Archival Tissue Research Articles

Abstract GS1-11: Sacituzumab Govitecan (SG) vs Treatment of Physician’s Choice (TPC): Efficacy by Trop-2 Expression in the TROPiCS-02 Study of Patients (Pts) With HR+/HER2– Metastatic Breast Cancer (mBC)

Abstract Background: HR+/HER2– mBC, the most common subset of breast cancer, is treated with sequential endocrine therapy + targeted agents followed by sequential single-agent chemotherapy (CT), with increasingly shorter benefit duration with each subsequent treatment. High Trop-2 expression is observed in breast cancer regardless of subtype. SG is a Trop-2-directed antibody-drug conjugate approved for pre-treated metastatic triple-negative breast cancer. In the phase 3 TROPiCS-02 study, SG showed both significant progression-free survival (PFS) benefit (HR, 0.66; P<0.001; median 5.5 vs 4.0 mo; JCO 2022) at the primary analysis and overall survival (OS) benefit (median 14.4 vs 11.2 mo; HR, 0.79; P=0.02; ESMO 2022) at the 2nd planned interim OS analysis vs TPC in pretreated HR+/HER2- mBC. Here we compare efficacy outcomes for SG and TPC by Trop-2 expression. Methods: Eligible pts had HR+/HER2- locally recurrent inoperable or mBC; received ≥1 prior taxane, endocrine therapy, a CDK4/6 inhibitor; and received 2-4 prior CT regimens for mBC. Pts were randomized 1:1 to receive SG (10 mg/kg IV on d 1 and 8, every 21 d) or TPC (eribulin, gemcitabine, capecitabine, or vinorelbine) until disease progression or unacceptable toxicity. The primary endpoint was PFS by independent review per RECIST v1.1; OS and objective response rate (ORR) were key secondary endpoints. ORR was assessed by blinded independent central review per RECIST v1.1. Membrane Trop-2 expression on archival tumor tissue was assessed by immunohistochemistry and expressed as a histochemical score (H-score; range, 0-300); efficacy outcomes were assessed in H-score <100 and ≥100 groups. The H-score <100 group was further divided into H-score ≤10 and >10- <100 subgroups to assess the activity of SG in pts with very low Trop-2 expression. Results: Data cut-off was January 3, 2022 for PFS (median follow-up, 10.2 mo) and July 1, 2022 for OS (median follow-up, 12.5 mo). In total, 543 pts were randomized to receive SG (n=272) vs TPC (n=271). Pts had a median of 3 prior CT regimens for mBC; 95% had visceral metastases. There were 238 (88%) vs 224 (83%) Trop-2-evaluable pts in the SG vs TPC groups, respectively; of these, 95% had tumors with Trop-2 H-score >0. Of Trop-2-evaluable pts, 192 (42%) and 270 (58%) had H-scores <100 and ≥100, respectively. Demographics and baseline characteristics were generally consistent across H-score groups. PFS and OS benefit was observed for SG vs TPC across both Trop-2 groups (Table). Median PFS was 5.3 vs 4.0 mo (HR, 0.77; 95% CI, 0.54-1.09) and 6.4 vs 4.1 mo (HR, 0.60; 95% CI, 0.44-0.81) in the H-score <100 and ≥100 groups; median OS was 14.6 vs 11.3 mo (HR, 0.75; 95% CI, 0.54-1.04) and 14.4 vs 11.2 mo (HR, 0.83; 95% CI, 0.62-1.11), respectively. Disease response was observed in the 34 pts with H-score ≤10 who received SG. In pts who received SG, those with H-score ≤10, >10- <100, and ≥100 had ORRs of 24%, 18%, and 23%, respectively. The safety profile for SG by Trop-2 H-score was consistent with previous reports. Conclusions: In this TROPiCS-02 post-hoc analysis, improved efficacy with SG vs TPC was observed regardless of Trop-2 expression, and there was no clear level of Trop-2 expression at which a better treatment effect for SG was observed. These results support SG as an effective novel treatment option for patients with pretreated, endocrine-resistant HR+/HER2- mBC, and reinforce that Trop-2 testing is not required for SG treatment. Table Citation Format: Hope Rugo, Aditya Bardia, Frederik Marmé, Javier Cortés, Peter Schmid, Delphine Loirat, Olivier Trédan, Eva Ciruelos, Florence Dalenc, Patricia Gómez Pardo, Komal Jhaveri, Monica Motwani, Oh Kyu Yoon, Hao Wang, Wendy Verret, Sara Tolaney. Sacituzumab Govitecan (SG) vs Treatment of Physician’s Choice (TPC): Efficacy by Trop-2 Expression in the TROPiCS-02 Study of Patients (Pts) With HR+/HER2– Metastatic Breast Cancer (mBC) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr GS1-11.

Abstract PD2-09: Characterization of the lymphovascular invasion microenvironment reveals immune response dichotomy

