-4.46) and 2.25 (1.34-3.78) for overall and progression-free survival, respectively (P < 0.001). In patients with esophageal cancer undergoing neoadjuvant CRT followed by esophagectomy, CAP-defined CRM positivity is an independent predictor of survival. Adjuvant therapy should be offered to patients with positive CRM.

Analytical performance verification of a high throughput system for Hepatitis B and Hepatitis C viral load quantifications

ObjectiveTo verify the analytical performance of cobas® HBV PCR and cobas® HCV PCR assays with Abbott m2000 RealTime System as the reference method. DesignDe-identified residual, archived patient specimens, and College of American Pathologists (CAP) proficiency testing samples were used. Analytical parameters verified were accuracy, precision, limit of detection (LOD), linear range, and cross-contamination. Experiments were designed in accordance with Clinical Laboratories Standards Institute (CLSI) guidelines and CAP standards. Analysis of accuracy was done through regression plots and Bland Altman analyses. Precision was analyzed through coefficient of variation and ANOVA; LOD through probit analysis; and linear range through polynomial fit analysis. ResultsThe regression plots for accuracy showed a slope nearing 1, with a y-intercept close to zero, while Bland Altman analyses also showed no systematic evidence of bias, though concordance of results was not perfect near the lower limit of quantification. Coefficients of variation were all below 15%, while ANOVA returned p-values above 0.99, indicating no statistically significant imprecision. The LOD verified were an order of magnitude higher than the manufacturer reported ones for both assays, while the linear range verified was more limited. Within the verified range, polynomial fit analysis showed line to be the best fit for the data. Conclusionscobas® HBV PCR and cobas® HCV PCR assays showed acceptable accuracy, acceptable precision, as well as no evidence of cross-contamination. The LOD verified were higher, and linear ranges more limited than those reported by the manufacturer. Verifications of these may be limited by availability of appropriate testing specimens.

HER2 Equivocal (Score = 2+) Breast Carcinoma Cases Identified by Immunohistochemistry at a South African Hospital. What is the Impact of Fluorescent In Situ Hybridization Testing?

The American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) guidelines are used for human epidermal growth factor receptor 2 (HER2) reporting in breast carcinoma. Cases that demonstrate weak to moderate complete membrane immunohistochemical staining in >10% of the tumor are scored as 2+ (equivocal). This study aimed to determine what proportion of HER2 immunohistochemistry (IHC) score = 2+ breast carcinomas were confirmed to be positive by HER2 fluorescent in situ hybridization (FISH). There were 241 HER2 IHC score = 2+ breast carcinomas included. Most (74.3%) carcinomas were estrogen and progesterone receptor-positive. Invasive breast carcinoma of no special type (89.2%) was the commonest histologic subtype. Most tumors were grade 2 (64.3%). As per the FISH report, at the time of diagnosis, 27 cases (11.2%) were HER2 FISH positive. All HER2 FISH equivocal cases and one FISH positive case assessed using the 2013 ASCO/CAP HER2 criteria were reclassified to HER2 FISH negative when the 2018 criteria were applied. There was a high level of agreement (κ = 0.979) between HER2 FISH results obtained using the 2013 and the 2018 criteria. This study provides insight into the frequency of HER2 FISH positivity (11.2%) among HER2 IHC score = 2+ breast carcinomas and the impact of modifications to the ASCO/CAP HER2 guidelines. Elimination of the HER2 FISH equivocal category by the 2018 guidelines has reduced the need for repeat testing and simplified clinical management. Reclassification of previous HER2 FISH positive to negative has resulted in some patients being ineligible for costly anti-HER therapy.

