Archival Tissue Research Articles

The immune microenvironment in hormone positive, HER2 negative breast cancer: A focus on tumour infiltrating lymphocytes, spatial phenotypes and tertiary lymphoid structures.

2522 Background: Hormone positive, HER 2 negative breast cancer (ER+/HER2-) has traditionally been considered an immune cold tumour subtype. Research to date indicates that TILs in ER+/HER2- breast cancer may be a negative prognostic marker. However, there is a paucity of data in this area regarding immune subpopulations and spatial phenotypes. Tertiary lymphoid structures (TLS) have been associated with improved prognosis and response to immunotherapy in other solid organ malignancies. We aim to explore the immune microenvironment in early stage ER+/HER2- breast cancer with a focus on tumour infiltrating lymphocytes, spatial phenotypes and tertiary lymphoid structures (TLS). Methods: Full face sections of archival tissue samples from patients with early stage ER+/HER2- breast cancer were stained with CD45 antibody (DAKA clone M0701) via the automated Leica Bond III system. Open source digital pathology analysis software, Qupath, was utilised to identify positive cells and calculate the percentage of positive CD45 staining cells (CD45%) within the tumour area. Spatial analysis was done to divide tumour area into three spatial phenotypes; Immune cold -lacking immune infiltrate;Immune excluded- immune cells restricted to the tumour periphery and immune hot - immune cells infiltrating the tumour core. A tissue micro array (TMA) was created and stained manually with a multiplex chromogenic immunohistochemistry panel for CD20/CD3/DCLamp to identify immune subpopulations associated with tertiary lymphoid structures. Results: A total of 409 patient samples were included in the analysis. 89% (n=367) of samples had a CD45% of 0-10%. CD45% stratification by 21-gene-recurrence score(RS) into Low, Intermediate and High showed a statistically higher CD45% in those with a High RS compared to Low RS (p value= 0.0015). We identified a trend towards worse disease free survival (DFS) in the CD45%high group which did not reach statistical significance. The immune hot spatial phenotype was associated with a worse DFS (p value = 0.034) with no statistically significant difference in DFS between immune cold and immune excluded phenotypes. The majority of samples had minimal immune infiltrate. Staining with a TLS panel revealed that samples with a significant infiltrate could broadly be divided into T cell only infiltrate or those with the presence of TLS, the significance of this is unclear. Conclusions: The majority of ER+/HER2- breast cancer samples had low numbers of TILs, however there is clear heterogeneity within this subgroup. Spatial elements and immune cell subpopulations may further guide the value of TILs in prognosis in this breast cancer subtype.

A comparative analysis of different DNA extraction methods for fish tissues

Nucleic acids extraction is the initial stage of molecular genetic research. The effectiveness of using a certain method of DNA extraction is determined by the amount of extracted DNA, the degree of probe degradation, the time consuming and laboriousness of the procedure. When researching rare, endangered species and populations, special attention is paid to extracting DNA from sources that do not cause destruction or death of individual. The current study was aimed at comparison the effectiveness of the most common methods of genomic DNA isolation from fish tissues for further use in the polymerase chain reaction. Essentially three DNA extraction methods were employed and compared for the quality of isolated DNA ‒ the phenol-chloroform extraction method, the salt extraction method, and the sodium perchlorate extraction method. Samples of fins, liver and caver taken from sterlet individuals fixed in ethanol served as material for research. The study of the effect of DNA probe quality on PCR course was carried out using two types of PCR ‒ site-specific, amplifying the fragment of cytochrome-b gene, and multilocus ‒ ISSR-PCR with a trinucleotide primer (AGC)6T. A comparison of DNA extraction methods confirmed that all of them enable obtaining high-quality DNA from both fresh and archival fish tissue samples for site-specific amplification. The yield of nucleic acids using different fish tissues (fins, liver and caver) is not the same. Therefore, when it is necessary to obtain the maximum amount of DNA, it is advisable to use the salt extraction method. In case of ISSR-PCR, different degrees of DNA purification affected the course of amplification. Additional purification by the method of sorption on Silica allows removing possible PCR inhibitors and obtaining clear spectra of electrophoretic separation of amplification products, regardless of primary DNA quality. Key words: DNA extraction methods, sterlet tissues, site-specific PCR, multilocus PCR.

Tumor-Infiltrating Lymphocytes and PD-L1 Expression in Pleomorphic Lobular Breast Carcinoma.

The prognostic and predictive role of stromal tumor-infiltrating lymphocytes (sTILs) is undetermined in pleomorphic invasive lobular cancer (pILC). The same applies for the expression of PD-1/PD-L1 in this rare breast cancer subtype. Here, we aimed to investigate the expression of sTILs and analyze the PD-L1 expression levels in pILC. Archival tissues from sixty-six patients with pILC were collected. The sTIL density was scored as a percentage of tumor area using the following cut-offs: 0%; <5%; 5-9%; and 10-50%. The PD-L1 expression was analyzed using IHC on formalin-fixed, paraffin-embedded tissue sections using SP142 and 22C3 antibodies. A total of 82% of the sixty-six patients were hormone receptor positive and 8% of cases were triple negative (TN), while 10% showed human epidermal growth factor receptor 2 (HER2) amplification. sTILs (≥1%) were present in 64% of the study population. Using the SP142 antibody, 36% of tumors demonstrated a positive PD-L1 score of ≥1%, and using the 22C3 antibody, 28% had a positive PD-L1 score of ≥1. There was no correlation between sTILs or PD-L1 expression and tumor size, tumor grade, nodal status, expression of estrogen receptor (ER), or amplification of HER2. Our data did not show any difference in survival between the three molecular subtypes of pILC with respect to sTILs and PD-L1 expression. This study shows that pILCs show some degree of sTILs and PD-L1 expression; however, this was not associated with a survival improvement. Additional large trials are needed to understand immune infiltration in lobular cancer, especially in the pleomorphic subtype.

Practical Utility of Liquid Biopsies for Evaluating Genomic Alterations in Castration-Resistant Prostate Cancer.

Traditional tissue-based assessments of genomic alterations in castration-resistant prostate cancer (CRPC) can be challenging. To evaluate the real-world clinical utility of liquid biopsies for the evaluation of genomic alterations in CRPC, we preemptively collected available plasma samples and archival tissue samples from patients that were being treated for clinically confirmed CRPC. The cell-free DNA (cfDNA) and tumor tissue DNA were analyzed using the AlphaLiquid®100-HRR panel. Plasma samples from a total of 87 patients were included in this study. Somatic mutations from cfDNA were detected in 78 (89.7%) patients, regardless of the presence of overt metastasis or concomitant treatment given at the time of plasma sample collection. Twenty-three patients were found to have known deleterious somatic or germline mutations in HRR genes from their cfDNA. Archival tissue samples from 33 (37.9%) patients were available for comparative analysis. Tissue sequencing was able to yield an NGS result in only 51.5% of the tissue samples. The general sensitivity of cfDNA for detecting somatic mutations in tissues was 71.8%, but important somatic/germline mutations in HRR genes were found to have a higher concordance (100%). Liquid biopsies can be a reasonable substitute for tissue biopsies in CRPC patients when evaluating genomic alterations.