Abstract Background: Metastasis is the leading cause of cancer related deaths in breast cancer patients. Lymphovascular invasion represents one of the earliest stages of metastasis wherein the cells are introduced to a very different and distinct microenvironment. Methods: We leveraged spatial techniques developed for limited specimens in archival tissue to study patient matched cross-sectional tumor samples from different stages of breast neoplasia including normal breast, ductal carcinoma in situ (DCIS), primary invasive carcinoma (IBC), lymphovascular invasion (LVI) and regional lymph node metastasis. We selected a set of 21 patients with ER+ breast cancer to generate cross-sectional samples of each of these stages, for a total of 331 samples. The areas of LVI were identified by a combination of H&E review and immunohistochemistry for podoplanin. We performed smart-3SEQ for gene expression profiling and light pass whole genome sequencing for DNA copy number alterations. Results: We profiled the spectrum of neoplasia for transcriptome-wide gene expression. Principal component analysis of all 252 DCIS, LVI, IBC, or metastasis samples using the top 500 genes with the highest variance demonstrated that clustering was roughly based on the diagnostic stage (i.e. DCIS, LVI, IBC, or metastasis). Differential gene expression profiling identified thousands of genes increased or decreased in expression across the transitional stages with the largest change in gene expression being the transition from normal breast to DCIS, dominated by gene expression down regulation. We next performed NMF clustering on 62 samples of LVI from 18 cases and identified two patterns of gene expression which define two subgroups. Gene ontology analysis revealed that one cluster was associated with increased proliferation and metabolism, whereas the second cluster was dominated by an immune response. When we analyzed the immune and proliferative LVI subgroups separately, we found that the immune profiles in the patient matched IBC and LVI samples from the LVI Immune cluster were similar, whereas the immune profiles in the patient matched IBC and LVI samples from the Proliferative cluster were significantly different. At the LVI stage, all immune cell populations estimated by CibersortX were decreased in the Proliferative LVI cluster. These changes were validated using immunofluorescence for proliferation (Ki67), T cells (CD3) and macrophages (CD68) on the same samples. Using the LVI centroids, we built a model that could predict the same clusters in the METABRIC IBC. Kaplan-Meier analysis showed a significant difference between groups, with the Proliferative-like IBC group having a worse prognosis than the Immune-like IBC group. Conclusions: We observed a dichotomy at the LVI stage with a more proliferative cluster that may escape the immune response and an immune cluster which has a microenvironment with a similar pattern to its primary IBC. The recognition of two groups of LVI, differing in immune association and proliferation, raises the possibility that the risk of metastasis could be different in these two groups, leading to different biological pathways of progression. Citation Format: Belén Rivero-Gutiérrez, Diego Mallo, Almudena Espín-Pérez, Sujay Vennam, Chunfang Zhu, Sushama Varma, Greg Scott, Joseph Foley, E Shelley Hwang, Carlo Maley, Robert West. Characterization of the lymphovascular invasion microenvironment reveals immune response dichotomy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD2-09.

Alliance A031902 (CASPAR): A randomized phase (ph) 3 trial of enzalutamide with rucaparib/placebo in first-line metastatic castration-resistant prostate cancer (mCRPC).

TPS277 Background: Despite a growing number of treatment options for first-line mCRPC, approximately 40% of patients (pts) have radiographic progression within the first year. Co-inhibition of androgen receptor (AR) and PARP is a promising therapeutic strategy that leverages synthetic lethality induced by impaired double-strand DNA repair. Two phase III studies have shown improvement in radiographic progression-free survival (rPFS) in HRR-mutant pts with abiraterone + PARPi combinations vs abiraterone alone. However, the results in HRR-wild type pts are conflicting, with only one of the studies demonstrating a benefit with the abiraterone + PARPi combination. ENZ + RUCA has shown an acceptable safety profile & no significant drug-drug interactions (S-DDI) in a phase 1b trial. This allows its evaluation in mCRPC. Methods: CASPAR (A031902) is a phase 3 study in which 984 pts will be randomized on a 1:1 basis to ENZ plus RUCA/PBO. HRR gene aberration is not required for enrollment. All pts will undergo next-generation targeted exome sequencing from archival tumor tissue (new biopsy only required if no archival tissue is available). Treatment will be continued until disease progression and crossover is not allowed. Key eligibility criteria are age ≥ 18 years, ECOG PS 0-2, biopsy-proven prostate adenocarcinoma, progressive (PSA or radiographic) disease per Prostate Cancer Working Group 3 guidelines, measurable or non-measurable disease per RECIST 1.1, no prior treatment for mCRPC (prior docetaxel, abiraterone, darolutamide, or apalutamide in non-mCRPC setting is allowed), no significant uncontrolled comorbidity, and no medications with S-DDI with ENZ/RUCA. Hierarchical co-primary endpoints are overall survival (OS) and rPFS. The OS analysis will be undertaken as a primary endpoint if the rPFS endpoint is met. For a one-sided logrank test with a type 1 error rate equal to 0.025, the study has 90% power to detect a hazard ratio (HR) of 0.71 in rPFS (median rPFS of 15 and 21 months in control and combination arms, respectively), and 80% power to detect an HR of 0.80 in OS (median OS of 32 and 40 months, respectively). Key secondary endpoints are rPFS and OS in pts with vs without pathogenic BRCA1, BRCA2, or PALB2 alterations; and differences in adverse events and quality of life (QOL) outcomes between the treatment arms. QOL assessments include Functional Assessment of Cancer Therapy–Prostate (FACT-P), Brief Pain Inventory Short Form (BPI-SF), and EQ-5D-5L. A key correlative endpoint is the sensitivity of ctDNA testing for HRR gene alterations. Enrollment began in July 2021 & the study is available for participation to all US-NCTN sites. Clinical trial information: NCT04455750 .

Alliance A032002 (ART): Phase II randomized trial of atezolizumab versus atezolizumab and radiation therapy for platinum-ineligible/refractory metastatic urothelial cancer.

