Wild-type Cohort Research Articles

Loss of Estrogen‐Related Receptor Alpha (ESRRA) Impairs Neuronal Mitochondrial Function and Results in Disturbed Sleep and Neural Oscillatory Patterns

ESRRA is an orphan nuclear receptor enriched in metabolically active tissues and has been known to affect the transcription of a set of genes that regulate mitochondrial bioenergetics in peripheral organs. Of note, ESRRA is also abundantly expressed in the brain, but the functional role of brain ESRRA remains poorly understood. To test the role of ESRRA on energy metabolism in neurons, we performed the Seahorse assay in mouse primary cortical neurons with or without ESRRA expression and found that neurons lacking ESRRA have significantly reduced cellular respiration compared to control neurons, indicating that loss of ESRRA decreases mitochondrial oxidative capacity and thus neuronal energy production. We have previously reported that global ESRRA null (KO) displayed markedly reduced locomotor activity (home cage vibration plate p<0.05, wheel running p<0.001), suggesting that these mice do display an energy deficit as a result of this manipulation. Based on the commonly appreciated role of sleep in the replenishment of energy stores for normal brain function, we hypothesized that reduced brain energy levels caused by genetic loss of ESRRA would lead to hypersomnia and diminished neural oscillations, which may underlie hypolocomotion seen in ESRRA KO mice. To test this hypothesis, wireless electroencephalography/electromyography (EEG/EMG) devices were implanted in a cohort of ESRRA KO and wildtype (WT) mice and 24hr sleep state was recorded and scored. Sleep architecture analysis from 24‐hour wireless EEG/EMG recording revealed that ESRRA KO mice exhibit significantly increased non‐rapid eye movement (NREM) (p<0.05) sleep as well as disrupted circadian pattern of rapid eye movement (REM) (p<0.01) sleep compared to WT littermates. Power spectral analysis further revealed that high range gamma (40‐100Hz) (p<0.001) and alpha (9‐13Hz) (p<0.05) oscillations are significantly decreased while low range gamma (25‐40Hz) (p<0.05) and beta (13‐25Hz) (p<0.05) oscillations are increased in ESRRA KO mice. Our findings not only reveal a previously unappreciated role of ESRRA in regulating neuronal mitochondrial function, sleep‐wake cycle, and neural oscillations, but also support a novel concept that a single molecular cause of diminished brain energy levels can prolong sleep duration and results in aberrant REM sleep pattern.

Memory B cells are a heterogenous population regulated by epigenetic programming

Abstract Memory B cells (MBC) are a heterogenous population that consist of immunoglobin class switched and non-class switched MBC, and these populations can arise via germinal center dependent or independent mechanisms. The timing of MBC development influences the fate outcome of MBC populations. These different populations of MBC are regulated by cell signaling, but it is not clearly defined what epigenetic factors influence MBC differentiation. EZH2 is an important histone methyltransferase that catalyzes H3K27me3 resulting in gene repression. EZH2 has been shown to regulate B cell differentiation into germinal centers and plasma cells; however, it is unknown if EZH2 regulates MBC development. To address this, a knockout model has been established where EZH2 is conditionally deleted using the CD19 and AICDA driven CRE expression. Here we used the influenza PR8 model to ascertain the kinetics of MBC differentiation and formation in the spleen, dLN, and lungs following a live infection in wild-type and EZH2-KO cohorts. Using B cell tetramers, antigen-specific MBC will be analyzed to define the kinetics of MBC development and determine the similarities and differences between the different populations of MBC. Overall, these data define the early kinetics of MBC establishment and the developmental timing that EZH2-dependent H3K27me3 remodeling is required for MBC formation. Supported by NIH/NIAID to CDS (R01 AI148471)

Natural History of Human Epidermal Growth Factor Receptor 2-Amplified and Human Epidermal Growth Factor Receptor 2 Wild-Type Refractory Metastatic Colorectal Cancer in US Clinical Practice.

