PD-L1 Amplification Research Articles

Abstract PO1-18-07: Phase I trial of combination pembrolizumab and ruxolitinib in metastatic triple negative breast cancer

Abstract BACKGROUND: The amplification of genes PDL-1, PDL-2, and JAK2 on chromosome 9p24, known as the PDJ amplicon, has been identified in 15% of triple negative breast cancer (TNBC) and is associated with tumor infiltrating lymphocytes and elevated JAK2 mRNA, suggesting a coordinated inflammatory response in TNBC. Adding pembrolizumab to chemotherapy in KEYNOTE trials 355 and 522 has validated the use of PD-1 blockade in TNBC and redefined the standard of care. Despite preclinical data suggesting a role for targeting the IL- 6/JAK2/STAT3 pathway, the JAK1/JAK2 inhibitor ruxolitinib has not shown efficacy as a single Agent in breast cancer. This is the first phase I clinical trial evaluating the combination of pembrolizumab and ruxolitinib in the treatment of metastatic TNBC. METHODS: We conducted a phase I clinical trial with accrual from 2018 to 2023 at Mayo Clinic Arizona. Patients (n=12) received 200 mg of IV pembrolizumab per cycle (every 3 weeks) with 3+3 dose escalation of oral ruxolitinib twice a day (dose level 0: 10 mg PO BID, level 1: 15 mg PO BID, level 2: 20 mg PO BID). The primary endpoint was to determine the maximum tolerated dose (MTD) of ruxolitinib and secondary endpoints were treatment safety and efficacy. Clinical response was assessed by computed tomography of the chest, abdomen, and pelvis every 3 cycles. PD-L1 expression in tumor was measured by immunohistochemistry (IHC). The effect of the combined therapies on T and B cell populations were assessed by flow cytometry and antibody profiling of 695 tumor antigens and 8 viral antigens by nucleic acid protein programmable array. RESULTS: Twelve female patients with chemotherapy refractory metastatic TNBC were treated on study. All received 200 mg IV pembrolizumab every cycle. Due to rapid disease progression of three patients early after treatment initiation, eligibility was restricted to prohibit patients with extensive pulmonary disease or elevated LDH. Those patients were excluded from efficacy or correlative analysis. The most common toxicities were transaminitis (n=12), anemia/leukopenia (n=12), and fatigue (n=9). The majority of adverse events were grade 1 and occurred at dose level 2. There were 5 adverse events grade 3 or higher: respiratory or thoracic complications (n=3), hypertension (n=1), and abdominal pain (n=1), but MTD was not established. The median number of cycles was 4 for each dose level (range n=2.0-9.0) and the median time to progression was 3 months (range n=1.4-6.3). No patient had tumor response by RECIST v1.1 criteria and there was no association of disease stability with PD-L1 tumor expression by IHC. Circulating antibodies to tumor antigens remained stable, and viral-specific antibodies mildly decreased by month 2 in all patients at dose levels 0 and 1. Of the 7 patients reactive to TRIM21 autoantibodies, 2 patients had a transient increase at 2 months. CONCLUSIONS: The combination of pembrolizumab with ruxolitinib was well tolerated at all dose levels, and the number but not the severity of adverse events increased with higher ruxolitinib dose. Two of six patients in dose level 0 and 1 had stable disease for six months on treatment, suggesting that the lower dose levels of ruxolitinib (i.e. 10 mg PO BID) may have more activity in combination with pembrolizumab. Citation Format: Anelia Kassi, Jacqueline Carmona, Sarah M. Green, Heidi Kosiorek, Michael T. Barrett, Barbara A. Pockaj, Donald Northfelt, Karen S. Anderson. Phase I trial of combination pembrolizumab and ruxolitinib in metastatic triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-18-07.

Molecular pathogenesis of adult T-cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive peripheral T-cell malignancy caused by human T-cell leukemia virus type-1 (HTLV-1) infection. Genetic alterations are thought to contribute to the pathogenesis of ATLL alongside HTLV-1 products such as Tax and HBZ. Several large-scale genetic analyses have delineated the entire landscape of somatic alterations in ATLL, which is characterized by frequent alterations in T-cell receptor/NF-κB pathways and immune-related molecules. Notably, up to one-fourth of ATLL patients harbor structural variations disrupting the 3'-UTR of the PD-L1 gene, which facilitate escape of tumor cells from anti-tumor immunity. Among these alterations, PRKCB and IRF4 mutations, PD-L1 amplification, and CDKN2A deletion are associated with poor prognosis in ATLL. More recently, several single-cell transcriptome and immune repertoire analyses have revealed phenotypic features of premalignant cells and tumor heterogeneity as well as virus- and tumor-related changes of the non-malignant hematopoietic pool in ATLL. Here we summarize the current understanding of the molecular pathogenesis of ATLL, focusing on recent progress made by genetic, epigenetic, and single-cell analyses. These findings not only provide a deeper understanding of the molecular pathobiology of ATLL, but also have significant implications for diagnostic and therapeutic strategies.