TPS589 Background: In patients (pts) with metastatic unresectable urothelial cancer, platinum-based chemotherapy remains the standard of care for first-line treatment followed by switch maintenance avelumab if disease control is achieved with chemotherapy. Outside of this setting, single agent immunotherapy is often used in pts that have recurrence after platinum-based chemotherapy or are platinum ineligible. Atezolizumab is a PD-L1 inhibitor currently approved for pts that have urothelial cancers expressing positive PD-L1 or pts ineligible for receiving platinum-based chemotherapy. Tumor-targeted radiotherapy can generate immune-stimulating effects without immune suppression as was previously thought. Moreover, it has become clear that radiotherapy can induce profound effects on tumor cells and the tumor microenvironment that can enhance or trigger an anticancer immune response. While numerous trials have investigated the abscopal effect, this trial will have specific parameters regarding drug type, radiation dose and administration. Methods: A032002 is a phase 2 trial addressing pts that are platinum ineligible or refractory to platinum-based chemotherapy. 144 pts will be randomized to receive either atezolizumab or atezolizumab and single site radiation therapy. The atezolizumab regimen is 1200 mg every 3 weeks. Administration of radiotherapy will occur to one non-target site (8 Gy x 3) for pts randomized to the atezolizumab + radiotherapy arm. All pts will undergo centralized PD-L1 testing (SP142 monoclonal primary antibody), which can be performed on archival tissue; a new biopsy is only required if no archival tissue is available. Key eligibility criteria include age ≥ 18 years, ECOG performance status 0-2, histologically confirmed metastatic urothelial cancer, having at least one measurable site per RECIST 1.1 to monitor for abscopal response, one site targetable for radiation, and tissue available for PD-L1 testing. The primary endpoint is tumor response within 6 months of randomization. Tumor response is defined as a complete response (CR) or partial response (PR) as assessed by the treating physician using RECIST 1.1 criteria. For a one-sided log rank test with a type 1 error rateof 0.10, the study has 90% power to detect a 20% increase in response rate. Key secondary endpoints include tumor response using iRECIST, progression-free survival and overall survival. Quality of life assessments include EORTC QLQ-C30, QLQ-BLM30 and PROMIS-Fatigue. Tissue, urine and blood samples will be collected and biobanked for future correlative science. Enrollment to ART began in December 2021. The study is available for participation at all US NCTN sites with a projected enrollment of 3 years. Support: U10CA180821, U10CA180882. Clinical trial information: NCT04936230 .

Phase II trial of lurbinectedin combined with avelumab as maintenance therapy for metastatic urothelial carcinoma with stable or responding disease following platinum-based chemotherapy.

TPS590 Background: Maintenance avelumab following stable/responding disease after 4-6 cycles of platinum-based chemotherapy is a conventional first-line therapy for metastatic urothelial carcinoma (mUC). Given the attrition of patients between lines of systemic therapy, there is a role for extending the benefits of maintenance avelumab by combinations with tolerable and active agents. Lurbinectedin (Lurbi) is an alkylating agent and selective inhibitor of oncogenic transcription. Lurbi is approved by the US FDA for treating small cell lung cancer with progression on platinum-based chemotherapy with typical dosing as 3.2 mg/m2 intravenously (IV) every 3 weeks. The activity of lurbi in the presence of DNA damage repair (DDR) alterations, multiple transcriptomic drivers and preclinical synergism with and feasibility of co-administration with immune checkpoint inhibitors (ICI) suggests potential activity of combination lurbi and ICI in mUC. Patients with stable or responding disease following platinum-based chemotherapy are phenotypically selected for deficiencies in DDR. Lurbi may be tolerable in this setting given mostly reversible myelosuppression and no serious non-hematologic toxicities especially neurotoxicity. Thus, the combination of lurbi with maintenance avelumab may warrant evaluation in mUC. Methods: A non-randomized multicenter (Advent Health Cancer Institute, Moffitt Cancer Center) phase II trial (n=36) is designed to evaluate combination avelumab (800 mg IV every 2 weeks) and lurbi (3·2 mg/m2 IV as a 1-h infusion every 4 weeks) for those with stable or responding disease following 4-6 cycles of platinum-based first-line platinum-based chemotherapy for mUC. Therapy is continued till progression of disease, intolerable toxicities or patient decision. Patients are seen for a clinical examination every 4 weeks (1 cycle) with laboratory evaluation every 2 weeks. Radiographic imaging is performed every 8 weeks for 6 months and then every 12 weeks thereafter to assess disease. Premedication for lurbi includes corticosteroids (dexamethasone 8 mg or equivalent) and serotonin antagonists. Mandatory granulocyte-colony stimulating factor (G-CSF) prophylaxis will be administered to all patients. Baseline archival tumor tissue and periodic blood is collected for correlative studies (tumor genomics/PD-L1 expression, circulating tumor DNA and immune markers). Given a median progression-free survival (PFS) with avelumab alone of 3.7 months, an alternative median PFS of 6 months is of interest warranting further development. We will enroll 33 evaluable patients to achieve 90% power at a 1-sided alpha level 0.1 to detect hazard ratio of 0.62. Given ~10% of enrolled patients may be inevaluable, we may enroll up to 36 patients. Clinical trial ID: NCT05574504. Clinical trial information: NCT05574504 .

Assessment of peritubular capillary rarefaction in kidneys of cats with chronic kidney disease.

Hypoxia is a key driver of fibrosis and is associated with capillary rarefaction in humans. Characterize capillary rarefaction in cats with chronic kidney disease (CKD). Archival kidney tissue from 58 cats with CKD, 20 unaffected cats. Cross-sectional study of paraffin-embedded kidney tissue utilizing CD31 immunohistochemistry to highlight vascular structures. Consecutive high-power fields from the cortex (10) and corticomedullary junction (5) were digitally photographed. An observer counted and colored the capillary area. Image analysis was used to determine the capillary number, average capillary size, and average percent capillary area in the cortex and corticomedullary junction. Histologic scoring was performed by a pathologist masked to clinical data. Percent capillary area (cortex) was significantly lower in CKD (median 3.2, range, 0.8-5.6) compared to unaffected cats (4.4, 1.8-7.0; P= <.001) and was negatively correlated with serum creatinine concentrations (r= -.36, P= .0013), glomerulosclerosis (r= -0.39, P= <.001), inflammation (r= -.30, P= .009), and fibrosis (r= -.30, P= .007). Capillary size (cortex) was significantly lower in CKD cats (2591 pixels, 1184-7289) compared to unaffected cats (4523 pixels, 1801-7618; P= <.001) and was negatively correlated with serum creatinine concentrations (r= -.40, P= <.001), glomerulosclerosis (r= -.44, P< .001), inflammation (r= -.42, P= <.001), and fibrosis (r= -.38, P= <.001). Capillary rarefaction (decrease in capillary size and percent capillary area) is present in kidneys of cats with CKD and is positively correlated with renal dysfunction and histopathologic lesions.