PURPOSEThe molecular heterogeneity of metastatic colorectal cancer (mCRC) presents a therapeutic challenge, with few trials focused on patients with human epidermal growth factor receptor 2 amplification (HER2-Amp). Our limited understanding of real-world patterns and outcomes by HER2 status of treatment-refractory patients leaves treatment decisions with little contextual information. We conducted a retrospective cohort study to describe the natural disease history of patients with refractory mCRC using an electronic health record–derived database with oncogenomic information.METHODSWe included patients with stage IV or recurrent mCRC diagnosed from January 2011 through December 2019 from a deidentified clinicogenomic database. Patients with ≥ 2 documented clinic visits, ≥ 2 lines of therapy (LOT) after mCRC diagnosis, and comprehensive genomic profiling were eligible. Patient records defined by treatment-refractory LOT were allocated to the HER2-Amp or HER2 wild-type (WT) cohort on the basis of comprehensive genomic profiling. Index date was defined as the start of any treatment-refractory LOT (≥ 2 LOT; patients could contribute multiple records). Descriptive statistics included demographic and clinical characteristics, treatments, laboratory values, and biomarkers. Overall survival (OS) was calculated as time (in months) from the index date until death from any cause and analyzed using Kaplan-Meier methodology. Sensitivity analyses were conducted to test the robustness of the primary findings.RESULTSA total of 576 patients were included (1,339 records); 63 (158 records) were HER2-Amp, and 513 (1,181 records) were HER2-WT. Demographics, clinical characteristics, biomarkers, and laboratory values were comparable between HER2 cohorts. OS was similar, with an unadjusted median OS of 11.2 months (95% CI, 8.6 to 15.1) and 9.9 months (95% CI, 8.3 to 10.9) across LOT for HER2-Amp and HER2-WT cohorts, respectively.CONCLUSIONThis study showed considerable treatment heterogeneity and poor outcomes among patients with treatment-refractory mCRC, emphasizing a substantial unmet therapeutic need.

Abstract PD14-01: Comprehensive molecular characterization of patients with metastatic invasive lobular carcinoma (ILC): Using real-world data to describe this unique clinical entity

Abstract Background: ILC is the second most common type of breast cancer and accounts for approximately 10% of all invasive breast cancers. A hallmark of ILC is the lack of E-cadherin (CDH1) expression, which is frequently used to discriminate between lesions with borderline ductal and lobular histologies. While the genomic landscape of primary ILCs has been well described, less is known about patients (pts) with metastatic-ILC (mILC). Better characterization of the genomic landscape associated with mILC is critical for identifying biomarkers that may provide new insight into ILC tumor biology and ultimately improve long-term outcomes in pts with mILC. Methods: We retrospectively analyzed de-identified next-generation sequencing (NGS) data from 150 advanced/metastatic pts with ILC and 51 with mixed lobular/ductal histology, defined using the histology of the sequenced biopsy. Diagnoses were abstracted from pathology reports submitted at the time of sequencing. We used the stage documented closest in time to biopsy collection, and samples were excluded if the staging date was unknown or exceeded 180 days after the biopsy date. Our dataset consisted of samples that were molecularly profiled using the Tempus xT solid tumor assay (DNA-seq of 595-648 genes at 500x coverage, full-transcriptome RNA-seq). The mutations identified for this study include somatic single-nucleotide variants and insertions/deletions. Furthermore, we examined the co-mutational landscape of CDH1-mutant disease and investigated transcript-level expression variation. Results: Mutations in CDH1 occurred in 65.3% of all mILC samples (98/150). CDH1 expression was similar between CDH1-mutant and WT mILC samples (Wilcoxon rank sum test, p=0.8). The median tumor mutational burden (TMB) score was significantly higher in CDH1-mutant samples (Wilcoxon rank-sum test, p=0.010). CDH1-mutant samples were more likely to have a high TMB (≥10 mutations/MB) when compared with the wild-type CDH1 cohort (10% vs. 6.2%), but this difference was not statistically significant (Fisher’s Exact test, p=0.5). Additionally, we observed that the ER+ subtype was more frequent in CDH1-mutant samples, although this difference was not statistically significant (97% vs 88%; Fisher’s exact test, p=0.063). PIK3CA mutations were enriched in CDH1-mutant mILC (Table 1). TBX3 and NCOR1 mutations were also mildly enriched in CDH1-mutant mILC, but these results were not significant when correcting for multiple testing (Table 1). CDH1-mutant mixed histology pts had lower CDH1 expression than WT pts (p<0.001, Wilcoxon rank sum exact test). PIK3CA mutations were enriched in CDH1-mutant mixed histology pts, but this difference was not statistically significant (50% vs. 31%; p=0.3, Fisher’s exact test). Log10 CDH1 expression across all mILC pts was lower than in mixed histology pts (3.01 vs 3.53; p<0.001, Wilcoxon rank-sum test). Conclusions: Our real-world dataset illustrates that the molecular landscape of CDH1-mutant mILC pts is distinct from CDH1-WT mILC pts. Additionally, mILC differs from mixed histology at a transcriptional level, with lower CDH1 expression regardless of CDH1 mutational status. Our findings suggest a use for CDH1 RNA expression levels in reclassifying mixed histology samples as mILC. Additionally, therapies targeting PIK3CA may be further investigated for their actionability in CDH1-mutant mILC cases. Table 1.Frequency of co-mutations in CDH1-mutant vs. WT mILC cohortsGenesCDH1-mutant (n=98)CDH1 WT (n=52)p-value1q-value2n (%)n (%)PIK3CA53 (54%)6 (12%)<0.001<0.001TBX313 (13%)0 (0%)0.0040.13NCOR111 (11%)0 (0%)0.0090.21Pearson’s Chi-squared test; Fisher’sexact test. 2Falsediscovery rate correction for multiple testing Citation Format: Andrew A Davis, Amir Behdad, Kayla Viets Layng, Firas Wehbe, Lorenzo Gerratana, Elizabeth Mauer, Alex Barrett, Ami N Shah, Paolo D’Amico, Lisa Flaum, William J Gradishar, Leonidas C Platanias, Massimo Cristofanilli. Comprehensive molecular characterization of patients with metastatic invasive lobular carcinoma (ILC): Using real-world data to describe this unique clinical entity [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD14-01.