Adenomyomas of the Gallbladder: An Analysis of Frequency, Clinicopathologic Associations, and Relationship to Carcinoma of a Malformative Lesion.

The nature and associations of gallbladder (GB) "adenomyoma" (AM) remain controversial. Some studies have attributed up to 26% of GB carcinoma to AMs. To examine the true frequency, clinicopathologic characteristics, and neoplastic changes in GB AM. Cholecystectomy cohorts analyzed were 1953 consecutive cases, prospectively with specific attention to AM; 2347 consecutive archival cases; 203 totally embedded GBs; 207 GBs with carcinoma; and archival search of institutions for all cases diagnosed as AM. Frequency of AM was 9.3% (19 of 203) in totally submitted cases but 3.3% (77 of 2347) in routinely sampled archival tissue. A total of 283 AMs were identified, with a female to male ratio = 1.9 (177:94) and mean size = 1.3 cm (range, 0.3-5.9). Most (96%, 203 of 210) were fundic, with formed nodular trabeculated submucosal thickening, and were difficult to appreciate from the mucosal surface. Four of 257 were multifocal (1.6%), and 3 of 257 (1.2%) were extensive ("adenomyomatosis"). Dilated glands (up to 14 mm), often radially converging to a point in the mucosa, were typical. Muscle was often minimal, confined to the upper segment. Nine of 225 (4%) revealed features of a duplication. No specific associations with inflammation, cholesterolosis, intestinal metaplasia, or thickening of the uninvolved GB wall were identified. Neoplastic change arising in AM was seen in 9.9% (28 of 283). Sixteen of 283 (5.6%) had mural intracholecystic neoplasm; 7 of 283 (2.5%) had flat-type high-grade dysplasia/carcinoma in situ. Thirteen of 283 cases had both AM and invasive carcinoma (4.6%), but in only 5 of 283 (1.8%), carcinoma arose from AM (invasion was confined to AM, and dysplasia was predominantly in AM). AMs have all the features of a malformative developmental lesion, and may not show a significant muscle component (ie, the name "adeno-myoma" is partly a misnomer). While most are innocuous, some pathologies may arise in AMs, including intracholecystic neoplasms, flat-type high-grade dysplasia or carcinoma in situ, and invasive carcinoma (1.8%, 5 of 283). It is recommended that gross examination of GBs include serial slicing of the fundus for AM detection and total submission if one is found.

Immunohistochemical expression of PTEN (phosphatase and tension homolog) in endometrial carcinoma

Background and objective: Different molecular alterations have been described in the pathogenesis of endometrial carcinoma, among themis mutation of the PTEN (phosphatase and tension homolog), a tumor suppressor gene. This study aimed to detect PTEN immunoexpression in endometrial carcinoma and to asses the association between PTEN immunoexpression with the clinicopathological parameters. Methods: This cross-sectional retrospective study was carried out between January 2016-December 2021 in Erbil city. A total of 112 formalin fixed, paraffin embedded, archival tissue blocks of total abdominal hysterectomy samples were collected in Erbilcity. The clinicopathological characteristics of the tumors were revised, and the specimens were analyzed immunohistochemically using monoclonal PTEN antibody clone 6H2.1 (Dako. Results: Seventy- seven cases (68.8%) were labeled as negative for PTEN immunoexpression. While 35 cases (31.2%) were labeled as positive for PTEN immunoexpression. PTEN immunoexpression was significantly associated with the tumor histological types (P = 0.001), while no significant association was found between PTEN immunoexpression and other clinicopathological parameters. Conclusion: Loss of PTEN immunoexpression frequently seen in endometrioid type of endometrial carcinoma than other types of endometrial carcinoma and is significantly associated with the histological types of endometrial carcinoma.

Utility of Banff Human Organ Transplant Gene Panel in Human Kidney Transplant Biopsies.

Microarray transcript analysis of human renal transplantation biopsies has successfully identified the many patterns of graft rejection. To evaluate an alternative, this report tests whether gene expression from the Banff Human Organ Transplant (B-HOT) probe set panel, derived from validated microarrays, can identify the relevant allograft diagnoses directly from archival human renal transplant formalin-fixed paraffin-embedded biopsies. To test this hypothesis, principal components (PCs) of gene expressions were used to identify allograft diagnoses, to classify diagnoses, and to determine whether the PC data were rich enough to identify diagnostic subtypes by clustering, which are all needed if the B-HOT panel can substitute for microarrays. RNA was isolated from routine, archival formalin-fixed paraffin-embedded tissue renal biopsy cores with both rejection and nonrejection diagnoses. The B-HOT panel expression of 770 genes was analyzed by PCs, which were then tested to determine their ability to identify diagnoses. PCs of microarray gene sets identified the Banff categories of renal allograft diagnoses, modeled well the aggregate diagnoses, showing a similar correspondence with the pathologic diagnoses as microarrays. Clustering of the PCs identified diagnostic subtypes including non-chronic antibody-mediated rejection with high endothelial expression. PCs of cell types and pathways identified new mechanistic patterns including differential expression of B and plasma cells. Using PCs of gene expression from the B-Hot panel confirms the utility of the B-HOT panel to identify allograft diagnoses and is similar to microarrays. The B-HOT panel will accelerate and expand transcript analysis and will be useful for longitudinal and outcome studies.