High Prevalence of MYD88 and CD79B Mutations and PD-L1 Alterations in Bone Marrow Large B Cell Lymphoma Associated with Hemophagocytic Lymphohistiocytosis

Introduction: Bone marrow large B cell lymphoma associated with hemophagocytic lymphohistiocytosis (HLH) is a rare disease with a highly aggressive clinical course. Until now, the molecular genomics of bone marrow large B cell lymphoma associated with HLH remains undetermined. Methods: Cases of bone marrow large B cell lymphoma with HLH were included. The inclusion criteria were as follows: (1) diagnosis of large B cell lymphoma presenting initially in the bone marrow; (2) no overt lymph node involvement or tumor formation; (3) fulfillment of five or more of the eight HLH-2004 diagnostic criteria. (4) cases with available samples. Genomic DNA was extracted from bone marrow cells or the plasma. A panel comprising 475 hematological malignancy-related genes were used for targeted gene sequencing. The prepared library was sequenced using Illumina HiSeq 4000. Molecular classification was predicted by using the LymphGen algorithm. Results: A total of 23 cases were included. The median age was 63. 71.4% of patients were the non-GCB subtype (10/14) based on the Hans algorithm and 50% (9/18) of cases showed CD5 expression. Somatic mutations were identified in all the cases. The most common disrupted genes were PIM1 (12/23, 52.2%), PD-L1 (9/23, 39.1%), MYD88 (8/23, 34.8%), BCL6 (6/23, 26.1%), CD79B (6/23, 26.1%), IRF4 (6/23, 26.1%) and TET2 (6/23, 26.1%) (Figure 1). For nine cases with PD-L1 aberrations, seven cases had PD-L1 structural variants (SVs) and one case showed PD-L1 amplification (Figure 2). All the six cases with BCL6 aberrations showed BCL6 SVs (Figure 2). For six cases with BCL6 SVs, four cases showed immunoglobulin heavy chain ( IGH)- BCL6 translocations and two cases harbored IKZF1- BCL6 translocations. Regarding molecular classification, eight cases (34.8%) were classified as the MCD subtype, two cases the BN2 subtype, two cases the ST2 subtype, and two cases the composite subtype. Conclusion: To the best of our knowledge, this is the first study exploring the molecular genetics of bone marrow large B cell lymphoma with HLH. Our study suggesting bone marrow large B cell lymphoma with HLH showed frequent MYD88/CD79B and PD-L1 alterations. Our study provided clues for the pathogenesis of bone marrow large B cell lymphoma with HLH and identified potential druggable targets for future clinical trials.

The role of concurrent amplification of PD-L1, PD-L2 and JAK2 in metastatic lung adenocarcinoma as a biomarker of immune checkpoint inhibitor response: a case report

Background: Immune checkpoint inhibitors (ICIs) have transformed the landscape of care for many malignancies, including non-small cell lung cancer (NSCLC). That said, ICIs are associated with significant immune related adverse events (IrAE) and most patients do not benefit. As a result, significant efforts have been devoted to identifying biomarkers able to predict response to ICIs.

Diagnostic Accuracy of Slow-Capillary Endobronchial Ultrasound Needle Aspiration in Determining PD-L1 Expression in Non-Small Cell Lung Cancer

The role of EBUS-TBNA in the diagnosis and staging of lung cancer is well established. EBUS-TBNA can be performed using different aspiration techniques. The most common aspiration technique is known as "suction". One alternative to the suction technique is the slow-pull capillary aspiration. To the best of our knowledge, no studies have assessed the diagnostic yield of slow-pull capillary EBUS-TBNA in PD-L1 amplification assessment in NSCLC. Herein, we conducted a single-centre retrospective study to establish the diagnostic yield of slow-pull capillary EBUS-TBNA in terms of PD-L1 in patients with NSCLC and hilar/mediastinal lymphadenopathies subsequent to NSCLC. Patients with hilar and/or mediastinal lymph node (LN) NSCLC metastasis, diagnosed by EBUS-TBNA between January 2021 and April 2022 at Pulmonology Unit of "Ospedali Riuniti di Ancona" (Ancona, Italy) were enrolled. We evaluated patient characteristics, including demographic information, CT scan/ FDG-PET features and final histological diagnoses, including PD-L1 assessment. A total of 174 patients underwent EBUS-TBNA for diagnosis of hilar/mediastinal lymphadenopathies between January 2021 and April 2022 in the Interventional Pulmonology Unit of the "Ospedali Riuniti di Ancona". Slow-pull capillary aspiration was adopted in 60 patients (34.5%), and in 30/60 patients (50.0%) NSCLC was diagnosed. EBUS-TBNA with slow-pull capillary aspiration provided adequate sampling for molecular biology and PD-L1 testing in 96.7% of patients (29/30); in 15/29 (51.7%) samples with more than 1000 viable cells/HPF were identified, whereas in 14/29 (48.3%) samples contained 101-1000 viable cells/HPF. These retrospective study shows that slow-pull capillary aspiration carries an excellent diagnostic accuracy, almost equal to that one reported in literature, supporting its use in EBUS-TBNA for PD-L1 testing in NSCLC.