Differences in Clinical Characteristics Between Delta Variant and Wild-Type SARS-CoV-2 Infected Patients.

Background: As delta variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prevailed in the current coronavirus disease 2019 (COVID-19) pandemic, its clinical characteristics with the difference from those of wild-type strains have been little studied.Methods: We reported one cohort of 341 wild-type patients with COVID-19 admitted at Wuhan, China in 2020 and the other cohort of 336 delta variant patients with COVID-19 admitted at Yangzhou, China in 2021, with comparisons of their demographic information, medical history, clinical manifestation, and hematological data. Furthermore, within the delta variant cohort, patients with none, partial, and full vaccination were also compared to assess vaccine effectiveness.Findings: For a total of 677 patients with COVID-19 included in this study, their median age was 53.0 years [interquartile range (IQR): 38.0–66.0] and 46.8% were men. No difference was found in age, gender, and percentage of patients with the leading comorbidity between wild-type and delta variant cohorts, but delta variant cohort showed a lessened time interval between disease onset to hospitalization, a reduced portion of patients with smoking history, and a lowered frequency of clinical symptoms. For hematological parameters, most values demonstrated significant differences between wild-type and delta variant cohorts, while full vaccination rather than partial vaccination alleviated the disease condition. This reflected the viremic effect of delta variant when vaccination succeeds or fails to protect.Interpretation: Delta variant of SARS-CoV-2 may cause severe disease profiles, but timely diagnosis and full vaccination could protect patients with COVID-19 from worsened disease progression.

Impact of p53 Disruption on AML & HR-MDS Treatment out-Come. a Single Centre Experience