The Pattern of Expression of Human Placental Lactogen Across Normal, Lactational, and Malignant Mammary Epithelium.

The immunoexpression of human placental lactogen (hPL) in mammary epithelium is not well studied in the literature. Our overall objective was to delineate the distribution pattern of hPL across mammary epithelia of varying levels of differentiation. This is the first research to study the level of expression of hPL in human lactational change epithelium. Immunohistochemistry (IHC) for hPL was performed on archival formalin-fixed paraffin-embedded tissue blocks of 97 cases. These consisted of 53 invasive ductal carcinomas, 21 lactational change cases, and 23 cases of normal mammary tissue. The results of this study show underexpression of hPL in malignant epithelium compared to normal and lactational groups individually and combined as a non-malignant group. However, a higher expression of hPL was noted in mammary carcinoma of axillary lymph node (ALN)-positive patients compared to ALN-negative cases. There was no statistically significant difference between hPL expression and tumor grade, estrogen receptors (ER), progesterone receptors (PR), or human epidermal growth factor receptor 2 (HER2) status. The comparison of the immunoexpression of hPL in malignant epithelium versus lactational change epithelium may provide the basis for future studies on the possible role of hPL in the protective mechanism of lactation tissue from carcinogenesis. Our results could be explained by the proposed mechanism in the literature, which is that breast cancer cells have a potential inhibitory effect on the translation of human chorionic somatotropin hormone (CSH) mRNA into hPL protein. Our results support the literature findings of a poorer prognostic outcome for breast malignancies when hPL is expressed but require further studies using a more comprehensive range of clinical parameters.

Acquisition of Genetic Aberrations During the Progression of High-Grade Intraepithelial Lesions/Micro-Invasive Squamous Cancers to Widely Invasive Cervical Squamous Cell Cancer.

Acquisition of genetic aberrations during cervical carcinogenesis in individual patients is poorly documented. To provide a comparative analysis of high-grade squamous intraepithelial lesions (n = 7) and pT1a squamous cancers (n = 1) and their recurrences, subsequent widely invasive cancers, and metastases developed during 1-24 years. Archival tissues of 8 patients were analyzed immunohistochemically for reserve-cell origin, human papillomavirus genotypes, mutations in 50 cancer genes, and chromosomal copy number variations. Intraepithelial lesions arose either from cytokeratin 17- or 7-expressing reserve cells. All preinvasive and invasive tumors carried human papillomavirus high-risk genotypes and lacked somatic mutations. Chromosomal copy number variations were identified in all intraepithelial lesions and invasive cancers. Four of 8 high-grade intraepithelial lesions progressed to invasive cancer after incomplete treatment, and 4 of 8 invasive cancers arose de novo after in sano resection. Four of 8 cancers carried mutations with high mutational frequency (PIK3CA E545K [n = 2]; PIK3CA and SMAD1 [n = 1]; HRAS, RB1, and EGFR [n = 1]), as did their corresponding regional metastases. One nonmetastasized cancer had a subclonal PIK3CA mutation, and an initially nonmutated, low-stage cancer developed ovarian metastases with PIK3CA amplification. One patient had neither mutations nor metastases. The patient with treated PIK3CA E545K-mutated pT1a cancer developed a subsequent nonmutated intraepithelial lesion that progressed to invasive cancer with a subclonal PIK3CA-H1047R mutation. Cancer-related deaths in 4 of 8 (50%) patients occurred independent of mutational status or metastatic disease. Recurrences arose after persistent or de novo human papillomavirus infection of residual reserve cells or squamous metaplasia. Activating driver mutations were identified in invasive cancers only. High mutational load correlated with metastases, which in turn represented clonal disease.

Association between artificial intelligence (AI) -assisted tumor AREG and EREG immunohistochemistry (IHC) and outcomes from anti-EGFR therapy during the routine management of metastatic colorectal cancer (mCRC): An observational cohort study.