Comprehensive genomic analysis of salivary gland carcinomas in a Chinese population.

e18094 Background: Salivary gland carcinomas (SGCs) represent a heterogeneous group of poorly characterized head and neck tumors, which are rare and challenging to treat. A strategy based on molecular screening and targeted therapy seems to be the best approach for treating patients with SGCs in the future, whereas the genomic mechanisms of SGCs are largely unclear. Methods: A total of 49 patients with histologically confirmed SGCs, including mucoepidermoid carcinoma (MEC, n = 10), adenoid cystic carcinoma (ACC, n = 20), carcinoma ex pleomorphic adenoma (cxPA, n = 10) and other pathological types (n = 9) were enrolled in our study. Tumor tissue specimens were collected and targeted next-generation sequencing for 450 cancer-related genes was performed in OrigiMed (Shanghai, China), a College of American Pathologists (CAP) accredited and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. Results: In all samples, the most frequently mutated genes were MYB (26.53%), TP53 (26.53%), ERBB2 (12.24%), KMT2D (12.24%), and TERT (12.24%). Actionable genes included ERBB2 (12.2%), PIK3CA (8.2%), ATM (4.1%), CDKN2A/B (4.1%), and EGFR (4.1%). Among these patients, female patients accounted for a higher proportion in ACC compared to that in MEC and cxPA (ACC: 65%, MEC: 30%, cxPA: 30%). Gene fusions/rearrangements were identified in 13 tumors, and 10 (76.9%) had evidence of MYB-NFIB fusion, which may represent an important new therapeutic target for ACC. Higher frequency of MYB (p &lt; 0.0001) and EP300 (p = 0.0445) mutations was also identified in ACC compared with that in other types. Mutational frequencies of TP53 (p = 0.0096) and ERBB2 (p = 0.0228) were obviously higher in cxPA, whereas the frequency of CCNE1 (p = 0.0433) mutations were significantly higher in MEC compared with those in other types. The gene mutations of Chinese SGCs were mainly enriched in RTK/RAS (56.7%), TP53 (40.8%), Notch (34.5%), and PI3K (30.5%) signaling pathways. Conclusions: This report revealed the molecular characteristics of Chinese SGC patients diagnosed with different pathological types, which would be helpful for the investigation of the underlying genomic mechanisms of SGC. Our result highlights the potential role of gene alterations, many of which could conceivably be actionable with targeted therapies.

Postoperative prognosis in patients with NSCLC with different EGFR mutation sites.

e20528 Background: Lung cancer can be divided into small cell lung cancer and non-small cell lung cancer. At the same time, non-small cell lung cancer can be divided into non-small cell lung cancer with different molecular mutations. Among them, the EGFR mutation accounts for about 50% of the patients with non-small cell lung cancer in China, which is a large group of patients. The EGFR mutation has many sites and many forms of mutational subtypes, including common mutations such as L858R, and rare mutations. Although the proportion of rare mutations is small, it is still different from common mutations in patients with TKI treatment and immunity. Methods: This study collected and analyzed the data from Formalin-Fixed Paraffin-Embedded (FFPE) tissues of 330 NSCLC patients which received surgery and underwent a targeted next-generation sequencing (NGS) assay performed by 3DMed Clinical Laboratory Inc., a College of American Pathologists (CAP) certified and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory of 3D Medicines Inc. between January, 2019 and June, 2022 in China, to obtain a comprehensive molecular profile of EGFR mutations. genomic alterations, tumor mutational burden (TMB) and PD-L1 expression were analyzed. Gene mutation and TMB level were analyzed by NGS, detected PD-L1 expression by using Dako PD-L1 IHC 22C3 pharmDx, Tumor Proportion Score (TPS) was used to determine expression of PD-L1. Finally, the postoperative follow-up data were also collected and analyzed in this study. Results: Therefore, we retrospectively analyzed the tissue samples of patients with non-small cell lung cancer who received surgical treatment, and carried out NGS detection and analysis, as well as postoperative follow-up. Among them, patients with EGFR mutations accounted for 51.2%. We further analyzed the TMB and PD-L1 expression levels of different EGFR mutation subtypes. First, we found that different EGFR mutation sites, whether common mutations such as L859R, 19del or unusual mutations such as S768I, There is no difference in the prognosis after operation, but it is very significant that our results show that there are two or more EGFR mutations at the same time. Conclusions: NSCLC with EGFR mutation is a very large group of patients, which should be further subdivided into different subtypes according to different EGFR mutation sites, and should perform individualized management. This study found that patients with two or more EGFR mutations and patients with EGFR single site mutations are different types of NSCLC, with different postoperative prognosis, and it may be related to the differences in the expression levels of TMB and PD-L1, which is a direction worthy of further exploration in the future.