Abstract CT188: A phase Ib study of sapacitabine (sapa) and olaparib (ola) in patients (pts) with BRCA1/2-mutated (BRCA1/2m) metastatic breast cancer (MBC)

Abstract Background: PARP inhibitors (PARPi) are superior to chemotherapy in pts with BRCA1/2m MBC, with median PFS (mPFS) of 7 months with single-agent ola. Sapa is an oral nucleoside analog; the active metabolite, CNDAC, generates single-strand DNA breaks that are converted to double-strand breaks (DSB) during subsequent replication, resulting in cell death. CNDAC-induced DSB repair is dependent on homologous recombination. Sapa combined with ola may augment DNA damage compared to ola alone in BRCA1/2m tumors. Methods: We conducted a phase Ib study of sapa/ola in pts with BRCA1/2m HER2-negative MBC who were not previously PARPi-exposed. Primary objective was to determine the RP2D of sapa with ola. Secondary objectives included safety, tolerability and mPFS. RAD51 staining was performed on archival tissue. Serial blood samples were collected for circulating tumor DNA (ctDNA) analysis using low-pass WGS with targeted sequencing via the DDR Discovery Panel (Resolution Bioscience). Results: 10 women (median age 46) were enrolled before the study was terminated for hematologic toxicity. Germline BRCA1 and BRCA2 mutations were present in 3 and 7 pts, respectively. 4 pts had received prior platinum. All pts initially received standard dose ola (300 mg twice daily) with 100 mg (n = 3), 150 mg (n = 4), or 200 mg (n = 3) sapa once daily on days 1-5 and 8-12 of an every 28-day cycle. The RP2D was not determined. Hematologic AEs were common, including anemia (80%), neutropenia (80%), and thrombocytopenia (40%), with 1 DLT of G4 neutropenia lasting more than 7 days. The ORR was 50% (95% CI: 18.7% - 81.3%) with mPFS of 9.7 months (95% CI: 8.02 - NA). 3 pts had clinical benefit of 15+ months, exceeding expectations with olaparib alone, including 2 with PR, and 2 who remained on trial for 36+ months. 3 patients had confirmed PRs of short duration. Of the remaining patients, one withdrew consent early and the other 3 had non-CR/non-PD or SD of 4, 7 and 9 months. At progression, 2 pts had evidence of BRCA reversion mutation associated with an MMEJ signature; ctDNA also demonstrated MRE11 and PTEN amplifications in 1 of these pts, suggesting multiple resistance mechanisms. 3 pts had no evidence of reversion but had acquired putative non-reversion mechanisms of resistance, including changes suggesting enhanced MRN complex function, FANCD2 and ATR amplifications and PIK3CA mutation. The majority of tumors (6/10) were RAD51-negative. Other tumors with RAD51 foci demonstrated replication stress evidenced by high levels of pKAP1 and pRPA, suggesting benefit associated with sapa. Conclusions: Sapa/ola produces high rates of hematologic toxicity. However, the ORR of 50%, mPFS of 9.7 months, and durability of 3+ years in 2 pts suggest possible combinatorial benefit. Further exploration with different sapa schedules or with substitution of a PARP1-selective inhibitor to decrease hematologic toxicity is warranted. Citation Format: Filipa Lynce, Noah Graham, Bose S. Kochupurakkal, Huy Nguyen, Philip D. Poorvu, Victoria Attaya, Raechel Davis, Kavita Garg, Lee P. Lim, Mark Li, Cloud P. Paweletz, Nabihah Tayob, Geoffrey I. Shapiro, Sara M. Tolaney. A phase Ib study of sapacitabine (sapa) and olaparib (ola) in patients (pts) with BRCA1/2-mutated (BRCA1/2m) metastatic breast cancer (MBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT188.

Abstract CT133: Trial in progress: a multicenter phase 1/1b dose escalation study of WTX-124 as a monotherapy and in combination with pembrolizumab in patients with selected advanced or metastatic solid tumors

Abstract Background: High-dose recombinant human IL-2 (HD IL-2) is an FDA-approved therapy that produces durable complete remissions in a subset of patients with advanced or metastatic cutaneous melanoma and renal cell carcinoma. However, the clinical benefit of HD IL-2 is counterbalanced by life-threatening toxicities such as capillary leak syndrome. WTX-124 is an engineered cytokine prodrug composed of a wild-type IL-2 fused via proprietary cleavable linkers to an inactivation domain and to a half-life extension domain. In the circulation and in normal tissues, WTX-124 is designed to remain inactive and not bind to high- (αβγ) and intermediate-affinity (βγ) IL-2 receptors. In contrast, proteolytic activation of WTX-124 in the tumor microenvironment (TME) is expected to liberate the IL-2 cytokine to stimulate antitumor immune responses. Preclinical data show that WTX-124 is activated by a range of dissociated human tumors in vitro but not by normal human cells or by patient serum. Moreover, WTX-124 demonstrates potent antitumor activity in multiple murine syngeneic tumor models with substantial expansion and activation of tumor-infiltrating lymphocytes. In the MC38 model, WTX-124 has a large therapeutic window of &amp;gt;19-fold compared to ~3-fold for recombinant human IL-2. Methods: This first-in-human, open-label phase 1/1b trial is investigating the safety, tolerability, pharmacokinetics, pharmacodynamics, immunogenicity, and antitumor activity of WTX-124 administered as a monotherapy or in combination with the anti-PD-1 antibody pembrolizumab to patients with advanced or metastatic solid tumors (NCT05479812). All patients must have ≥ 1 measurable lesion per RECIST 1.1 and a tumor type for which immune checkpoint inhibitor (ICI) therapy is indicated. Dose escalation uses a modified toxicity probability interval-2 study design with WTX-124 administered intravenously (IV) every two weeks in 28-day cycles. If the safety of WTX-124 as a monotherapy is established in the first three cohorts, the study will then initiate dose escalation of WTX-124 in combination with pembrolizumab 400mg IV every six weeks. Dose expansion is anticipated to include four arms that will enroll patients with either advanced or metastatic cutaneous melanoma or renal cell carcinoma to receive WTX-124 monotherapy or combination therapy (N=20 patients per arm). Patients with melanoma assigned to the WTX-124/pembrolizumab combination arm may be naïve to all prior systemic therapy for advanced disease, but all other patients in expansion must have previously received a standard of care ICI regimen. Pre- and on-treatment tumor biopsies, archival tumor tissue, and blood samples will be used to investigate multiple exploratory biomarkers. As of January 12, 2023, WTX-124 monotherapy dose escalation is proceeding. Citation Format: Ildefonso Ismael Rodriguez-Rivera, Justin C. Moser, Mateusz Opyrchal, Brendan Curti, Mehmet A. Bilen, Saero Park, Marissa Bruno, Paul Windt, Kulandayan K. Subramanian, Sameer S. Chopra, Randi Isaacs. Trial in progress: a multicenter phase 1/1b dose escalation study of WTX-124 as a monotherapy and in combination with pembrolizumab in patients with selected advanced or metastatic solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT133.