203 Background: AREG and EREG are ligands of EGFR. AI assisted IHC evaluation of tumor AREG/EREG expression predicted benefit from anti-EGFR therapy in a retrospective analysis of the PICCOLO trial of second-line irinotecan ± panitumumab. Here, we sought to validate those findings through an analysis of patients who received anti-EGFR therapy during routine care for mCRC. Methods: Patients (pts) with available archival FFPE tumor tissue who received panitumumab or cetuximab ± chemotherapy at any time for treatment of mCRC at 8 UK cancer centers were eligible. Central RAS testing by next generation sequencing (NGS) was performed for pts where extended RAS testing had not been previously undertaken. RAS-mutant (mut) and RAS-unknown pts were excluded from the primary analysis population. AREG and EREG positive tumor cells were identified by IHC, performed locally at 6 of 8 sites. Pathologists annotated tumor areas on digital images of glass slides. AI algorithms calculated the percentage of tumor cells staining positive for AREG and EREG within tumor areas. More than 20% AREG and/or EREG tumor cell positivity was regarded as high biomarker expression, the optimal cut-point identified in PICCOLO. Study endpoints were progression-free survival (PFS), overall survival (OS), and locally reported response rate (RR) and disease control rate (DCR). Results: Of 541 pts recruited, 494 (91.3%) had adequate archival tissue for analysis. Central RAS testing was successfully performed in 255 of 393 (64.9%) pts without existing extended RAS results, leading to 45 exclusions, leaving 449 pts in the primary analysis population. 26 (5.8%) pts were BRAF-mut. 110 (24.5%) received concomitant FOLFOX and 304 (67.7%) FOLFIRI. After adjustment for additional prognostic factors, high AREG/EREG expression (n = 360; 80.2%) was associated with significantly prolonged PFS (HR 0.73; 95% CI, 0.56-0.95; p = 0.02), OS (HR 0.66 [0.50-0.86]; p = 0.002), and DCR (OR 1.92 [1.05-3.54]; p = 0.04), but not RR (unadjusted OR 1.39 [0.83-2.33]; p = 0.21). Median PFS in the high vs low biomarker groups was 8.5 vs 4.4 months; median OS 16.4 vs 8.9 months. The significant difference in OS (high vs low) was maintained in the subgroup with right-sided primary tumor location (n = 107; 23.8%) (HR 0.56 [0.37-0.86]; p = 0.007). Conclusions: High tumor AREG/EREG expression was associated with significantly prolonged PFS, OS and DCR among a cohort of pts treated with anti-EGFR therapy during routine care of mCRC. The prognostic effect observed validates the predictive effect of AREG/EREG seen in the PICCOLO trial, where AREG/EREG had no prognostic effect in patients receiving chemotherapy alone. A prospective biomarker-led trial is in set-up to support the use of AREG/EREG IHC in clinical practice.

Time trends in prevalence of p16 positivity and combined HPV/p16 positivity in a large cohort of Danish vulvar cancer patients.

Vulvar cancer is rare, but causes substantial morbidity in affected patients. A subset of vulvar cancers is caused by high-risk human papillomavirus (hrHPV), which primarily exerts its oncogenic effect through upregulation of tumor suppressor protein p16. Tumors positive for both hrHPV and p16 (double positive) are assumed to be HPV-driven, but only few large studies have investigated the combined prevalence of hrHPV and p16 positivity in vulvar cancer over time. In this Danish cross-sectional study, we assessed the prevalence of p16 positivity and double positivity for hrHPV and p16 in a large sample of vulvar squamous cell carcinomas (VSCCs) diagnosed during 1990 to 2017. In a nationwide register, we identified VSCCs from 13 hospitals across Denmark, and collected archival tumor tissue for hrHPV testing with INNO-LiPA and immunohistochemical p16 staining. We calculated the prevalence of hrHPV, p16 positivity and double positivity according to time, age and histological subtype and evaluated time trends through estimated annual percentage changes. We included 1278 VSCCs. Overall, 35.0% (95% confidence interval [CI]: 32.4-37.6) were positive for p16 and 31.0% (95% CI: 28.4-33.5) were positive for both hrHPV and p16. The prevalence of p16 positivity and double positivity increased over time, both in women aged ≤59 and ≥60 years. The double positive prevalence was higher in nonkeratinizing (60.7%) and warty/basaloid VSCCs (67.5%) than in keratinizing (16.1%) and verrucous VSCCs (5.0%). These results indicate that approximately one-third of vulvar cancers were caused by hrHPV infection, supporting a substantial preventive potential of the HPV vaccine.

The Gastric Cancer Registry Genome Explorer: A tool for genomic discovery.

434 Background: The Gastric Cancer Registry (GCR) collects clinical questionnaire data and biospecimens from gastric cancer (GC) patients and individuals at high risk for GC (through family history of GC or a germline CDH1 mutation). Its purpose is to facilitate research into better detection and treatment strategies for GC. In 2020, the GCR Genome Explorer (GCR-GE), a publicly accessible and interactive database of clinical and genomic data, was launched to meet this goal. Methods: We generated genomic datasets from participants when GC status and archival gastric tumor tissue were available. We processed the tumor tissue (and paired normal tissue when possible) for whole genome, whole exome, and bulk RNA sequencing. The following genomic features were identified: copy number variations, gene mutations, gene expression, microbiome composition, estimation of tumor-infiltrating immune cells, and tumor neoantigens. We populated (1) all genomic alterations identified from GCR sequencing data, and (2) demographic and pathologic data regarding the tumor that were compiled from participant questionnaires and medical records into the GCR-GE. In addition, sequencing files from the Cancer Genome Atlas (TCGA) were similarly analyzed and uploaded as external datasets. Features of the GCR-GE include cross-study comparisons, study summaries, and queries down to the individual gene, neoantigen, and patient level. Results: In total, 243 GC patients donated tumor samples. The new GCR-GE release contains genomic datasets for 214 of these tumors, as well as datasets for 443 gastric tumors and 185 esophageal tumors from TCGA. Data generation was possible thanks to 756 subjects who enrolled in the GCR from 2011-2022. Most subjects were diagnosed with GC only (N=487), but interestingly, some met multiple criteria. For instance, 10 GC patients had a family history; 10 had a germline CDH1 mutation; and 21 patients had GC, a family history, and a germline CDH1 mutation. Efforts to upload additional datasets are ongoing. All data in the GCR-GE is downloadable. Conclusions: The GCR-GE is a comprehensive resource of genomic and clinical data. Given its accessibility, ease of use, and large cohort sizes, the GCR-GE presents a highly valuable tool for accelerating GC research.

Association of punctate TAK1 expression with mortality in patients with microsatellite-stable colorectal cancer.