Multi-omics study of the genomic features and clinical characteristics of hepatic sarcomatoid carcinoma located at different sites.

e15153 Background: Hepatic sarcomatoid carcinoma (HSC) is a rare and highly malignant tumor, which accounts for less than 2% of hepatic carcinoma. HSC can occur in the liver (Sarcomatoid hepatocellular carcinoma, SHCC) or in the intrahepatic bile duct (Sarcomatoid intrahepatic cholangiocarcinoma, SiCCA). We aimed to compare the clinical characteristics and genomic features between SHCC and SiCCA. Methods: In this study, we recruited 20 patients who were pathologically diagnosed with HSC from January 2018 to February 2021 in Hunan Provincial People’s Hospital. Whole exome sequencing and PD-L1 expression detected by immunohistochemistry were performed on the qualified tissue samples obtained from18 (14 with SHCC and 4 with SiCCA) of the enrolled patients. Tests were performed in OrigiMed, a College of American Pathologists (CAP) accredited and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. Fisher test was used to determine the association between two categorical variables, and P&lt; 0.05 was considered statistically significant. Results: A total of 15 cases of SHCC and 5 cases of SiCCA were enrolled in this study, with median age of 59 years old (ranged from 34 to 75 years old). Among them, 15 (75%) were diagnosed as AJCC stage III-IV. Compared with SiCCA, we observed a significant higher proportion of undifferentiated samples in SHCC ( P&lt; 0.001) (Table 1). The most commonly mutated genes of HSC included TP53 (64% in SHCC and 75% in SiCCA) and TTN (43% in SHCC and 100% in SiCCA). The most commonly mutated genes in SHCC also included TERT (43%), CCDC168 (36%), and LATS1 (36%), and that in SiCCA also included CDKN2A (50%), KRAS (50%), and CDKN2B (50%). All these genes were mainly enriched in cell cycle and ERBB pathway. In addition, amplifications of HYDIN, C4A, LRRC37A and deletions of CDKN2A, NTRK2, NTRK3, NRG1, PAX3, OR4K5, TPP2 and KRTAP4-6 were detected in the SHCC cohort, and amplifications of ZDHHC11, PMS2 and deletions of NRG1 and CDKN2A were detected in the SiCCA cohort. We also assessed TMB and PD-L1 status among these two cohorts. We found that 86% (12/14) of SHCC and 100% (4/4) of SiCCA had TMB of lower than 10 mutations/Mb. The expression of PD-L1 was positive (CPS &gt; = 1) in 86% (12/14) of SHCC and 75% (3/4) of SiCCA. Conclusions: We are the first to discover the significant differences in tumor differentiation between SHCC and SiCCA. We also profiled the mutational characteristics, TMB status, and PD-L1 status of HSC, shedding light on the precision medicine of HSC. [Table: see text]

Frequency and spectrum of clinically significant molecular alterations detected by tumor gene panel testing for pediatric cancer patients in the Texas KidsCanSeq study.