Abstract CT254: Trial in progress: a first-in-human, phase 1, multicenter dose escalation and dose expansion study of WTX-330 in adult patients with advanced or metastatic solid tumors or non-Hodgkin lymphoma

Abstract Background: Interleukin-12 (IL-12) is a potent proinflammatory cytokine with activating effects on both innate and adaptive immune cells. Though recombinant IL-12 (rIL-12) causes tumor regression with antitumor immune memory formation in a range of immunocompetent mouse models, clinical development of rIL-12 was limited by serious toxicities resulting in several patient deaths. WTX-330 is an engineered cytokine prodrug composed of a wild-type IL-12 heterodimer fused via proprietary cleavable linkers to an inactivation domain and to a half-life extension domain. In the circulation and in normal tissues, WTX-330 is designed to remain inactive and not bind to high-affinity IL-12 receptors. In contrast, proteolytic activation of WTX-330 in the tumor microenvironment is expected to liberate the IL-12 cytokine to stimulate antitumor immunity. Preclinical data show that WTX-330 is activated by a range of dissociated human tumors in vitro but not by normal human cells or by patient serum. Moreover, a WTX-330 murine surrogate molecule has potent antitumor activity as a monotherapy in multiple syngeneic mouse models, including in those resistant to anti-PD-1 therapy. In the MC38 model, the WTX-330 surrogate has a large therapeutic window of 49-fold compared to 5-fold for recombinant chimeric IL-12. Methods: This first-in-human, open-label phase 1 trial is investigating the safety, tolerability, pharmacokinetics, pharmacodynamics, immunogenicity, and antitumor activity of WTX-330 administered as a monotherapy to patients with advanced or metastatic solid tumors or non-Hodgkin lymphoma (NCT05678998). All patients must have ≥ 1 measurable lesion per RECIST 1.1 and be refractory to all standard of care therapies for their indication. Dose escalation utilizes a Bayesian logistic regression model with overdose control with WTX-330 administered intravenously every two weeks in 28-day cycles. Dose expansion is anticipated to include two arms (A, B) of 20 patients each. Patients eligible for Arm A will have tumor types for which immune checkpoint inhibitors (ICIs) are indicated but demonstrate either primary or secondary resistance to this therapy. Primary resistance is defined as disease progression or SD &amp;lt; 6 months as the best response after at least 6 weeks of exposure to PD-(L)1 inhibitors. Secondary resistance is defined as disease progression ≥ 6 months after initiation of PD-(L)1 inhibitors in patients who received clinical benefit (i.e., CR, PR, or SD &amp;gt; 6 months). Patients eligible for Arm B will have any solid tumor for which ICI therapy is not indicated, or non-Hodgkin lymphoma. Pre- and on-treatment tumor biopsies, archival tumor tissue, and blood samples will be assayed for a range of exploratory biomarkers. As of January 12, 2023, the trial is opening sites and recruiting patients. Citation Format: Ildefonso Ismael Rodriguez-Rivera, Justin C. Moser, Mateusz Opyrchal, Aparna R. Parikh, Saero Park, Marissa Bruno, Paul Windt, Kulandayan K. Subramanian, Sameer S. Chopra, Randi Isaacs. Trial in progress: a first-in-human, phase 1, multicenter dose escalation and dose expansion study of WTX-330 in adult patients with advanced or metastatic solid tumors or non-Hodgkin lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT254.

Abstract CT082: Next-generation sequencing (NGS) and cytokine assessment from a phase III study of copanlisib in combination with rituximab in patients with indolent non-Hodgkin lymphoma (iNHL) - associations with survival endpoints

Abstract Introduction: The PI3K inhibitor copanlisib (C) plus the anti-CD20 antibody rituximab (R) was superior to R plus placebo (P) in patients (pts) with relapsed iNHL (Matasar et al. Lancet Oncol 2021). We previously reported that C+R improved median progression-free survival (PFS) in the iNHL, follicular lymphoma (FL), and non-FL groups, with a statistically significant improvement in the PTEN-positive population (Shalini et al., AACR 2022). Here we conducted NGS analysis from pts treated with either C+R or P+R for possible impact on PFS outcomes. We also determined if there was an association between plasma cytokine levels at baseline with survival. Methods: Adult pts with relapsed B-cell iNHL were randomly assigned 2:1 to either C+R or P+R; standard dosing on a 28-day cycle applied. Either fresh or archival tumor tissues were collected for central pathology review and biomarker analysis. NGS was conducted using the TSO500 platform for 476 genes. Analyses included EZH2 and BCL2 mutation status in FL subset, and other genes for FL subsets and overall iNHL, as prognostic and predictive markers. The effect of mutations on PFS and overall survival (OS) outcomes was assessed. Cytokines from baseline plasma were assessed using the multiplex MSD platform (71-plex). Association between cytokine levels and PFS and OS were explored. Results: Baseline tumor samples were evaluable by NGS from 200 pts; 127 C+R and 73 P+R. Variant alleles with frequencies &amp;gt;10% were analyzed. One quarter (25.7%) of FL pts were determined to have EZH2 mutations. C+R treatment significantly improved PFS for FL pts with in mutant EZH2 (p=0.0066) compared to P+R. FL pts with mutant BCL2 had a statistically improved PFS compared to WT BCL2 when receiving C+R treatment. Plasma samples were available for 71-plex assessment in 373 pts; 246 C+R and 127 P+R. Pts were assigned as high or low baseline cytokine groups based on the median values. For majority of cytokines, the low cytokine group correlated with better PFS for all pts, FL and Other iNHL pts in the C+R arm. For OS, there were too few events at the time of primary data cutoff to make any associations. However, with longer follow up (2 years from primary completion) there was a significant improvement in OS in iNHL pts with low IL2 values at baseline receiving C+R compared to pts receiving P+R; HR 0.495 (95%CI: 0.270,0.908), p=0.0230. This association was only seen for pts with low (or undetected) levels of IL2 at baseline. Conclusions: Mutation status was associated with improvements in PFS in FL pts with mutant BCL2 or mutant EZH2 treated with C+R. iNHL pts with low plasma levels of IL2 levels treated with C+R showed a significant improvement in OS. Citation Format: Shalini Chaturvedi, Anke Weispfenning, David Bauer, Rong Du, Tine Descamps, Sara Bellinvia, Teresa Lunt, Lidia Mongay Soler, Barrett H. Childs, Pier Luigi Zinzani. Next-generation sequencing (NGS) and cytokine assessment from a phase III study of copanlisib in combination with rituximab in patients with indolent non-Hodgkin lymphoma (iNHL) - associations with survival endpoints [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT082.