Introduction: Acute myeloid leukaemia (AML) is a heterogeneous disorder that arises from clonal expansion of malignant hematopoietic precursor cells. Somatic mutations of the p53 gene have been reported in 5-10% of AML, with a higher incidence therapy-related disease and elderly patients. Alteration or loss of p53 is one of the most powerful independent indicators of poor outcome.Methods: This is a retrospective analysis of AML & high risk-Myelodysplastic syndrome (HR-MDS) patients treated over the study period (2006-2020). Informed consent was obtained. Initial presentation, treatment response, and survival were analysed. Percentage of p53 expression (a surrogate marker for TP53 mutations) by immunohistochemistry (IHC) (>30% cut-off) on BM trephines was analysed and its impact on treatment outcome was evaluated.Results: We identified 114 patients (AML=104 & HR-MDS=10), the median age was 70 years (Range 36-85) with male predominance (M=73 vs. F=41). The AML group included, 57 patients (50%) with Denovo AML and 47 patients (41%) with Secondary AML (MDS=27, MPN=12 / JAKII mutation =7, t-AML=8). The median age was 70 years (Range 36-85) and the median blast count was 70% (Range 25-95%). Cytogenetic analysis reports were available on 78 patients (75%)(Poor risk=40 vs. Intermediate risk=38). NPM1 (22 patients) and FLT3 mutations analysis (33 patients )were available and reported as positive in 4(18%) and 5(15%) patients respectively.The HR-MDS is defined by patients who fall into higher-risk group categories in the original or revised IPSS (N=10), the median age was 74 years (Range 63-83) with male predominance (M=7 vs. F=3). The median blast count was 15% (Range 11-18%). Cytogenetic risk prognostic subgroup results were available on 6 patients (60%), 2 patients (2%) had intermediate-risk and 4 patients (4%) had poor-risk.Ninety-nine patients (87%) were eligible for treatment, 48 patients (42%) were treated with intensive induction chemotherapy, and 51 patients (45%) received less intensive therapy (HMA / LD-Ara C). Five patients (4%) consolidated with allogeneic stem cell transplant (Allo-SCT) post-induction therapy. The remaining (15 patients) were on supportive care. Forty-one AML patients (36%) relapsed post CR1, 3 patients had Allo-SCT in CR2. Table 1At the time of study analysis (31/01/2021), 25 patients (22%) are still alive including 23 patients (20%) with AML (Denovo AML=18 & Sec AML=5) and 2 patients (4%) with HR-MDS. The median age of the survival group was 69 years (Range 51-83). The whole study cohort median OS was 12 months. (Figure. 1) The median OS & PFS for AML patients were 15 & 13 months respectively. According to AML sub-type, t-AML associated with shortest median OS of 2.5 months (P.value 0.0190). (Figure. 2) The median OS was more favourable for intermediate-risk than the high-risk cytogenetic AML, 24 and 10 months respectively (P<00016).(Figure. 3) The HR-MDS group median OS was 22 months.One hundred and four patient's BM trephines (91%) were available and screened for p53 IHC expression (AML= 96 & HR-MDS =8). Eight samples (8%) showed p53 over-expression, all these samples with underlying AML diagnosis. The remaining 96 samples (92%) were negative for high p53 expression. The majority of p53 over-expressed AML harboured complex cytogenetics, 4 patients with Sec AML, 3 patients with Denovo AML, and 1 patient with t-AML. Most of the patients (5 patients) in this sub-set had a refractory disease to induction therapy, of whom 2 patients (25%) had Allo-SCT consolidation in CR2. Only one patient (12%) is still alive in this sub-group.The median OS & PFS in p53 wild-type AML cohort was 16 & 13 months respectively, which compared favourably to 12 & 8 months in those with p53 disruptions. The P. values were 0.134 & 0.193, respectively. The impact of the p53 alterations was more pronounced on the AML sub-group treated with intensive chemotherapy (42%), the median OS & PFS in wild-type p53 AML were 24 & 20 months, while in p53 disrupted cohort were 12 & 10 months respectively (P= 0.030)(95%CI of ratio 0.1755 to 1.425) & (P=0.049)( 95%CI of ratio 0.7015 to 5.702).(Figure.4)Conclusion: In this study we demonstrated the potential role for p53 expression by IHC, which is readily available in routine practice, in assessing AML prognosis. Our real-life data confirms the dismal prognosis of p53 alterations in this disease. Participation in clinical trials based on genetic risk stratification is warranted. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Impact of p53 Disruption on Mantle Cell Lymphoma (MCL) Treatment out-Come, Multi-Centre Retrospective Study

Background: MCL is a rare type of non-Hodgkin lymphoma (NHL) with an aggressive clinical course. MCL is associated with poor prognosis and incurable disease in the majority of patients. However, a minority of patients will not require any treatment for many years. TP53 mutations continue to confer a dismal prognosis in MCL with a median survival of 1.3 years. However, targeted therapy and, more recently, promising CAR-T treatment have revolutionised the outcome.Methods: Patients were identified through our pathology database, and informed consent was obtained. The patient's demographic characteristics, clinical features, laboratory findings, MIPI score, initial / subsequent treatment and survival were analysed. p53 expression percentage and its concordance with the mutational status were evaluated in a subset of patients.Results: One hundred and one patients were included in this study, with a male predominance (80%), median age 67 years (Range 37-89 years) and majority with advanced disease. Fifty-one patients (50%) presented with extra-nodal disease. Most of the patients presented with Classical MCL ( 75%) histological sub-type, 20 patients (20%) with blastoid variant and 6 patients (6%) with indolent disease. Seventy-four patients (73%) had a high MIPI risk score, while intermediate and low scores were noted in 17 patients (17%) and 10 patients (10%), respectively. Chemo-immunotherapy (CIT) was the main initial treatment modality in our cohort (79 patients / 78%). Watch and wait approach was applied in 6 patients with the indolent disease, 4 patients (4%) were not fit for any form of therapy. Forty-four patients (44%) had Rituximab maintenance and autologous stem cell transplant (Auto-SCT ) consolidation was used in 12 patients (12%). Five patients (5%) had allogeneic SCT (Allo-SCT) post-remission.Initially, 29 patient samples were used to validate immunohistochemistry (IHC) p53 expression percentage and its correlation with TP53 mutational data from genomic sequencing (SS and NGS). p53 expression of > 30% had 100% concordance with TP53 mutational status.A total of 128 patient samples, including 27 samples with relapsed disease, were screened for TP53 alterations using IHC p53 expression as a surrogate marker. Twenty samples (16%) showed p53 overexpression (15/12% diagnostic & 5/19% relapsed samples).At the final data compilation (31/01/2021), 44 patients were still alive, and 57 patients had died. The OS and PFS for the whole cohort were 69 and 47 months, respectively. (Figure 1)The prognostic impact of age (<65 years), MIPI score & disease sub-type were confirmed in this cohort with a P-value of P.00046, P.0.036 and P.0.0192, respectively. In the treated cohort, p53 disruption revealed a dismal prognosis and poor treatment outcome, with a median OS and PFS in the p53 wild-type cohort of 95 months and 50 months. In contrast, in patients harbouring p53 disruption, the median OS & PFS were 38months (P.0323) and 25 months (P.0383 ), respectively. (Figure 2)ConclusionThis study reflects real-life MCL experience and the potential use of p53 expression using IHC in routine practice in assessing MCL disease prognosis. It also confirms the dismal outcome of MCL patients with TP53 mutations. Participation in clinical trials based on genetic risk stratification is warranted. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