S3633 Metastatic Gastric Adenocarcinoma in 23-Year-Old Female

Introduction: Gastric cancer is the fourth most common malignancy in the USA. Since there are no screening recommendations in place, most gastric cancers are detected at a later stage in the USA. The average age of occurrence of gastric cancer is >40 years. Some of the most common risk factors include Helicobacter pylori infection, atrophic gastritis, ethnicity, and dietary factors. Case Description/Methods: A 23-year-old female with a past medical history of attention deficit hyperactivity disorder presented with complaints of worsening cough and exertional dyspnea for 2 months. The patient also reported experiencing poor appetite, occasional nausea, vomiting, and unintentional weight loss of 10 pounds in 2 months with low back pain. The patient does not smoke cigarettes and drinks alcohol socially. No significant family history. Physical examination was unremarkable. Labs were normal except for mildly elevated ALP. Chest x-ray showed bilateral infiltrates. CT chest showed a miliary pattern of the lung parenchyma with multifocal patchy infiltrates and mediastinal and hilar lymphadenopathy. CT abdomen and pelvis (Figure) showed a calcified mass at the lesser curvature measuring up to 36.4 mm, with mixed attenuation lesion in the left hepatic lobe along with portal, celiac, and retroperitoneal lymphadenopathy; metastatic lesion within the L2 vertebral body with mild superior endplate compression and soft tissue extension to epidural space along with left portal venous thrombosis. Upper GI endoscopy (Figure) showed a large, infiltrative, ulcerated, noncircumferential mass on the lower curvature of the gastric body. Biopsy of the gastric mass revealed infiltrative adenocarcinoma, poorly differentiated with mucous and signet-ring features. Tumor cells were negative for E-cadherin expression and HER-2 immunohistochemical staining and positive for PD-L1 and MYC amplification. The plan was to perform palliative chemotherapy because of the advanced nature of the disease. Discussion: Significant progress has been made in understanding gastric adenocarcinomas, but the pathways leading to the development of gastric cancer are still unclear. As gastric cancer exhibits heterogeneity and molecular expressions, it makes it more difficult to understand. Improvements in micro-array-based gene expression profiling can help us to better delineate the tumor behavior and help us with treatment.Figure 1.: a. CT abdomen - partially calcified gastric mass; b. EGD - A large infiltrative and ulcerated, noncircumferential mass on the lesser curvature of the gastric body; c. & d. Histopathology - H&E stain (20 x): gastric mucosa infiltrated by carcinoma cells with a signet-ring morphology.

Immune Checkpoint Blockade for the Treatment of Hodgkin Lymphoma.

Classical Hodgkin lymphoma is biologically different than other lymphomas. The cancer cells only occupy a small amount of the lymph node and evade the immune system by amplification of PD-L1 and PD-L2. Therefore, checkpoint inhibitors are a logical treatment option for Hodgkin lymphoma patients to unlock the immune system. Checkpoint inhibitors have shown high response rates in clinical trials in advanced-stage Hodgkin lymphoma. The two most commonly used checkpoint inhibitors are pembrolizumab and nivolumab, both FDA approved as third-line therapy. There is increasing interest in the use of checkpoint inhibitors with combination chemotherapy or with other targeted agents in the second-line or even frontline setting. In this review, we will highlight the clinical trials that led to approvals of checkpoint inhibitors for Hodgkin lymphoma.

CD274 (PD-L1) Copy Number Changes (Gain) & Response to Immune Checkpoint Blockade Therapy in Carcinomas of the Urinary Tract.

Immune checkpoint inhibitors are an important therapeutic option for urothelial carcinoma, but durable responses are achieved in a minority of patients. Identifying pre-treatment biomarkers that may predict response to these therapies or who exhibit intrinsic resistance, is of paramount importance. To explore the prevalence of PD-L1 copy number alteration in urothelial carcinoma and correlate with response to immune checkpoint inhibitors. We analyzed a cohort of 1050 carcinomas of the bladder and upper urinary tract that underwent targeted next generation sequencing, prospectively. We assessed PD-L1 protein expression, copy number status (next generation sequencing/FISH), and detailed treatment response. We identified 9 tumors with PD-L1 amplification and 9 tumors with PD-L1 deletion. PD-L1 protein expression was the highest in PD-L1 amplified tumors. Of the 9 patients whose tumors harbored PD-L1 amplification, 6 received immunotherapy with 4 deriving clinical benefit, and two achieving durable response. Of the 9 patients whose tumors had PD-L1 copy number losses, 4 received immunotherapy with 3 experiencing disease progression. PD-L1 copy number alterations may serve as potential biomarkers of response to immunotherapy in urothelial carcinoma patients, if validated in larger cohorts.

Targeting BRAF Activation as Acquired Resistance Mechanism to EGFR Tyrosine Kinase Inhibitors in EGFR-Mutant Non-Small-Cell Lung Cancer.

Osimertinib has become a standard of care in the first-line treatment of advanced-stage non-small-cell lung cancer (NSCLC) harboring exon 19 and 21 activating mutations in the EGFR gene. Nevertheless, the 18.9-month median progression-free survival emphasizes the fact that resistance to osimertinib therapy is inevitable. Acquired resistance mechanisms to osimertinib in EGFR-driven NSCLC include MET amplification, EGFR C797S mutation, neuroendocrine differentiation, small-cell lung carcinoma histologic transformation, PD-L1 and KRAS amplifications and ESR1-AKAP12 and MKRN1-BRAF translocations, as well as BRAF V600 mutation. This last one represents 3% of the acquired resistance mechanisms to osimertinib. In this review, we discuss the rationale for EGFR/BRAF/MEK co-inhibition in the light of a clinical case of EGFR-mutant NSCLC developing a BRAF V600 mutation as an acquired resistance mechanism to osimertinib and responding to the association of osimertinib plus dabrafenib and trametinib. Additionally, we discuss the acquired resistance mechanisms to osimertinib plus dabrafenib and trametinib combination in that context.