Abstract CT278: ERBB2 amplification detected in ctDNA as a surrogate for tumor tissue FISH analysis of HER2 status in a phase 1 study with zanidatamab for the treatment of locally advanced or metastatic HER2 expressing cancers

Abstract Background: HER2-targeted therapies have substantially improved outcomes for patients with HER2-positive breast and gastric cancers. Several other cancers exhibit HER2 expression and/or amplification of its gene (ERBB2), suggesting that HER2-targeted agents may have broader therapeutic utility. Zanidatamab is a humanized, novel bispecific antibody directed against two non-overlapping domains of HER2. The aim of this Phase 1 dose-escalation and expansion study (NCT02892123) was to evaluate the safety and efficacy of zanidatamab across a range of solid tumors. Parallel to drug development, there has been rapid advancements in NGS technologies including the Guardant360 assay that can specifically sequence ctDNA and detect amplifications of the ERBB2 gene, which can lead to overexpression of HER2. FISH, the current gold standard for HER2 amplification detection, is a tissue-based assay that assesses the raw ERBB2 copy number as well as ratio of ERBB2 to a centromeric protein of chromosome 17 where the ERBB2 gene resides. We evaluated concordance of the FISH and Guardant360 assays to detect ERBB2 amplification in plasma samples. Unlike gene copy number in tissue analysis, the observed plasma copy number (pCN) is also a function of the tumor burden and rate of tumor shedding of ctDNA into the bloodstream. Methods: HER2 status was determined from a fresh tumor biopsy or in archival FFPE tissue samples by IHC and FISH according to ASCO-CAP guidelines from the Phase 1 study with zanidatamab in multiple cancer types (cholangiocarcinoma [21], colorectal carcinoma [27], all other [87]). Plasma samples were collected prior to the first cycle of zanidatamab and on-treatment for testing with Guardant360, 74 gene ctDNA NGS-based assays. Results: A concordance of 82% was observed in ERBB2/HER2 amplifications between the Guardant360 and FISH assays. An exploratory adjustment method based on tumor DNA shedding was developed by Guardant using the maximum mutant allele fraction (maxVAF) as a surrogate for tumor content. Majority of patients experienced a decrease in HER2 pCN post treatment, with 9 PD patients having the least and 21 PR patients the largest changes in ctDNA fraction (maxVAF). Conclusion: These results indicate that ERBB2 amplification detected by the Guardant360 assay could be used as a surrogate for FISH analysis in lieu of invasive surgical procedures. Citation Format: Diana Shpektor, Daryanaz Dargahi, Antonios Samiotakis, Sara Wienke, Ali Livernois, Arielle Yablonovitch, Geethika Yalamanchili, Elaina Gartner, Funda Meric-Bernstam. ERBB2 amplification detected in ctDNA as a surrogate for tumor tissue FISH analysis of HER2 status in a phase 1 study with zanidatamab for the treatment of locally advanced or metastatic HER2 expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT278.

Lesional Antibody Synthesis and Complement Deposition Associate With De Novo Antineuronal Antibody Synthesis After Spinal Cord Injury.

Spinal cord injury (SCI) disrupts the fine-balanced interaction between the CNS and immune system and can cause maladaptive aberrant immune responses. The study examines emerging autoantibody synthesis after SCI with binding to conformational spinal cord epitopes and surface peptides located on the intact neuronal membrane. This is a prospective longitudinal cohort study conducted in acute care and inpatient rehabilitation centers in conjunction with a neuropathologic case-control study in archival tissue samples ranging from acute injury (baseline) to several months thereafter (follow-up). In the cohort study, serum autoantibody binding was examined in a blinded manner using tissue-based assays (TBAs) and dorsal root ganglia (DRG) neuronal cultures. Groups with traumatic motor complete SCI vs motor incomplete SCI vs isolated vertebral fracture without SCI (controls) were compared. In the neuropathologic study, B cell infiltration and antibody synthesis at the spinal lesion site were examined by comparing SCI with neuropathologically unaltered cord tissue. In addition, the CSF in an individual patient was explored. Emerging autoantibody binding in both TBA and DRG assessments was restricted to an SCI patient subpopulation only (16%, 9/55 sera) while being absent in vertebral fracture controls (0%, 0/19 sera). Autoantibody binding to the spinal cord characteristically detected the substantia gelatinosa, a less-myelinated region of high synaptic density involved in sensory-motor integration and pain processing. Autoantibody binding was most frequent after motor complete SCI (grade American Spinal Injury Association impairment scale A/B, 22%, 8/37 sera) and was associated with neuropathic pain medication. In conjunction, the neuropathologic study demonstrated lesional spinal infiltration of B cells (CD20, CD79a) in 27% (6/22) of patients with SCI, the presence of plasma cells (CD138) in 9% (2/22). IgG and IgM antibody syntheses colocalized to areas of activated complement (C9neo) deposition. Longitudinal CSF analysis of an additional single patient demonstrated de novo (IgM) intrathecal antibody synthesis emerging with late reopening of the blood-spinal cord barrier. This study provides immunologic, neurobiological, and neuropathologic proof-of-principle for an antibody-mediated autoimmunity response emerging approximately 3 weeks after SCI in a patient subpopulation with a high demand of neuropathic pain medication. Emerging autoimmunity directed against specific spinal cord and neuronal epitopes suggests the existence of paratraumatic CNS autoimmune syndromes.

Abstract 2173: A multi-institutional prospective biomarker study of durvalumab after concurrent chemoradiation therapy in patients with unresectable stage III non-small cell lung cancer (WJOG11518L/SUBMARINE)