NF1-mutated melanomas reveal distinct clinical characteristics depending on tumour origin and respond favourably to immune checkpoint inhibitors

BackgroundNF1-mutated tumours represent a small subset (10–15%) of melanomas, not sufficiently analysed in large clinical cohorts. This study investigated the largest multicentre collection of NF1-mutated melanomas to date. MethodsThis study analysed a multicentre tumour tissue sample cohort from 266 patients with NF1-mutated melanoma. Targeted next-generation sequencing of the TERT promoter and 29 relevant melanoma genes was performed. Survival was compared with NF1 wild-type cohorts from the Tissue Registry in Melanoma project (n = 432). ResultsMost NF1-mutated melanoma arose in the head-and-neck region of patients >60 years. NF1 alterations were frequently inactivating, primarily non-sense, less frequently truncating mutations. Non-inactivating NF1 mutations more frequently co-occurred with activating BRAF and RAS mutations. NF1-mutated tumours had higher numbers of gene mutations and UV signature C>T and CC>TT transitions than BRAF, RAS and triple wild-type melanomas. NF1-mutated acral and mucosal melanomas harboured a different mutation signature and were frequent in women (69% and 83%, respectively), differing from non-acral cutaneous NF1-mutated melanomas (men 73%, women 27%). Overall survival in stage IV disease was comparable for patients with NF1-mutated or wild-type melanoma. However, in patients receiving first-line immune checkpoint inhibitor treatment, better median overall survival (mOS) was observed for NF1-mutated than wild-type tumours (mOS = not reached vs mOS = 25.82, p = 0.0154, n = 80 and 432, respectively). ConclusionsCutaneous, acral and mucosal NF1-mutated melanomas vary in clinical and genetic characteristics and demonstrate a favourable outcome on immune checkpoint inhibition therapy.

Breast Irradiation is Well Tolerated in Carriers of a Pathogenic ATM Variant

<h3>Purpose/Objective(s)</h3> There is mixed and limited scientific evidence regarding radiotherapy (RT) tolerance in carriers of a pathogenic, germline <b>ATM</b> variant. The ASTRO-ASCO-SSO guidelines on management of hereditary breast cancer suggest offering RT to these patients if clinically indicated. We investigate RT toxicity for carriers of either a pathogenic <b>ATM</b> variant, or variant of unknown significance (VUS), in <b>ATM</b>. <h3>Materials/Methods</h3> Patients with a breast cancer history who had hereditary genetic testing and were found to carry either a pathogenic variant or VUS in <b>ATM</b> were identified. For those who received RT at our institution, toxicity metrics were recorded including dermatitis (scored per CTCAE version 4.0), moist desquamation, and cosmetic outcome (scored per Harvard scale). Fisher's exact test for count data was performed to compare toxicity between the two cohorts. Wilcoxon rank-sum testing was performed to assess for differences in cohort patient characteristics. <h3>Results</h3> Forty-four carriers of a pathogenic <b>ATM</b> variant, and 163 carriers of an <b>ATM</b> VUS, were identified. Fifteen pathogenic and 56 VUS patients, respectively, received RT at our institution and had available toxicity records. The cohorts did not statistically differ in race, medical comorbidities, clinical or pathologic T-stage, hormone receptor status, receipt of chemotherapy, fractionation schedule, or receipt of a boost. No patients had a known collagen vascular disease. 77% of patients (55/71) had breast conservation. 62% received whole breast RT, 7% received chest wall RT, 4.2% received partial breast RT, and 26.8% received whole breast or chest wall RT with regional nodal RT. 58% received conventionally-fractionated RT, and 38% received hypofractionated RT. Median toxicity follow up was 11.6 months. 29% (4/15) of pathogenic carriers and 26.4% (14/56) of VUS carriers developed moist desquamation (<i>P</i> = 0.7). The rates of grade 2+ acute dermatitis were 71% in pathogenic carriers and 57% in VUS carriers (<i>P</i> = 0.4). 100% of pathogenic carriers and 91% of VUS carriers had a good/excellent cosmetic outcome at first follow up (<i>P</i> > 0.9). No patients with a pathogenic variant had a fair or poor cosmetic outcome at last follow up. <h3>Conclusion</h3> RT is well tolerated in carriers of a pathogenic <b>ATM</b> variant, with rates of grade 2+ dermatitis and moist desquamation comparable to a matched VUS cohort. High rates of good or excellent cosmetic outcome were seen following treatment. Further analyses including an <b>ATM</b> wild type cohort and continued detailed toxicity documentation/analysis is on-going to elucidate longer term morbidity.