Clinical Impact of Somatic Copy Number Alterations in Circulating Tumor DNA from Diverse Lymphoma Subtypes

Introduction:Somatic copy number alterations (SCNAs) constitute a major class of genomic aberrations known to have functional significance across cancers, including prognostic value in lymphoma. For example, diffuse large B-cell lymphoma (DLBCL) patients with tumors harboring SCNAs in MYC, BCL2, or TP53 tend to have inferior outcomes (Quesada, ASH 2016). However, detection of copy number alterations without invasive tissue biopsies remains difficult. We applied Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq), a targeted high-throughput sequencing platform, to noninvasively detect and monitor genome-wide SCNAs in patients with DLBCL, primary mediastinal B-cell lymphoma (PMBCL), and classical Hodgkin lymphoma (cHL).Methods: We profiled pre-treatment blood samples from 209 patients with diverse lymphomas including 168 DLBCL, 20 cHL, and 21 PMBCL, using a custom 314kb gene panel targeting recurrently altered regions in these cancers. Additionally, we sequenced 139 tumor biopsies to use as matched tissue gold standards for SCNA confirmation. In each sample, we interrogated the sequencing read distribution to summarize focal and broad SCNAs directly from plasma. All thresholds were tuned for 95% specificity, which were empirically verified in independent withheld healthy controls. Results were orthogonally validated through tissue karyotyping, fluorescence in situ hybridization (FISH), and whole genome sequencing (WGS) (Fig 1A and Fig 1B).Results: We determined our limit of detection (LOD) for detecting SCNAs in plasma at ~1% circulating tumor fraction with 77% sensitivity; we observed higher sensitivity above 5% circulating tumor fraction (sensitivity = 95%). In our cohort, 70% of patients had over 1% circulating tumor burden with representation from all clinical stages. In DLBCL patients, we detected gains in MYC (12%), BCL2 (24%), BCL6 (19%), and PD-L1 (23%), as well as losses in TP53 (27%) and CDKN2A (19%). SCNAs detected in plasma were 85% concordant with those detected in matched tumor biopsies. Additionally, PD-L1 copy number gains were more frequently detected in both PMBCL (63%, p= 0.026) and cHL (71%, p= 0.012) than in DLBCL (23%), consistent with prior reports (Chapuy B, Blood 2016; Roemer MG, J. Clin. Oncol. 2016).We also demonstrate that noninvasive detection of copy number gains in MYC and BCL2, as well as loss of TP53, are negatively prognostic for survival (p < 0.0001, p= 0.0013, p < 0.0001, respectively) (Fig 1C). Furthermore, we identified associations between DLBCL cell-of-origin classification and specific SCNAs, with non-GCB DLBCL enriched for BCL2, BCL6, and PD-L1 copy number gains (39% vs 17%; 31% vs 15%, and 39% vs 25%, respectively). Circulating tumor burden was not statistically different between GCB and non-GCB patients. TP53 losses were more frequently observed in relapsed/refractory patients compared to treatment naïve patients (22% vs 45%, p= 0.023).We performed a preliminary analysis monitoring disease over time in three patients with clear SCNAs detected in plasma at diagnosis. Across longitudinal blood samples (n= 42), SCNA-based disease detection demonstrated 95% concordance with mutation-based disease detection, with a LOD down to ~0.1% circulating tumor burden. Our results suggest noninvasive SCNA profiling could provide an independent method for detecting residual disease, particularly in tumors characterized by structural alterations. Further validation and expansion of this monitoring method will be explored and presented.Conclusions: Noninvasive identification of genome-wide SCNAs is feasible via targeted next generation sequencing. Our approach allows for both genotyping and monitoring of copy number gains and losses from plasma, without requiring matched germline or tissue specimens. This strategy not only allows for the noninvasive genotyping of tumor SCNAs, but also provides an orthogonal method for monitoring residual disease from liquid biopsies using next generation sequencing.Figure 1: A) Matched tissue FISH staining confirms arm-level PD-L1 amplification (red) detected in plasma. D9Z1 (green) was used as an assay control. B) SCNAs detected in plasma were concordant with karyotype analysis of a matched tumor biopsy. C) Copy number alterations in MYC, BCL2, and TP53 are adversely prognostic for survival in R-CHOP treated DLBCL patients. [Display omitted] DisclosuresHüttmann:Bristol-Myers Squibb, Takeda, Celgene, Roche: Honoraria; Gilead, Amgen: Other: Travel cost. Gaidano:Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Westin:Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Apotex: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Advani:Bayer Healthcare Pharmaceuticals: Research Funding; Gilead: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Celgene: Research Funding; Janssen: Research Funding; Genentech: Research Funding; Seattle Genetics: Research Funding; Nanostring: Consultancy; Spectrum: Consultancy; Millennium: Research Funding; FortySeven: Research Funding; Pharmacyclics: Research Funding; Regeneron: Research Funding; Agensys: Research Funding; Infinity: Research Funding; Juno Therapeutics: Consultancy; Kura: Research Funding; Merck: Research Funding; Pharmacyclics: Consultancy; Cell Medica: Research Funding; Sutro: Consultancy. Diehn:Varian Medical Systems: Research Funding; Roche: Consultancy; Novartis: Consultancy; Quanticel Pharmaceuticals: Consultancy. Alizadeh:Genentech: Consultancy; Celgene: Consultancy; Gilead: Consultancy; Roche: Consultancy; CiberMed: Consultancy.