220 Background: Microsatellite stable (MSS) colorectal cancer continues to have limited options for personalised therapeutic targets. The NFKB pathway is known to play an important role in inflammation-related carcinogenesis but has yet to be translated into therapies for the clinical patient. The aim of this study was to investigate the expression of cytoplasmic and punctate TAK1 (transforming growth factor β-activated protein kinase 1) in colorectal cancer and its relationship to immune checkpoint expression and prognosis. Methods: Patients undergoing primary colorectal cancer resection between 1997 and 2007 at Glasgow Royal Infirmary (UK) were studied for clinicopathological data and immunohistochemistry (IHC) performed on archival FFPE tissue from resected specimens. Antibodies for TAK1, PD-1, PD-L1, IKK alpha and other proteins were used for IHC, with digital analysis (QuPath) for quantification of cytoplasmic staining and punctate score for juxta-nuclear TAK1 assessment. Kaplan-Meier curves were created with log-rank test to determine survival. Cox-proportional hazards regression were used to determine multivariate hazard ratios (HR) and 95% confidence intervals (CI). Results: A total of 898 patients who underwent colorectal resection were identified. Higher TAK1 punctate expression was observed in left colon and rectal cancer, compared with right sided disease (p = 0.045). MMR proficient tumours had higher frequencies of high TAK1 punctate expression (p &lt; 0.001). Both cytoplasmic and punctate TAK1 expression correlated with IKK expression (p &lt; 0.050). High cytoplasmic TAK1 expression was associated with increased PD-1 and PD-L1 expression (p &lt; 0.001). Punctate TAK1 expression was associated with worse survival (p = 0.037). These differences were accentuated in patients with MSS status (p = 0.016). On multivariate analysis, high punctate TAK1 expression remained a predictor of worse cancer-specific survival (HR 1.843, CI 1.129-2.956, p = 0.011). Conclusions: TAK1 expression was associated with MSI status, and higher TAK1 expression correlates with upregulated PD-1 and PD-L1 expression. High punctate TAK1 expression predicted cancer-specific survival. In subgroup analysis of MSS patients, high punctate TAK1 expression was associated with poor survival. Further interrogation into this pathway may identify inflammation-related therapeutic targets in MSS patients with colorectal cancer.

Analysis of the role of tumor microenvironment on clinical outcomes of chemotherapy in patients with advanced gastric cancer using high plex digital spatial profiling.

456 Background: Tumor consists of not only cancer cells but also various immune cells or stromal cells. And tumor microenvironment (TME) is known to be associated with response and resistance to treatment. However, the role of TME in the response to chemotherapy in advanced gastric cancer (AGC) remains unclear. We evaluated whether TME can affect the survival in patients with AGC who received first-line chemotherapy using high plex digital spatial profiling (DSP). Methods: Patients with AGC who received first-line chemotherapy were registered. Archival tumor tissue before first-line chemotherapy were obtained and tissue microarrays (TMAs) were made. Regions of interest (ROIs) were selected on each TMA and high plex DSP including RNA transcript was performed in all ROIs using GeoMx DSP (Whole Transcriptome Atlas) after masking by cytokeratin (CK) immunofluorescence. Differentially expressed gene (DEG), geneset enrichment analysis (GSEA), gene ontology (GO) and cell subsets were analyzed separately in CK-positive and CK-negative areas, using EdgeR, mSigDb, DAVID and CIBERSORT. Results: Thirty-three tumor tissues from 33 patients and 5 normal tissues were analyzed. All patients received fluoropyrimidine and platinum doublet chemotherapy. HER2+ was 15.2% and all with HER2+ AGC received trastuzumab plus chemotherapy. Five received nivolumab plus chemotherapy. Response rate was 48.5% and progression-free survival (PFS) was 9.8 months (95% CI, 6.85-NA). In CK-positive area, high expression of PIGR (polymeric Immunoglobulin receptor gene) (FDR q=0.013) and low expression of IFNE (FDR q=0.027) were associated with long PFS. Response to interferon beta (FDR q=0.044), cytokine-cytokine receptor interaction (FDR q=0.014) and antigen processing and presentation pathway (FDR q=0.015) were less activated in patients with long PFS in GO and GSEA. Although the density of CD8+ T cell did not affect PFS (6.9 vs. 10.8, p=0.25), high density of CX3CR1+memory CD8+T cells (Tmem) showed longer PFS (18.3 vs. 6.6 months, p=0.01), compared with low density of CX3CR1+CD8+Tmem. In CK-negative area, low density of CD8+ T cells were associated with longer PFS (6.3 vs. 18.3 months, p= 0.008). Conclusions: TME particularly in CK-positive area might affect survival for chemotherapy in AGC. PIGR and CX3CR1+CD8+Tmem were suggested as good candidate markers for predicting clinical outcomes of chemotherapy, indicating further analysis of TME will be needed especially in patients with AGC who received immune checkpoint inhibitors.

HER2 testing in colorectal cancer: Concordance analysis between breast and gastric scoring algorithms from the MOUNTAINEER trial.