P60.12 Prevalence of c-Met overexpression (c-Met+) and Impact of Prior Lines of Treatment on c-Met Protein Expression in NSCLC

Teliso-V, an anti–c-Met antibody conjugated with monomethyl auristatin E, is currently being investigated as monotherapy in c-Met+ 2nd/3rd-line metastatic NSCLC in a phase 2 study (NCT03539536). Here we present prevalence of c-Met+ NSCLC based on histopathology and EGFR mutation status. c-Met expression change from archival (diagnostic (Dx)/post-Dx biopsy) vs fresh (tumor biopsy at time of study prescreening) is also presented for patient subsets. In this study, patients were prospectively selected for c-Met–positive expression by immunohistochemistry (central assay; SP44 antibody). Archival or fresh tumor tissue was eligible for c-Met expression testing and enrollment consideration. Per protocol, if archival tissue was c-Met negative, patients could provide fresh tumor tissue for reassessment of c-Met expression. As of December 2020, 841 patients evaluable for c-Met expression have been prescreened (Table 1). c-Met+ rates were lower in EGFR wild-type (25%) vs mutant (37%) non-squamous cohorts. Squamous histology had a 39% positivity rate. c-Met+ rates were higher in fresh vs archival tumor tissue. Similar trends were observed when EGFR wild-type and mutant cohorts were separated based on c-Met–high or –intermediate groups (Table 2). Sixteen patients (non-squamous cohorts: EGFR mutant, N=8; EGFR wild-type, N=8) initially reported c-Met–negative status based on archived tissue and submitted fresh tissue for c-Met reassessment. Tumors from EGFR mutant (50%) and wild-type (12.5%) cohorts had changes in c-Met status from negative to positive, potentially due to prior anti-EGFR or immuno-oncology/chemotherapy treatments, degradation of the epitope over time in the sample, or heterogeneity within tumor lesions.Table 1Patientsc-Met Positive, %a (n)NSQ EGFR MU24537 (90)NSQ EGFR WT44625 (122)Squamous15039 (58)aThe c-Met–positive cutoffs (by immunohistochemistry using SP44 antibody) were derived from retrospective receiver operating characteristic analysis of phase 1 (NCT02099058) Teliso-V monotherapy data. Cutoffs were established as membrane staining: non-squamous (≥25% 3+) and squamous (≥75% 1+) NSCLC. MU, mutant; NSQ, non-squamous; WT, wild-type. Open table in a new tab aThe c-Met–positive cutoffs (by immunohistochemistry using SP44 antibody) were derived from retrospective receiver operating characteristic analysis of phase 1 (NCT02099058) Teliso-V monotherapy data. Cutoffs were established as membrane staining: non-squamous (≥25% 3+) and squamous (≥75% 1+) NSCLC. MU, mutant; NSQ, non-squamous; WT, wild-type. Based on defined cutoffs, 25%–39% of NSCLC tumors were c-Met+. Consistent with prior observations, an increase in c-Met expression was observed in tumor tissue after anti-EGFR treatments. The increase in c-Met expression following immuno-oncology/chemotherapy treatments was a new observation. Additional data are needed to make conclusive remarks.