Precision Medicine in Oncology: A Review of Multi-Tumor Actionable Molecular Targets with an Emphasis on Non-Small Cell Lung Cancer.

Precision medicine is essential for the modern care of a patient with cancer. Comprehensive molecular profiling of the tumor itself is necessary to determine the presence or absence of certain targetable abnormalities or biomarkers. In particular, lung cancer is a disease for which targetable genomic alterations will soon guide therapy in the majority of cases. In this comprehensive review of solid tumor-based biomarkers, we describe the genomic alterations for which targeted agents have been approved by the United States Food and Drug Administration (FDA). While focusing on alterations leading to approvals in a tumor-agnostic fashion (MSI-h, TMB-h, NTRK) and on those alterations with approvals in multiple malignancies (BRAF, ERBB2, RET, BRCA, PD-L1), we also describe several biomarkers or indications that are likely to lead to an approved drug in the near future (e.g., KRAS G12C, PD-L1 amplification, HER2 overexpression in colon cancer, HER2 mutations in lung cancer). Finally, we detail the current landscape of additional actionable alterations (EGFR, ALK, ROS1, MET) in lung cancer, a biomarker-rich malignancy that has greatly benefitted from the precision oncology revolution.

Clinical Applications of Genomic Alterations in ATLL: Predictive Markers and Therapeutic Targets.

Simple SummaryIn this review paper, we aim to summarize recent findings of genomic alterations found in adult T-cell leukemia/lymphoma (ATLL), which is an incurable disease induced by a virus; human T-cell leukemia virus type 1 (HTLV-1). Genomic alterations of ATLL have been comprehensively analyzed and the identified alterations and HTLV-1 infection synergistically act for ATLL development. As HTLV-1 is an endemic disease, ATLL frequently occurs in the endemic areas. Current clinicogenomic analyses suggest the existence of regional difference in ATLL pathophysiology. From a clinical perspective, several studies identified alterations that act as predictive markers and that a part of the alterations can be targetable in ATLL. The alterations can be leveraged to improve ATLL prognosis.Adult T-cell leukemia/lymphoma (ATLL) is a peripheral T-cell lymphoma (PTCL) caused by human T-cell leukemia virus type 1 (HTLV-1). Recent comprehensive genomic analyses have revealed the genomic landscape. One of the important findings of genomic alterations in ATLL is that almost all alterations are subclonal, suggesting that therapeutic strategies targeting a genomic alteration will result in partial effects. Among the identified alterations, genes involved in T-cell receptor signaling and immune escape mechanisms, such as PLCG1, CARD11, and PD-L1 (also known as CD274), are characteristic of ATLL alterations. From a geographic perspective, ATLL patients in Caribbean islands tend to be younger than those in Japan and the landscape differs between the two areas. Additionally, young Japanese ATLL patients frequently have CD28 fusions, compared with unselected Japanese cases. From a clinical perspective, PD-L1 amplification is an independent prognostic factor among every subtype of ATLL case. Recently, genomic analysis using deep sequencing identified a pre-ATLL clone with ATLL-common mutations in HTLV-1 carriers before development, indicating that genomic analysis can stratify cases based on the risks of development and mortality. In addition to genomic alterations, targetable super-enhancers have been identified in ATLL. These data can be leveraged to improve the prognosis of ATLL.

Abstract PS5-04: Therapeutic considerations in microsatellite instability high (MSI- H) breast cancers (BC) identified by comprehensive genomic profiling (CGP)