Abstract Background: Programmed cell death-ligand 1 (PD-L1) inhibitor durvalumab has been found to improve disease control in patients with unresectable stage III non-small cell lung cancer (NSCLC) treated with definitive concurrent chemoradiation therapy (dCCRT), but approximately a half of patients eventually experience disease progression within one year after the start of durvalumab. The mechanism of this resistance at such an early timing is poorly identified. Therefore, we conducted a prospective biomarker study to explore the resistance mechanisms to durvalumab after CCRT (Trial Identifier: UMIN000035916). Methods: A total of 139 patients with unresectable stage III NSCLC enrolled into this study after dCCRT were treated with durvalumab for up to 1 year. 135 patients who fulfilled the predetermined selection criteria were included for this primary biomarker analysis. Archival tumor tissues collected before the treatment were analyzed by immunohistochemistry for several key biomarkers such as PD-L1, CD8, and CD73, DNA sequencing for tumor-mutation burden (TMB) and genomic profile, as well as gene expression profiling (GEP). Peripheral blood mononuclear cells (PBMC) were also collected before and after durvalumab for flow-cytometry (FCM) analysis where available. Progression-free survival (PFS) was compared based on these biomarkers. Results: Median follow-up time was 14.4 months. Median PFS was 14.9 months (95% confidence interval [CI], 11.3-not reached), and 1-year PFS rate was 56.3% (95% CI, 47.3-64.3). CD8+ tumor-infiltrating lymphocytes (TILs), PD-L1 tumor proportion score (TPS), TMB, and CD73 on tumor cells were associated with PFS (hazard ratios [HRs], 0.40 [95% CI, 0.22-0.73] for CD8+TILs; 0.67 [95% CI, 0.40-1.11] for PD-L1-TPS ≥1%; 0.59 [95% CI, 0.34-1.02] for high TMB; 1.45 [95% CI, 0.86-2.43] for high CD73). Common pathogenic gene alterations such as EGFR, KRAS, and TP53 were not associated with PFS. Multivariable analysis including key clinical factors as covariables indicated that CD8+TILs and the CD73-expressing tumor cells were independently associated with durvalumab outcome (HRs, 0.26 [95% CI, 0.08-0.89] for CD8+TILs; 5.83 [95% CI, 1.33-25.62] for CD73). The significance of CD73-expressing tumor cells as a resistance factor was more obvious in patients with high CD8+TILs (HR, 2.35 [95% CI, 1.14-4.86]). GEP of tumor tissue and FCM of PBMC suggested that CD73-expressing tumor cells prevented effective reinvigoration of antigen-reactive CD8+ T cells by durvalumab due to the immunosuppressive activity. Conclusions: Low preexisting CD8+TILs or high CD73-expressing tumor cells were suggested to be primary resistance mechanism to durvalumab after CCRT in stage III NSCLC. Further development of next treatment approach will be prompted based on these findings. Citation Format: Koji Haratani, Atsushi Nakamura, Nobuaki Mamesaya, Kenji Sawa, Yasuto Yoneshima, Ryota Saito, Tomohiro Ozaki, Kenjiro Tsuruoka, Akito Hata, Kosuke Tsuruno, Tomohiro Sakamoto, Shunsuke Teraoka, Masahide Oki, Hiroshi Watanabe, Tomoyuki Otani, Kazuko Sakai, Shuta Tomida, Yasutaka Chiba, Akihiko Ito, Kazuto Nishio, Nobuyuki Yamamoto, Kazuhiko Nakagawa, Hidetoshi Hayashi. A multi-institutional prospective biomarker study of durvalumab after concurrent chemoradiation therapy in patients with unresectable stage III non-small cell lung cancer (WJOG11518L/SUBMARINE) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2173.

Abstract 125: Archival single cell sequencing reveals persistent subclones over years to decades of DCIS progression

Abstract Ductal carcinoma in situ (DCIS) is a common precursor of invasive breast cancer (IBC), yet the genomic progression to recurrent disease remains poorly understood. A main contributor to this gap in knowledge arises from technical challenges with genomic profiling of formalin-fixed paraffin-embedded (FFPE) materials. To address this challenge, we developed Arc-well, the first high-throughput method that can perform single cell DNA sequencing of thousands of cells from FFPE materials and frozen tissues. Using Arc-well, we profiled genomic copy number in 27,851 single cells from 26 archival FFPE tissues that were stored for 3-31 years. Analysis of genomic evolution in 10 patients with matched DCIS and recurrent cancers (DCIS or IBC) separated by 2-16 years showed that many primary DCIS lesions had already undergone whole-genome-doubling and had extensive clonal diversity, similar to the paired recurrence. The data from most patients (8/10) suggest an evolutionary bottleneck model of progression, in which a single subclone persisted during the progression to the recurrent disease, revealing copy number aberrations associated with invasion and recurrence. Citation Format: Kaile Wang, Tapsi Kumar, Junke wang, Darlan Minussi, Emi Sei, Jianzhuo li, Tuan Tran, Aatish Thennavan, Min Hu, Anna Casasent, Zhenna Xiao, Shanshan Bai, Yuehui Zhao, Amado Zurita, Ana Aparicio, Brian Chapin, Jie ye, Jianjun Zhang, Don Gibbons, Andrew Futreal, Lorraine King, Jeffrey Marks, E. Shelley Hwang, Vandna Shah, Ellinor Sawyer, Petra Kristel, Jelle Wesseling, Esther H. Lips, Nicholas Navin. Archival single cell sequencing reveals persistent subclones over years to decades of DCIS progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 125.

Abstract 4692: Comparison of interassay similarity and cellular deconvolution in spatial transcriptomics data using Visum CytAssist

Abstract Next-generation sequencing (NGS) of bulk cell populations is a useful and ubiquitous tool for the molecular characterization of clinical tumor samples. Bulk NGS reveals transcript abundance within a tumor sample and can further infer cell populations via deconvolution algorithms (PMID:31570899). However, it can’t ascribe the cellular context for a given gene’s expression or elucidate the spatial organization of tumor microenvironments. These additional features are critical to our understanding of tumor biology and are key to the development of immuno-oncology therapeutics. Spatial Transcriptomics (ST) is an emerging technology that characterizes gene expression within the spatial context of tissue. ST data can be generated directly from archival formalin fixed paraffin embedded samples, enabling the study of spatial gene expression in real-world clinical settings. We have studied a dataset comprising 6 samples from non-small cell lung cancer (NSCLC) patients and 1 core needle biopsy from a tumor of unknown origin. We used the 10X Visium CytAssist platform to generate ST data and additionally generated paired bulk RNAseq data. To test the interassay reliability of CytAssist on archival FFPE tissue sections, we compared ST results across 3 sample preparation conditions. We further studied the state of the tumor microenvironment by applying state-of-the-art computational approaches to deconvolve immune cell populations and produce super-resolution ST maps, validated using multiplex immunofluorescence (IF) via CODEX (PMID:30078711). We find key quality control metrics and spatial biomarkers are consistent across all 3 sample preparation conditions. When comparing deconvolution results between bulk and spatially-resolved methods we observe modest correlations for many cell types despite differences in sample preparation, supporting the idea that bulk and spatial samples contain complementary transcriptomic information. However, within samples, we find many of the correlations observed in bulk do not show strong spatial correlation. These comparisons indicate the importance of considering spatial context when studying the tumor immune microenvironment. Finally, we find an agreement between super-resolution ST and multiplex IF across key spatial biomarkers. These results demonstrate clinical archival FFPE samples yield high interassay reliability via the CytAssist platform. Results were consistent through 3 different H&amp;E staining protocols and findings were consistent when superresolution deconvolution was utilized which further strongly correlated with high-resolution multiplex IF. Our findings demonstrate the feasibility and translational utility of ST to discover spatial signatures and the cellular context in retrospective clinical cohorts to empower discovery and translational efforts in precision oncology and therapeutic development. Citation Format: Mario G. Rosasco, Chi-Sing Ho, Tianyou Luo, Michelle M. Stein, Luca Lonini, Martin C. Stumpe, Jagadish Venkataraman, Sonal Khare, Ameen A. Salahudeen. Comparison of interassay similarity and cellular deconvolution in spatial transcriptomics data using Visum CytAssist. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4692.