P59.16 Characterizing the Tumour-Immune Microenvironment in EGFR Mutant NSCLC

Non-small cell lung cancer (NSCLC) comprises around 80% of lung cancers, with mutations in epidermal growth factor receptor (EGFR) effecting approximately 26%. These constitutive activating mutations promote tumour cell proliferation and survival, and are inevitably refractory to tyrosine kinase inhibitors (TKIs) as well as current classes of immune checkpoint immunotherapy (ICI). Third generation TKI Osimertinib offers prolonged disease-free survival, however the reasons for the lack of efficacy of ICI in EGFR mutant tumours remain unclear. The cellular immune composition of the tumour microenvironment is increasingly being recognised as a major factor in host-tumour responses. The use of multispectral immunohistochemistry (IHC) is a valuable tool to investigate the cellular composition of these tumours. Here we compare a cohort of EGFR exon 19 deletion (LREA) tumours to stage matched EGFR wild type (WT) tumours by multispectral IHC. Automated staining was performed for CD8, FoxP3, PD-1, CD68, PD-L1 and cytokeratin markers using MOTiF™ PD-1/PD-L1 lung cancer kit, and slides imaged by Vectra®Polaris™. Image analysis was performed using QuPath, and marker counts were obtained within tumour and adjacent microenvironment compartments Immune cells marked by CD8, FoxP3, PD-1, CD68 composed 5%-30% of total cells in the WT tumours, while LREA tumours contained 5% to 15% of total cells (t-test p = 0.17). While these immune cells trended to be lower in LREA tumours, further investigation into their distribution in a larger cohort of patients is required. The LREA cohort displayed poorer progression free survival (PFS) following resection and platinum therapy than the WT cohort (468 days vs 622 days, p=0.2). Neighbourhood mapping suggested cellular populations within the tumour microenvironment which associated with the different tumour profiles. Our data suggests that tumour intrinsic mechanisms may result in poorer host-tumour responses. One such mechanism is the immunosuppression of the local microenvironment, and we aim to expand this work to encompass a wider cohort of patient tumours and cellular markers.

IBCL-165: Tazemetostat in Relapsed/Refractory (R/R) Follicular Lymphoma (FL): Propensity Score-Matched Analysis of E7438-G000-101 Trial Outcomes

Context Tazemetostat, an oral enhancer of zeste homolog 2 (EZH2) inhibitor, received FDA approval based on a phase 2 study (NCT01897571); in this study, the wild-type (WT) EZH2 cohort included more patients with poor-risk features than the mutant (MT) EZH2 cohort. Objective Assess outcomes in patients with WT versus MT EZH2 R/R FL after minimizing baseline characteristic differences by creating a matched sample of comparable WT and MT patients. Design In a post hoc analysis of NCT01897571, we generated propensity scores for each WT (n=54) and MT (n=45) EZH2 patient based on the likelihood of being selected, given their baseline characteristics. Characteristics prognostic for clinical outcomes with appreciable baseline differences between cohorts included ECOG performance status, number of prior anticancer therapies, progression of disease within 24 months, double-refractory status, and prior hematopoietic stem cell transplant. Patients were matched using a 1:1 nearest-neighbor approach. Baseline covariates between the matched groups were sufficiently balanced. Setting Thirty-eight global clinics/hospitals. Patients Adults with R/R FL who received ≥2 prior systemic therapies. Interventions Tazemetostat 800 mg orally in continuous 28-day cycles. Main Outcome Measures Objective response rate (ORR) estimates were measured for the matched groups. Progression-free survival (PFS) was described using Kaplan–Meier analyses. Results The propensity-matched sample included 56 patients (28 WT, 28 MT). Before matching, ORR was 35% (95% CI [22%, 48%]) in WT and 69% (95% CI [55%, 83%]) in MT EZH2 groups; after matching, ORR was 50% (95% CI [31%, 69%]) and 71% (95% CI [54%, 88%]), respectively. Median PFS was 11.1 months (95% CI [5.4, 16.7]) in WT and 13.8 months (95% CI [11.1, 22.1]) in MT prior to matching, and 14.3 months (95% CI [11.1, inf]) and 14.8 months (95% CI [10.7, inf]) in WT and MT matched groups, respectively. Conclusion As expected, efficacy remained higher in the MT group; however, after adjustment, ORR and PFS improved in the WT group. This hypothesis-generating analysis suggests that outcomes in patients with WT EZH2 R/R FL treated with tazemetostat may have been more similar to those with MT EZH2 in the phase 2 trial if baseline disease characteristics were more equally matched.

Histone H3.3 G34-mutant Diffuse Gliomas in Adults.