Abstract Background: Non-colorectal MSI-H tumors are increasingly identified by CGP. Rare types such as MSI-H BC remain poorly defined with an evidence gap on how to optimally sequence or combine with standard of care treatment. MSI can be measured by either IHC, PCR, or CGP and can be caused by both sporadic and germline variants within different tumor types. Prior studies in BC have shown evidence of dMMR by IHC cases MSS based on PCR. This could be due to intra-tumor heterogeneity, specific microsatellite loci evaluated, or penetrance of germline, somatic, or epigenetic alterations. Published data suggests carriers of germline pathogenic MMR variants have a BC risk equivalent to the normal population and currently germline testing is recommended only for BRCA. Currently in advanced BC, standard tumor biomarker testing includes IHC, PCR, and FISH; however, with increasing use of CGP we demonstrate additional actionable biomarkers as well as potential germline variants in MSI-H BC. Methods: DNA was extracted and hybrid capture CGP was performed on 29,160 BC cases. TMB was determined on 0.8-1.2 Mb of DNA and MSI status on 95-114 loci. Genomic LOH was also evaluated. Comparative analysis was done with 101 MSI-H BC, 841 MSS BC and 4,988 non-breast MSI-H cancers. Histological subtype was obtained from the pathology along with orthogonal testing for ER/PR/HER2 status. Somatic-germline-zygosity (SGZ) status was predicted using a published research use algorithm. Select case reports with clinical outcomes will be presented. Results: We identified 101 (0.35% of total) MSI-H BC cases: 29 ER+/HER2-, 5 HER2+, 29 TNBC, and 28 unknown. Amongst BC cases with known subtype, TNBC was enriched for MSI-H vs MSS (53.4 vs 35.8%, p=0.005). The median TMB in MSI-H BC (26.1 mut/Mb, IQR 17.4;42.8) was significantly lower than that of MSI-H colon (46.1mut/MB) and higher than that of MSI-H uterine tumors (22.6mut/Mb) in our comparison group (p&amp;lt;0.001 for both, Kruskal-Wallis test). Pathogenic variants in an MMR gene were found in 61.4% of MSI-H BC with MLH1 loss being the most common (13.6%) and much higher vs. the non-breast MSI-H cohort (2.4%, p&amp;lt;0.0001). Germline mutations in MMR genes in BC are rare yet 5/52 MMR short variants identified in 101 MSI-H BCs were predicted to be germline, 34 somatic, and 13 could not be determined. We identified 21 MSI-H BC patients with a total of 25 pathogenic BRCA1/2 alterations of which 4 were likely germline, 10 were homozygous, and were enriched in TNBC. These were mainly frameshift mutations, including BRCA2 T3033fs* in 5/18 (28%) cases; however, 7/25 were deletions, rearrangements, or nonsense mutations. Median gLOH was significantly higher in BRCA altered (19.7%) compared to BRCA wild-type MSI-H BC cases (9.6%) (p=0.007, Wilcox test). Additional potentially targetable biomarkers included 26 CDx eligible PIK3CA mutations, 11 ERBB2 activating point mutations in the TKD or ECD domain, 1 FGFR2 rearrangement, and 6 AKT1 E17K mutations. Four cases also had concurrent (CD274) PD-L1 amplifications. Conclusion: MSI-H BC is rare but CGP can identify additional therapeutic options for rational combination with targeted therapies such as PI3K, PARP, and HER2 inhibitors. BRCA alterations may be of germline or somatic origin and they may be targetable, as demonstrated by gLOH, rather than passenger mutations. Further characterization of these tumors and comparison to both MSS BC and non-breast MSI-H tumor types, combined with treatment outcomes, can provide insights on rationale combinations and/or sequencing of therapeutic agents. Citation Format: Kimberly McGregor, Natalie Danzinger, Jeffrey S. Ross, Kyle Gowen, Alexa B. Schrock, Garrett M. Frampton, Dean C. Pavlick, Jan W. Davis, Carl R. Gray, Jeffrey M. Venstrom. Therapeutic considerations in microsatellite instability high (MSI- H) breast cancers (BC) identified by comprehensive genomic profiling (CGP) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-04.

Abstract PO058: Targeted next-generation sequencing (NGS) of 105 cancer-related genes in circulating tumor DNA (ctDNA) from patients with advanced cancers treated with immune checkpoint inhibitors (iCPI)

Abstract Plasma-derived ctDNA provides easily accessible minimally invasive material for molecular testing. There is increasing evidence that underlying genotypes including microsatellite instability (MSI-H), tumor mutation burden, PD-L1 expression or amplification and other molecular changes can be associated with distinct response patterns to iCPI. We isolated ctDNA from plasma samples collected from patients with advanced cancers before starting therapy with iCPI and tested them using a targeted NGS panel (Tempus xF) at average depths of 20,000x(raw reads)/5,000x(unique reads) to detect single nucleotide variants, insertions, deletions in 105 genes; copy number amplifications in 6 genes; copy number deletions in 2 genes; and gene rearrangements (translocations) in 7 genes spanning ~0.3 Mb of genomic space. Variants of unknown significance were excluded. MSI-H status was also tested. Results were compared to molecular testing of archival tumor tissue to assess agreement with ctDNA, and to treatment outcomes. Twenty patients with advanced cancers (breast 5, colorectal 2, cholangiocarcinoma 2, uveal melanoma 2, other 9) dispositioned to start therapy with iCPI (PD-L1/PD1 antibody, n = 18; LAG3 antibody, n = 2) were enrolled. Of 20 patients, 19 had ctDNA results, which passed quality control. The rates of complete detection of all molecular alterations present in archival tumor tissue was 47% (n = 9) and of partial (key driver alterations) detection 32% (n = 6) with a total complete or partial detection rate of 79%. Individual detection rates in ctDNA compared to tissue per patient ranged from 0%-100% (median, 78% in 19 patients). Seventeen patients had a total of 38 alterations (TP53, n = 14; NF1, n = 5; PIK3CA, n = 2; KRAS, n = 2; ESR1, n = 2; FBXW7, n = 2; others [including PD-L1 amplification], n = 11) in ctDNA not reported in archival tissue. Patients with lower amounts of ctDNA (&amp;lt;median variant allele frequency) had longer median time-to-treatment failure (TTF) on iCPI (3.6 vs. 1.9 months, P = 0.018) and longer overall survival (OS; not reached vs. 4.7 months, P = 0.015) compared to patients with higher amounts of ctDNA. In conclusion, targeted NGS testing of pretreatment ctDNA samples demonstrated a high rate of detection for driver alterations previously noted in archival tumor tissue. Higher quantity of ctDNA was associated with shorter TTF on iCPI and shorter OS compared to lower quantity of ctDNA. Citation Format: S. Greg Call, Helen J. Huang, Jordi Rodon, Daniel D. Karp, Apostolia M. Tsimberidou, Aung Naing, Filip Janku. Targeted next-generation sequencing (NGS) of 105 cancer-related genes in circulating tumor DNA (ctDNA) from patients with advanced cancers treated with immune checkpoint inhibitors (iCPI) [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO058.

Prognostic significance of PD-L1 protein expression and copy number gains in locally advanced cervical cancer.