Abstract 5559: Exploratory analysis of hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) PIK3CA mutated (mut) advanced breast cancer (aBC) treated with taselisib (TAS) and/or fulvestrant (FUL) reveals increased CCNE2 expression with poor outcome to placebo (PBO) + FUL but not TAS + FUL in SANDPIPER

Abstract PIK3CA mutations occur in ~40% of patients (pts) with HR+/HER2- aBC. TAS is a potent and selective β-sparing PI3K inhibitor that has shown modest clinical benefit when combined with FUL endocrine therapy in patients (pts) with PIK3CAmut aBC. To better understand the molecular features associated with response to TAS and/or FUL, we examined the gene expression profiles of archival tissue from pts enrolled in SANDPIPER (NCT02340221). Most patients did not have prior treatment with a CDK4/6i. RNA sequencing was performed on baseline tissue samples from 456 pts with PIK3CAmut tumors. Exploratory biomarker analyses revealed 28 genes associated with worse outcome. Of these, CCNE2 [HR=1.11 (1.04-1.19), p=0.002], RRM2 [HR=1.14 (1.07-1.22), p&amp;lt;0.0001] and TUB4A [HR=1.15 (1.05-1.26), p=0.004] are involved in the regulation of mitotic cell cycle. CCNE2 encodes CYCLIN E2, a G1 cyclin that binds and activates CDK2. Elevated expression of CCNE2 has been associated with poor outcome in ER-positive breast cancer, and has been identified as part of gene signatures that predict disease progression in tamoxifen-resistant breast cancer or advanced breast cancer. Since CCNE2 expression may be a marker of more proliferative tumors, we assessed whether the inclusion of CCNE2 gene expression would add value to a prognostic model that already includes cell cycle as a factor. Analysis of deviance after sequential addition of variables into Coxph models with stratification factors showed that elevated CCNE2 expression is significant, in addition to cell cycle signature scores, for association with worse PFS (likelihood ratio test p = 0.001). When we grouped patients based on CCNE2 expression split by quartile with the highest values (Q4) grouped as CCNE2 high vs lower values (Q1-Q3), we found that the CCNE2 high group does worse in the PBO + FUL treatment arm. Median PFS for CCNE2-high group in the placebo arm was 3.75 months compared to the other groups (log-rank p &amp;lt; 0.0001; PBO, CCNE2-low 7.3 months, TAS, CCNE2-high 7.6 months; TAS, CCNE2-low 7.3 months). A significant statistical interaction was observed between high CCNE2 gene expression and PBO + FUL treatment arm HR=1.51 (1.03-2.20), p=0.04], suggesting that CCNE2 expression may be associated with poor PFS to in 2L ER+/HER2- PIK3CAmut aBC patients that receive PBO + FUL. Our analysis revealed an association between high CCNE2 expression and worse PFS in patients that received PBO + FUL. Further, this association was not observed in the patients that received the combination of TAS+FUL, which may inform future rational combination strategies for the clinical development of PI3K inhibitors in ER+/HER2- PIK3CAmut aBC. Citation Format: Radia M. Johnson, Bonnie Liu, Javier Cortés, Susan Dent, Nadia Harbeck, Ian E. Krop, William Jacot, Robert L. Yauch, Thomas J. Stout, Frauke Schimmoller, Steven Gendreau, Timothy R. Wilson. Exploratory analysis of hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) PIK3CA mutated (mut) advanced breast cancer (aBC) treated with taselisib (TAS) and/or fulvestrant (FUL) reveals increased CCNE2 expression with poor outcome to placebo (PBO) + FUL but not TAS + FUL in SANDPIPER. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5559.

Abstract 3405: Changes in HER3 expression associated with chemotherapy for pancreatic cancer

Abstract Background: Currently, intense efforts towards the development of targeted therapy, including anti-HER3 strategies for cancer treatment, are being made. Recently, HER3 activation was linked to resistance to targeted therapies (EGFR/HER2/MEK/RAF) and hormonal therapy by upregulating HER3 expression after those treatments in several cancer types. However, HER3 expression profile in pancreatic cancer treatment is still unknown. Here, we evaluated the status of HER3 expression after chemotherapy for pancreatic cancer. Methods: Patients with pancreatic cancer who underwent chemotherapy between January 2010 and June 2020 and from whom post-treatment archival tissue specimens were collected were included in this study. All resectable and locally advanced cases were evaluated using resected specimens, and all metastatic cases were evaluated using biopsy specimens. In patients from whom both pre- and post-chemotherapy specimens were collected, changes in HER3 expression status before and after treatment were immunohistochemically evaluated by using HER3/ErbB3 (D22C5) XP Rabbit mAb (Cell Signaling Technology) as a primary antibody. IHC scoring (0, 1+, 2+, 3+) of membranous staining intensity was performed according to HER2 IHC gastric scoring guideline. Results: Post-chemotherapy HER3 expression was evaluable in 41 patients, comprising 12, 14, 14, and 1 case as HER3 3+, 2+, 1+ and 0, respectively. In the HER3 3+/2+ and 1+/0 groups, tumor localization was 69%/67% in the pancreatic head, 23%/13% in the pancreatic body, and 8%/20% in the pancreatic tail; tumor stage was resectable (post-neoadjuvant status) 46%/20%, locally advanced 42%/47%, and metastatic 12%/33%; the median tumor size was 30 mm/35 mm; CEA elevation was 23%/20% and CA19-9 elevation was 69%/73%; treatment regimens were FOLFIRINOX 23%/40%, gemcitabine + nab-paclitaxel 19%/0%, gemcitabine + S-1 (neoadjuvant setting) 42%/27%, and the other chemotherapy 4%/20%, respectively. HER3 expression was able to be evaluated before and after chemotherapy in 5 cases: HER3 IHC score changed from 3+/2+ to 1+/0 for 1 case, from 1+/0 to 3+/2+ for 1 case and maintained at 3+/2+ for 3 cases. The median overall survival was 19.6 and 20.5 months in the HER3 3+/2+ and HER3 1+/0 groups, respectively (P = 0.965). Conclusion: HER3 expression with 3+/2+ is observed in 63% of pancreatic cancer patients, and HER3-expressing pancreatic cancer is of great interest as a therapeutic target. HER3 expression status may change before and after cytotoxic combination therapy in some cases of pancreatic cancer. Citation Format: Tomoyuki Satake, Chigusa Morizane, Mao Okada, Mariko Nishioka, Nobuyoshi Hiraoka, Satoshi Nara, Tomoya Kakegawa, Maki Kobayashi, Kumiko Koyama, Minoru Esaki, Takuji Okusaka. Changes in HER3 expression associated with chemotherapy for pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3405.