198 Background: HER2 overexpression/amplification (HER2+) occurs in 3%-5% of patients (pts) w/ metastatic colorectal cancer (mCRC). Rates of HER2+ can increase to ~10% in pts w/ RAS/BRAF wild-type mCRC tumors. The MOUNTAINEER trial (NCT03043313) evaluated the efficacy and safety of the investigational combination of tucatinib with trastuzumab in pts with HER2+ and RAS wild-type mCRC. Established regional guidelines for mCRC recommend HER2 testing and HER2-directed treatment options; however, there is currently no established best practice for HER2 testing and interpretation in mCRC. Here, we present data from a concordance analysis comparing breast and gastric HER2 testing algorithms in the mCRC setting. Methods: The MOUNTAINEER trial enrolled pts w/ HER2+ mCRC identified using ≥1 method: tissue-based local immunohistochemistry (IHC), in situ hybridization (ISH), and/or next-generation sequencing (NGS) testing. Archival or fresh tumor tissue was submitted to a sponsor-designated central laboratory for confirmatory HER2 testing w/ IHC/FISH per the package insert of the FDA approved assay and scored by both the breast and gastric algorithms for HER2 IHC. A positive result per the breast scoring criteria for IHC requires circumferential membrane staining for HER2, while the gastric criteria allows for circumferential, basolateral, or lateral staining patterns. Results: A total of 114 pts were enrolled with HER2+ tumors per ≥1 local testing methods; 69 pts were HER2+ by NGS, 46 by IHC 3+, and 36 by ISH. Of 105 pts who had tissue available for central HER2 testing w/IHC/FISH, 98 had valid HER2 results; 82/98 (83.7%) of pts had tumors centrally confirmed as HER2+ using both the breast and gastric algorithms. Tissue samples from pts in the MOUNTAINEER trial had 100% concordance between breast and gastric algorithms in HER2 status and 99% concordance in HER2 IHC score. Conclusions: Central pathology testing using both the breast and gastric criteria showed high concordance between these two commonly used algorithms. A high central confirmation rate of local HER2+ results was also observed. These data support the use of either the breast or gastric algorithms to identify HER2+ mCRC tumors until an FDA-approved HER2 assay is available for mCRC. Clinical trial information: NCT03043313 . [Table: see text]

NRG GI008: Colon adjuvant chemotherapy based on evaluation of residual disease (CIRCULATE-US).

TPS260 Background: Currently, there are no biomarkers validated prospectively in randomized studies for resected colon cancer (CC) to determine need for adjuvant chemotherapy (AC). However, circulating tumor DNA (ctDNA) represents a highly specific and sensitive approach (especially with serial monitoring) for identifying minimal/molecular residual disease (MRD) post-surgery in CC patients (pts), and may outperform traditional clinical and pathological features in prognosticating risk for recurrence. CC pts who do not have detectable ctDNA (ctDNA-) are at a much lower risk of recurrence and may be spared the toxicities associated with AC. Furthermore, for CC pts with detectable ctDNA (ctDNA+) who are at a very high risk of recurrence, the optimal AC regimen has not been established. We hypothesize that for pts whose CC has been resected, ctDNA status may be used to risk-stratify for making decisions about AC. Methods: In this prospective phase II/III trial, up to 1,912 pts with resected stage III A, B (all pts) and stage II, IIIC (ctDNA+ only) CC will be enrolled. Based on the post-operative ctDNA status using personalized and tumor-informed assay (Signatera™, bespoke assay), those who are ctDNA- (Cohort A) will be randomized to immediate AC with fluoropyrimidine (FP) + oxaliplatin (Ox) for 3-6 mos per established guidelines vs . serial ctDNA monitoring. Patients who are ctDNA+ post-operatively or with serial monitoring (Cohort B) will be randomized to FP+Ox vs . more intensive AC with addition of irinotecan (I) for 6 mos. The primary endpoints for Cohort A are time to ctDNA+ status (phase II) and disease-free survival (DFS) (phase III) in the immediate vs . delayed AC arms. The primary endpoint for Cohort B is DFS in the FP+Ox vs FP+Ox+I arms for both phase II and phase III portions of the trial. Secondary endpoints include prevalence of detectable ctDNA post-operatively, time-to-event outcomes (overall survival and time to recurrence) by ctDNA status, and the assessment of compliance to adjuvant therapy. Biospecimens including archival tumor tissue, as well as post-operative plus serial matched/normal blood samples, will be collected for exploratory correlative research. Active enrollment across the NCTN started in June, 2022. Support: U10-CA-180868, -180822; UG1CA-189867; Natera, Inc. Clinical trial information: NCT05174169 .

The Application of Guanidinium to Improve Biomolecule Quality in Fixed, Paraffin-embedded Tissue.

Neutral buffered formalin (NBF) is the most common fixative in clinical applications. However, NBF damages proteins and nucleic acids, limiting the quality of proteomic and nucleic acid-based assays. Prior studies have demonstrated that BE70, a fixative of buffered 70% ethanol, has many benefits over NBF but the degradation of proteins and nucleic acids in archival paraffin blocks remain a challenge. Thus, we evaluated the addition of guanidinium salts to BE70 with the hypothesis that this may protect RNA and protein. Guanidinium salt supplemented BE70 (BE70G)-fixed tissue is comparable with that of BE70 via histology and immunohistochemistry. Western blot analysis also revealed that HSP70, AKT, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression signals in BE70G-fixed tissue were higher than those in BE70-fixed tissue. The quality of nucleic acids extracted from BE70G-fixed, paraffin-embedded tissue was also superior, and BE70G provides improved protein and RNA quality at shorter fixation times than its predecessors. The degradation of proteins, AKT and GAPDH, in archival tissue blocks is also decreased with the addition of guanidinium salt to BE70. In conclusion, BE70G fixative improves the quality of molecular analysis with more rapid fixation of tissue and enhanced long-term storage of paraffin blocks at room temperature for evaluation of protein epitopes.

Labyrinthin Expression Is Associated with Poor Prognosis in Patients with Non-Small-Cell Lung Cancer.

To determine Labyrinthin (LAB) expression in non-small-cell lung cancer (NSCLC), we immunostained and scored for LAB immunohistochemistry (IHC) expression on sections of tissue microarrays (TMAs) prepared from 256 archival tissue blocks of NSCLC. Propensity-score-weighted Kaplan-Meier curves and weighted Cox models were used to associate LAB expression with overall survival. LAB mRNA expression was assessed in The Cancer Genome Atlas (TCGA) and correlated with clinical phenotype and outcome. Positive LAB IHC expression (>5% of tumor cells) was detected in 208/256 (81.3%) of NSCLC samples, and found in both lung adenocarcinomas (LUAD) and lung squamous cell cancer (LUSC). LAB positivity was associated with poor overall survival (HR = 3.56, 95% CI: 2.3-5.4; p < 0.0001) and high tumor differentiation grade or metastasis compared with negative LAB expression. Univariant and multivariate survival analyses demonstrated LAB expression as an independent prognostic factor for NSCLC patients. LAB RNA expression in TCGA-LUAD was higher in primary and advanced-stage tumors than in normal tissue, and was associated with poorer overall survival. No significant differences or associations were found with LAB RNA expression in TCGA-LUSC. The LAB IHC assay is being used to identify candidate cancer patients for the first-in-human phase I trial evaluating the LAB vaccines (UCDCC#296, NCT051013560).