Although immune checkpoint inhibitors against programmed death-1 (PD-1) and its ligand (PD-L1) have demonstrated promising results in several solid malignancies, including cervical cancer, there are some limitations to using PD-L1 immunohistochemical expression as a predictive biomarker for selecting patients who may benefit from such therapy. To examine the protein expression and genetic status of PD-L1 with clinical outcomes in locally advanced cer- vical cancer. We investigated the PD-L1 gene copy number gains assessed by fluorescence in situ hybridization (FISH) and PD-L1 expression using immunohistochemistry in 123 patients with locally advanced cervical cancers between December 2008 and December 2016. The prevalence of PD-L1 immunohistochemical expression was detected in 103/123(83%) cases. PD-L1 gene am- plification and polysomy were detected in 7% and 40% of cases, respectively. PD-L1 gene amplification and polysomy were associated with positive PD-L1 immunostaining (score 1+ to 3+) in 88% and 68% of cases, respectively. Clinically, PD-L1 immunopositivity was associated with parametrial invasion at diagnosis. In contrast, PD-L1 polysomy was associated with parametrial invasion and FIGO stages III-IV, whereas PD-L1 amplification was associated with nodal metastasis. In multi- variate analysis, PD-L1 amplification was predictive of worse RFS (HR, 5.68; 95%CI, 1.98-16.28; p = 0.001), whereas PD-L1 polysomy was predictive of worse LRR (HR, 4.13; 95%CI, 1.63-10.49; p = 0.003). PD-L1 immunohistochemical expression was not associated with worse outcomes in Cox models. Our results showed that an increase in PD-L1 gene copy number could be a novel prognostic and possible predictive biomarker for anti-PD-1/PD-L1 therapy in locally advanced cervical cancer.

Targeting macrophage priming by polyphyllin VII triggers anti-tumor immunity via STING-governed cytotoxic T-cell infiltration in lung cancer

Stimulator of interferon genes (STING) controlled innate immune pathway is essential for host defense against pathogenic infection and effective anti-tumor adaptive immunity initiation. Although macrophages transformed across diverse phenotypes play crucial roles in anti-tumor immune response, events determining this transformation and the host-intrinsic role of STING in this process remain controversial. Here we report how STING signaling acts as a key switch to dominate the gene expression patterns of macrophage transformation for promoting priming and releasing immunosuppression. Furthermore, polyphyllin VII, a potential STING agonist, exerts anti-tumor efficacy upon macrophages priming and subsequent cytotoxic T lymphocytes intratumoral infiltration. Meanwhile, the simultaneous PD-L1 amplification on macrophages in response to PP VII is also ruled by STING, thus PP VII may benefit immune-checkpoint blockade therapy for combining. Moreover, PP VII suppresses carcinogenesis upon restraining the immunosuppressed macrophage transformation. This is due to the boosted STING that negatively regulates a STAT3 propagated crosstalk between immune cells and tumor cells. Overall, PP VII-stimulated STING in macrophages provides a paradigm for anti-tumor, and if possible, anti-infection immunotherapy.

Molecular events in the pathogenesis of vulvar squamous cell carcinoma.

Vulvar squamous cell carcinomas (VSCC), which constitute over 90% of vulvar malignancies in adults, are classifiable into 2 subgroups that are mostly clinicopathologically distinct, a classification that is fundamentally based whether or not the tumors are HPV-mediated. In this review, we aim to summarize the recent advances in the understanding of molecular events in the pathogenesis of VSCC, including common and targetable mutations, copy number alterations, epigenetics, noncoding RNAs, and tumor immune microenvironment, which may provide insight into the future management of the disease. These events show substantial differences between the 2 subgroups, although significant areas of overlap exist. Recurrent, driver mutations appear to be substantially more prevalent in HPV(-) VSCC. TP53 mutations are the most common somatic mutations in VSCC overall, and are notably predominant in the HPV(-) VSCC, where 30-88% show a mutation. TP53 mutations are associated with worse patient outcomes, and co-mutations between TP53 and either HRAS, PIK3CA or CDKN2A appear to define subsets with even worse outcomes. A wide variety of other somatic mutations have been identified, including a subset with different mutational frequencies between HPV(+) and HPV(-) VSCC. CDKN2A mutations are common, and have been identified in 21 to 55% of HPV(-) VSCC, and in 2 to 25% of HPV(+) VSCC. Hypermethylation of CDKN2A is the most frequently reported epigenetic alteration in VSCC and the expression of some microRNAs may be associated with patient outcomes. The PTEN/PI3K/AKT/mTOR pathway is commonly altered in HPV(+) VSCC, and is accordingly potentially targetable. HPV-positivity/p16 block expression by immunohistochemistry has been found to be an independent prognostic marker for improved survival in VSCC, and may have some predictive value in VSCC patients treated with definitive radiotherapy. 22-39.3% and 68% of VSCC show EGFR amplification and protein overexpression respectively, although the prognostic and predictive value of an EGFR alteration requires additional study. Recurrent chromosomal gains in VSCCs have been found at 1q, 2q, 3q, 4p, 5p, 7p, 8p, 8q, and 12q, and there may be differential patterns of alterations depending on HPV-status. At least one-third of VSCC patients may potentially benefit from immune checkpoint inhibition therapy, based on a high frequency of PD-L1 expression or amplification, or a high tumor mutational burden. Additional studies are ultimately required to better understand the global landscape of genetic and epigenetic alterations in VSCC, and to identify and test potential targets for clinical application.