10018 Background: Tumor sequencing has the potential to inform diagnosis, prognosis, and treatment of childhood cancer patients. Prospective data are needed to evaluate the clinical utility of these tests. Methods: Patients less than 18 years of age with extracranial solid tumors (ST), central nervous system tumors (CNS), lymphomas and histiocytic disorders (L/H) were enrolled in the Texas KidsCanSeq clinical genomics study at six Texas institutions. Available tumor samples from a subset of patients designated as high risk (based on poor prognosis at diagnosis or tumor recurrence) were sequenced using pediatric cancer-focused targeted DNA (initially 124 genes; expanded to 169 genes) and RNA (81 genes) panels developed at Texas Children’s Hospital. DNA variants (single nucleotide variants, insertion/deletions, splice site variants, copy number alterations) and fusion genes detected by RNA testing were analyzed. Tumor alterations were classified by molecular pathologists and oncologists as carrying strong (Tier 1) or potential (Tier 2) clinical significance following consensus guidelines from the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists. Results: A total of 626 patients were enrolled between 2018 and 2021, of whom 380 were designated as high risk. Tumor samples adequate for molecular testing were available from 342 of the 380 (90%) high risk patients (196 ST [57%], 132 CNS [39%] and 14 L/H [4%]). Clinically significant alterations were identified in 246 of 342 (72%) tumors: 165 (48%) from DNA testing only, 34 (10%) from RNA testing only, and 47 (14%) from both modalities. The frequency of clinically significant alterations was similar across tumor cohorts (74% ST, 68% CNS, 71% L/H). Alterations relevant to therapy-choice were reported in 127 of 342 (37%) tumors (35% ST, 26% CNS, 57% L/H); 87 of these 127 (69%) were not known from prior clinical testing. The most frequently altered therapeutic gene alterations (Table) involved BRAF (n=25), CDKN2A/B (n=20), SMARCB1 (n=17), FGFR1/2/3/4 (n=14), NF1 (n=13), PIK3CA (n=11), ALK (n=10), CDK4 (n=9), and RET (n=7). One hypermutated tumor was identified: a high-grade glioma (tumor mutation burden &gt;190 mut/Mb) in a patient with Lynch syndrome due to a germline MSH6 variant. Conclusions: Tumor alterations of clinical significance according to consensus guidelines were reported in a large proportion of children with diverse high risk or recurrent cancer diagnoses, the majority of which were not detected by other standard pathologic analysis. These data further support broader adoption of tumor molecular profiling for these patients. [Table: see text]

Microsatellite instability in sarcomas: Which tests are ideal for diagnosis, and a systematic review.

e23519 Background: There is a lack of clinically relevant biomarkers to inform immunotherapy for patients with advanced sarcoma. High levels of microsatellite instability (MSI-H) is a histology-agnostic biomarker of response to pembrolizumab. However, the frequency of MSI-H in sarcoma is low - ranging from &lt; 0.5% to 4%. MSI status can be assessed through Next Generation Sequencing (NGS), immunohistochemistry (IHC), or PCR followed by fragment analysis with capillary electrophoresis (PCR). To date, both the College of American Pathologists and the European Society of Medical Oncology have not determined the best method to evaluate MSI in sarcomas. We performed a systematic review to evaluate the methods to diagnose MSI-H in sarcomas and to evaluate the impact of this biomarker in treatment with pembrolizumab. Methods: We performed a literature search using the PubMed database to identify all English full-text available articles between January 2020 and December 2022, with the terms “sarcoma and microsatellite instability” and “sarcoma and pembrolizumab”. One independent reviewer screened the studies, two independent reviewers analyzed the selected studies, and disparities were resolved with a fourth reviewer. Results: Concerning the diagnosis of MSI-H in sarcomas, 187 abstracts were found, and 31 articles were identified for full analysis. MSI status was reported for 7958 patients - 7762 with a single test and 196 with more than one test. NGS was used for 6649, IHC for 1222, and PCR for 285. MSI-H tumors were 25 (0.4%), 38 (3.1%), and 21 (7.33%) for each technique, respectively. Two investigators performed the three available tests to evaluate concordance, with one concordant case and one discordant case. 194 cases were evaluated for both IHC and PCR, with 4 discordant results. No article that described NGS results performed a second test. To analyze the results of pembrolizumab therapy in MSI-H sarcomas, we analyzed 199 abstracts of which we selected 13 prospective and 31 retrospective papers for full analysis. The K cohort of Keynote-158 is the only prospective trial concerning therapies guided by MSI-H in sarcoma (n = 14 patients, ORR = 33%). Regarding retrospective data, we identified 2 case reports of the use of pembrolizumab in patients with advanced sarcoma – both achieved a response to therapy. Conclusions: There is limited data to establish the best diagnostic test for MSI-H in patients with sarcoma. The concordance between NGS, IHC, and PCR seems to be high, but discrepancies have been described and need to be further evaluated. There is only one prospective trial that enrolled patients with sarcomas utilizing MSI-H as a biomarker. Albeit only a small number of patients were enrolled (n = 14), efficacy is consistent with that seen in other histologies, and scarce retrospective data corroborate the potential role of MSI-H as a predictive biomarker of pembrolizumab efficacy in sarcoma.