Using EGFR amplification to stratify recurrent glioblastoma treated with immune checkpoint inhibitors.

While immune checkpoint inhibitors (ICI) have had success with various malignancies, their efficacy in brain cancer is still unclear. Retrospective and prospective studies using PD-1 inhibitors for recurrent glioblastoma (GBM) have not established survival benefit. This study evaluated if ICI may be effective for select patients with recurrent GBM. This was a single-center retrospective study of adult patients diagnosed with first recurrence GBM and received pembrolizumab or nivolumab with or without concurrent bevacizumab. Archival tissue was used for immunohistochemistry (IHC) and targeted DNA next-generation sequencing (NGS) analysis. Median overall survival (mOS) from initial diagnosis was 24.5months (range 10-42). mOS from onset of ICI was 10months (range 1-31) with 75% surviving > 6months and 46% > 12months. Additional IHC analysis on tumors from eight patients demonstrated a trend of longer survival after ICI for those with elevated PD-L1 expression. NGS of samples from 15 patients identified EGFR amplification at initial diagnosis and at any time point to be associated with worse survival after ICI (HR 12.2, 95% CI 1.37-108, p = 0.025 and HR 3.92, 95% CI 1.03-14.9, p = 0.045, respectively). This significance was corroborated with previously tested EGFR amplification via in situ hybridization. ICI did not extend overall survival for recurrent GBM. However, molecular sequencing identified EGFR amplification as associated with worse survival. Prospective studies can validate if EGFR amplification is a biomarker of ICI resistance and determine if its use can stratify responders from non-responders.

Investigation of a herpesvirus outbreak in mixed breeds of adult domestic ducks using next generation sequencing.

This report characterizes the first lethal outbreak of Marek's disease on a large farm of mixed-breed adult ducks (>18,000) and identifies the pathogen that resulted in high mortality (35%). Clinical signs included inappetence, respiratory distress, depression, muscle weakness, and ataxia. Post mortem revealed enlarged fragile liver mottled with miliary whitish spots and an enlarged spleen. Histopathology revealed hepatocellular necrosis with eosinophilic intra-nuclear inclusion bodies, necrosis of splenic follicles and degeneration/necrosis of renal tubules. The disease was tentatively diagnosed as a herpesvirus infection, confirmed by virus isolation from the liver. DNA was isolated from 15-year-old archival formalin-fixed tissues from infected ducks and subjected to next generation sequencing (NGS). Despite highly degraded DNA, short stretches of G- and C-rich repeats (TTAGGG and TAACCC) were identified as telomeric repeats frequently found in herpesviruses. Megablast and further investigative bioinformatics identified presence of Marek's disease virus (MDV), a Gallid alphaherpesvirus type 2 (GAHV-2), as the cause of the acute fatal infection. The source of infection may be attributed to a dead migratory flamingo found close to the duck enclosures three days prior to the outbreak; hence, GAHV-2 may also be responsible for the fatal infection of the flamingo accentuated by heat stress. Considering the possible spread of this highly contagious and lethal virus from a flamingo to the ducks, and the increasing zoonosis of animal viruses into humans, such as monkey B alphaherpesvirus transmission from macaques to humans with ~80% fatality, this observation has important ramifications for human health and safety of the poultry industry.

Targeted surveillance detected novel beaked whale circovirus in ten new host cetacean species across the Pacific basin

The first cetacean circovirus, beaked whale circovirus (BWCV), was recently reported in a Longman’s beaked whale (Indopacetus pacificus) stranded in Hawai‘i and represents an emergent disease with unknown population impacts. In other species, circovirus infection may cause mortality or opportunistic co-infection by other pathogens. We report on a targeted surveillance of stranded cetaceans in the Pacific basin, including archived beaked whale species, strandings where pathological findings suggested disease presence, mass stranded animals, and additional individuals to represent a broad range of Hawaiian cetacean species. Archived tissues primarily from the brain, kidney, liver, lung, spleen, and lymph nodes of individuals stranded between 2000 and 2020 (n=30) were tested by PCR for the presence of BWCV. Suspect positive tissue amplicons were confirmed as BWCV through sequencing. Of the screened individuals, 15 animals tested positive in one or more tissues, with a single striped dolphin (Stenella coeruleoalba) testing positive in all six tissues. The highest rate of detection among positive cases was found in the brain (69%), followed by lymph (67%) and lung tissues (64%). Additionally, co-infections of cetacean morbillivirus (n=3), Brucella ceti (n=1), and Toxoplasma gondii (n=1) were found among the positive cases. These results expand the potential host range for BWCV into ten additional odontocete species. New host species include a dwarf sperm whale (Kogia sima) that stranded on O‘ahu in 2000, predating the initial case of BWCV. The results broaden the known geographic range of BWCV to Saipan in the Western Pacific, and American Samoa in the South Pacific, where stranded Cuvier’s beaked whales (Ziphius cavirostris) tested positive. Although the clinical significance is currently unknown, this study demonstrates that BWCV has a high prevalence within targeted cetacean screening efforts. Infectious diseases pose a major threat to cetaceans and BWCV may represent an important emerging disease within populations spanning the central, Western, and South Pacific.