Abstract 387: Analysis of FFPE human tumor tissues using CODEX with signal amplification

Abstract Analysis of FFPE human tumor tissues using CODEX with signal amplification Characterizing the complexities of the tumor microenvironment is fundamental to understanding disease mechanisms. The spatial relationships between infiltrating immune cells and the remodeling of the cellular matrix is widely recognized as a key component to defining tumor heterogeneity. Current methodologies for studying cells within the context of tissue architecture, like traditional immunofluorescence (IF) and immunohistochemistry (IHC), are limiting–allowing the assessment of only a few parameters at a time. The CODEX technology has overcome this limitation through a DNA-based labeling strategy, involving adding and removing dye-labeled oligonucleotides (reporters) across multiple cycles to oligonucleotide-labeled antibodies. In this manner, tens of markers can be analyzed on the same tissue. Additionally, CODEX interfaces with existing inverted microscopes and provides a cost-effective, fully automated platform for highly multiplexed imaging. Recently, we expanded our workflow to amplify the fluorescent signal intensities of low expressing biomarkers by combining Tyramide Signal Amplification (TSA) with CODEX. This approach is associated with key markers used to analyze the tumor microenvironment, including PD-L1 and PD-1. We demonstrated signal amplification of more than 50X compared with non-amplified signal. Here, we present the analysis of CODEX data for a panel of over 20 biomarkers on human FFPE tissue including signal amplification of PD-L1, PD-1 and foxp3. Through amplification of key markers as the final cycle of a CODEX experiment, low expression can be measured and used to identify key cell types, including Treg cells and PD-L1 positive tumor and stromal regions. Citation Format: Sejal Mistry, Gajalakshmi Dakshinamoorthy, Jessica Yuan, Pieter Noordam, Joseph Kim, Won-Mean Lee, Julia Kennedy-Darling. Analysis of FFPE human tumor tissues using CODEX with signal amplification [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 387.

Immunogenomic profiling determines responses to combined PARP and PD-1 inhibition in ovarian cancer

Combined PARP and immune checkpoint inhibition has yielded encouraging results in ovarian cancer, but predictive biomarkers are lacking. We performed immunogenomic profiling and highly multiplexed single-cell imaging on tumor samples from patients enrolled in a Phase I/II trial of niraparib and pembrolizumab in ovarian cancer (NCT02657889). We identify two determinants of response; mutational signature 3 reflecting defective homologous recombination DNA repair, and positive immune score as a surrogate of interferon-primed exhausted CD8 + T-cells in the tumor microenvironment. Presence of one or both features associates with an improved outcome while concurrent absence yields no responses. Single-cell spatial analysis reveals prominent interactions of exhausted CD8 + T-cells and PD-L1 + macrophages and PD-L1 + tumor cells as mechanistic determinants of response. Furthermore, spatial analysis of two extreme responders shows differential clustering of exhausted CD8 + T-cells with PD-L1 + macrophages in the first, and exhausted CD8 + T-cells with cancer cells harboring genomic PD-L1 and PD-L2 amplification in the second.

PD-L1 expression, tumor mutational burden, and microsatellite instability status in 746 pancreas ductal adenocarcinomas.

757 Background: Pancreas ductal adenocarcinomas (PDA) has a 5-year survival rate of 6% with a need for new therapeutic options. The approval of pembrolizumab for some gastrointestinal cancers shows the potential of immunotherapy (IMT) in PDA. We evaluated the IMT-associated biomarkers of PD-L1 expression, tumor mutational burden (TMB), microsatellite instability (MSI) and PD-L1 amplification in PDAs. Methods: 746 formalin-fixed paraffin embedded samples were evaluated for PD-L1 IHC using the Dako 22C3 pharmDx assay and scored using tumor proportion score (TPS). The cases had comprehensive genomic profiling (CGP) via DNA sequencing, using a hybrid-capture next-generation sequencing assay (FoundationOne and FoundationOneCDx) for genomic alterations (GAs), TMB, and MSI. Results: PD-L1 was positive (TPS ≥ 1%) in 29% (214/746) and negative in 71% (532/746). 43/214 (20%) of positive cases were high positive (TPS ≥ 50%). TMB (590 cases) had a mean of 3.20, 3.46, and 3.61 mutations/Mb for PD-L1 negative, positive, and high positive groups. 3 hypermutated (TMB ≥ 20) were negative for PD-L1 expression. 3/581 cases were MSI-high with a high TMB score (average 23.53 mutations/Mb). 2 MSI-high cases were negative for PD-L1 and 1 was high positive. PD-L1 amplification was not detected (0/746). Only BCOR was significantly different between PD-L1 high positive and PD-L1 negative tumors (Table). Conclusions: Of 729 PDA cases, 29% were positive (TPS ≥ 1%) for PD-L1 expression while only 6% of all cases showed a high level of PD-L1 expression on tumor cells. TMB high (3/729) and MSI-High (3/729) cases were rare. Only 2 of the TMB high cases were also MSI-high. PD-L1 amplification was not detected. Comparing GAs in PD-L1 high positive vs negative cases was only significantly different for BCOR. Further investigation is needed to see if a combined positive score of PD-L1 expression may identify a subset of patients with PDA who are more likely to respond to IMT. [Table: see text]