Acid sphingomyelinase expression is associated with survival in resectable pancreatic ductal adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is one of the most common cancers worldwide. Unfortunately, the prognosis of PDAC is rather poor, and for instance, in the USA, over 47,000 people die because of pancreatic cancer annually. Here, we demonstrate that high expression of acid sphingomyelinase in PDAC strongly correlates with long-term survival of patients, as revealed by the analysis of two independent data sources. The positive effects of acid sphingomyelinase expression on long-term survival of PDAC patients were independent of patient demographics as well as tumor grade, lymph node involvement, perineural invasion, tumor stage, lymphovascular invasion, and adjuvant therapy. We also demonstrate that genetic deficiency or pharmacological inhibition of the acid sphingomyelinase promotes tumor growth in an orthotopic mouse model of PDAC. This is mirrored by a poorer pathologic response, as defined by the College of American Pathologists (CAP) score for pancreatic cancer, to neoadjuvant therapy of patients co-treated with functional inhibitors of the acid sphingomyelinase, in particular tricyclic antidepressants and selective serotonin reuptake inhibitors, in a retrospective analysis. Our data indicate expression of the acid sphingomyelinase in PDAC as a prognostic marker for tumor progression. They further suggest that the use of functional inhibitors of the acid sphingomyelinase, at least of tricyclic antidepressants and selective serotonin reuptake inhibitors in patients with PDAC, is contra-indicated. Finally, our data also suggest a potential novel treatment of PDAC patients with recombinant acid sphingomyelinase.Key messagesPancreatic ductal adenocarcinoma (PDAC) is a common tumor with poor prognosis.Expression of acid sphingomyelinase (ASM) determines outcome of PDAC.Genetic deficiency or pharmacologic inhibition of ASM promotes tumor growth in a mouse model.Inhibition of ASM during neoadjuvant treatment for PDAC correlates with worse pathology.ASM expression is a prognostic marker and potential target in PDAC.

Pathologic Tumor Regression is Associated With Improved Survival in Pancreatic Cancer.

The advent of effective chemotherapy regimens has increased the use of neoadjuvant multiagent chemotherapy in pancreatic cancer. However, the effect of tumor downstaging with neoadjuvant treatment on survival is unclear. Retrospective study included all resected patients with pancreatic adenocarcinoma who underwent neoadjuvant chemotherapy with FOLFIRINOX or gemcitabine/Abraxane. Downstaging was quantified using (1) difference between presenting AJCC clinical and final pathologic stage and (2) College of American Pathologists (CAP) Tumor Regression Grading Schema. Eighty-seven patients met inclusion criteria. FOLFIRINOX was the most common regimen, 63.2% vs 21.8%. Change in regimen occurred in 15% of patients. Downstaging based on a difference in AJCC stage group occurred in only 4.6%. In contrast, 45.2% were classified as downstaged by the CAP Tumor Regression of 0-2. Downstaging was similar for FOLFIRINOX gemcitabine/Abraxane (64.7 vs 53.6, P = .12) using the CAP criteria. On univariate analysis, treatment regimen (gemcitabine/Abraxane vs FOLFIRINOX, median survival 27 vs 29 mo; HR 1.57, P = .2) had similar survival. Downstaging by the AJCC stage was not associated with improved survival (HR 1.51, P = .4). However, there was a survival benefit for those downstaged by the CAP Tumor Regression Grading Schema, the median survival of 41 mo vs 25 mo; HR 3.05, P = .009. Improved survival 3.32 (1.35-8.16), P = .009) was maintained on multivariate analysis. Survival is significantly improved in those downstaged, as assessed by the CAP Tumor Regression Schema. Downstaging is an important prognostic variable that can help with joint decision making for clinicians and patients.