Abstract 3404: Single nuclei RNAseq analysis of recurrent high-grade glioma tumors from patients in the phase 0/1 ‘trigger’ trial of ribociclib plus everolimus

Abstract Background: The RB-CDK4/6 and mTOR signaling pathways are deregulated in high-grade glioma (HGG) and mTOR activation is a potential mechanism of resistance to CDK4/6 inhibition. This study evaluates the tumor pharmacokinetics (PK) and pharmacodynamics (PD) of combined CDK4/6 and mTOR inhibition in recurrent HGG patients. Furthermore, single nuclei RNA sequencing (snRNAseq) provides further insight on the transcriptomic and cell state change in all patients enrolled in the Phase 0 trial. Methods: Eligible patients had recurrent HGG with (1) RB+, (2) CDKN2A/B deletion or CDK4/6 amplification, and (3) PTEN loss or PIK3CA mutations. Six patients received five days of ribociclib (400mg QD) plus everolimus (2.5mg QD) and underwent tumor resection at 2, 8 or 24 hours following the last dose. Six dose-escalation cohorts (n=3 each) reached a dose-level of ribociclib (600mg QD) plus everolimus (70mg QW). Tumor tissue, CSF, and plasma were collected. Total and unbound drug concentrations were determined using validated LC-MS/MS methods. Tumor PD effects were compared to matched archival tissue. A PK ‘trigger’ (i.e., unbound concentration &amp;gt; 5-fold biochemical IC50) and a PD ‘trigger’ (&amp;gt;30% decrease in both pRB and pS6) in Gd-nonenhancing tissue were set to qualify patients for expansion. Single nuclei cell analysis was performed on 21 fresh frozen surgical tumor samples to capture transcriptional and cell state changes. Results: 24 patients with WHO Grade III (n=2) and Grade IV (n=22) gliomas were enrolled. No dose-limiting toxicities were observed. Following presurgical drug, all patients demonstrated marked decrease in Gd-enhancement on preoperative MRI. In Gd-nonenhancing tumor regions, the median unbound concentration of ribociclib was 561 nM whereas no unbound everolimus concentrations were detected. Across all dose-levels, 57% (13/23) and 43% (10/23) of tumors demonstrated RB and S6 phosphorylation decrease, respectively. snRNAseq analysis revealed significant downregulation of mTOR signaling only in endothelial cells at higher dose levels of everolimus. Amongst the tumor cells, the neural progenitor-like cell state was decreased whereas oligodendrocyte progenitor-like cell state was increased compared to standard-of-care cohort, suggesting a possible inhibition of CDK4-driven neural progenitor-like cell state. Conclusion: In adult HGG, ribociclib achieves pharmacologically-relevant concentrations in Gd-nonenhancing tumor. Everolimus exhibits limited penetration into human glioma tissue. Phase 0 surgical tumor tissue is shown to be great resource for identifying pharmacodynamic treatment effect at the single nuclei level. Our study supports further development of ribociclib, but not everolimus, for the treatment of glioma patients. Citation Format: Nader Sanai, An-Chi Tien, Kevin Johnson, Jun Jiang, Yu-Wei Chang, Chelsea Montgomery, Anita DeSantis, Yoko Fujita, Seongho Kim, Jing Li, Roel Verhaak, Shwetal Mehta. Single nuclei RNAseq analysis of recurrent high-grade glioma tumors from patients in the phase 0/1 ‘trigger’ trial of ribociclib plus everolimus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3404.

Abstract 559: Expression of molecular targets for antibody-drug conjugates in patients with NSCLC and RET fusions

Abstract Background RET fusions are found in 1-2% of patients (pts) with advanced non-small cell lung cancer (NSCLC). RET inhibitors are highly effective, but resistance eventually occurs. Antibody-drug conjugates (ADC) are promising treatments in NSCLC, that could be employed at time of resistance. Data on the membranous expression of ADC targets, such as EGFR/HER2/HER3 and the possible impact of novel therapeutic agents in RET+ NSCLC are lacking. Methods We conducted a single-center observational study, collecting tissue specimens from 19 patients (pts) affected by RET+ NSCLC. The expression of EGFR/HER2/HER3 proteins on the cellular membrane was evaluated by immunohistochemistry (IHC). EGFR expression was assessed by using the DAKO EGFR pharmDx kit (Dako, Glostrup, Denmark); HER2 staining (4B5 clone by Roche clone) was evaluated using ASCO/CAP guidelines in scale from 0 to 3+; HER3 staining (D22C5 clone by Cell Signaling) was categorized by intensity as 0, 1+, 2+, and 3+, according to the same criteria used for HER2 evaluation. All IHC analyses were performed at Gustave Roussy. Results Out of 19 pts, 18 were evaluable for EGFR expression, 18 for HER2, 17 for HER3. All analyses were conducted on archival tissue samples from tumor biopsies performed predominantly in treatment-naïve pts (17/19). Pts had adenocarcinoma in 5/19 cases, KIF5B fusion partner in 13/19 cases. Median PD-L1 expression in evaluable pts was 15% (IQR 1- 60.00). EGFR expression was high, moderate, and low in 4 (22%), 2 (11%) and 5 (27%) cases, respectively. HER3 expression was 2+ in 4 (24%) cases, 1+ in 5 (29%) pts and 0 in the other 47%. None had HER2 membranous expression. EGFR expression was more frequently found in pts with uncommon fusion partners (6/11 vs 0/7, p=0.038) and non-adenocarcinoma histology (5/11 vs 0/7, p=0.10). HER3 expression evaluated at 2+ were identified in 3 cases with KIF5B-RET fusion and adenocarcinoma and 1 case with ANKRD26-RET fusion and undifferentiated histology. PD-L1 expression did not show significant difference between pts with positive vs negative EGFR or HER3 staining (p=1). Conclusions EGFR and HER3 had variable expression in RET+ NSCLC, deserving further exploration for novel treatment strategies. HER2 expression was not found in our RET cohort. Citation Format: Arianna Marinello, Maria Rosa Ghigna, Anas Gazzah, Karine Godefroy, Ludovic Lacroix, Damien Vasseur, Jordi Remon-Masip, Fabrice Barlesi, David Planchard, Jean-Yves Scoazec, Benjamin Besse, Mihaela Aldea. Expression of molecular targets for antibody-drug conjugates in patients with NSCLC and RET fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 559.