The characteristics of H3.3 G34-mutant gliomas in adults have yet to be specifically described. Thirty adults with H3.3 G34-mutant diffuse gliomas were retrospectively reviewed for clinical and pathologic information. Molecular profiling using next-generation sequencing was performed in 29 of the 30 H3.3 G34-mutant patients with 1 patient lacking available tumor samples, as well as 82 IDH/H3 wild-type adult diffuse glioma patients. The age at diagnosis of H3.3 G34-mutant diffuse gliomas was significantly younger than IDH/H3 wild-type gliomas (24 vs. 57 y, P<0.001). Overall, 19 of the 30 patients were diagnosed of glioblastoma with the primitive neuronal component, and 8 were glioblastoma. The molecular profiling analysis revealed higher frequencies of Olig-2 loss of expression, TP53 mutation, ATRX mutation, PDGFRA mutation, and MGMT promoter methylation (P<0.05) in H3.3 G34-mutant gliomas than IDH/H3 wild-type gliomas. No TERT promoter mutation and only 1 case of EGFR amplification were detected in the H3.3 G34-mutant cohort, the frequencies of which were significantly higher in the IDH/H3 wild-type cohort. A dismal prognosis was observed in H3.3 G34-mutant patients comparing to IDH/H3 wild-type cohort (overall survival: 14 vs. 22 mo; P=0.026). Univariate and multivariate analyses showed that the extent of resection and TP53 mutation were independently affecting prognosis. The distinct pathologic and molecular features of H3.3 G34-mutant diffuse gliomas in adult patients demonstrated the clinical importance of detecting H3.3 G34R/V mutations. The dismal prognosis of this rare high-grade glioma disease we reported here would further promote the investigation of dedicated therapeutic strategies.

NUAK2 and RCan2 participate in the p53 mutant pro-tumorigenic network

Most inactivating mutations in TP53 gene generates neomorphic forms of p53 proteins that experimental evidence and clinical observations suggest to exert gain-of-function effects. While massive effort has been deployed in the dissection of wild type p53 transcriptional programme, p53 mutant pro-tumorigenic gene network is still largely elusive. To help dissecting the molecular basis of p53 mutant GOF, we performed an analysis of a fully annotated genomic and transcriptomic human pancreatic adenocarcinoma to select candidate players of p53 mutant network on the basis their differential expression between p53 mutant and p53 wild-type cohorts and their prognostic value. We identified NUAK2 and RCan2 whose p53 mutant GOF-dependent regulation was further validated in pancreatic cancer cellular model. Our data demonstrated that p53R270H can physically bind RCan2 gene locus in regulatory regions corresponding to the chromatin permissive areas where known binding partners of p53 mutant, such as p63 and Srebp, bind. Overall, starting from clinically relevant data and progressing into experimental validation, our work suggests NUAK2 and RCan2 as novel candidate players of the p53 mutant pro-tumorigenic network whose prognostic and therapeutic interest might attract future studies.

Response to the EZH2 Inhibitor Tazemetostat Is Independent of Cell of Origin Determined Via Hans Imunohistochemistry or Nanostring Lymphoma Subtyping Test in EZH2 Wild-Type DLBCL Patients

Response to the EZH2 Inhibitor Tazemetostat Is Independent of Cell of Origin Determined Via Hans Imunohistochemistry or Nanostring Lymphoma Subtyping Test in EZH2 Wild-Type DLBCL Patients

Immune Checkpoint Profiling of TP53 Mutant and Wild-Type Myeloid Malignancies: TP53 Mutations Direct Immune Tolerance Via an Immunosuppressive Phenotype

Background Augmenting adaptive immunity through immune checkpoint blockade introduced a paradigm shift in the treatment of cancer patients. Although aberrant mRNA upregulation of several immune checkpoints (e.g. PDL1) has been described in CD34+ cells of myelodysplastic syndrome (MDS) patients compared to normal controls (Yang et al ., 2014), clinical investigations of checkpoint inhibitors in myeloid malignancies to-date have shown low response rates to monotherapy with a lack of response biomarkers. MDS is genetically heterogeneous evidenced by the array of somatic gene mutations that may impact differential checkpoint display. In concordance, our interrogation of the AML TCGA dataset identified TP53 mutant (MT) AML as an immunologically distinct subset with checkpoint upregulation. Therefore, our goal was to comprehensively characterize checkpoint molecules by lineage and stage of maturation in MDS to discern relationships with immune cell subsets in molecularly defined cohorts.Patients and Methods Utilizing the Moffitt Cancer Center tissue core and MDS database, bone marrow mononuclear cells were obtained from genetically profiled MDS (n=23), CMML (n=5) and secondary AML patients (n=22). NGS of up to 54 genes was performed at the time of sample acquisition with 5% VAF threshold and 500x minimum depth of coverage. Multi-color flow cytometry acquisition of immune checkpoints with a myeloid and lymphoid panel (Table 1) was performed using a BD LSRII, and analysis was conducted using FlowJo v10.3. Clinical variables and outcomes of patients were characterized at the time of sample procurement. Mann-Whitney and Kruskal-Wallis tests were used for comparative analyses. Median values of each checkpoint were calculated to compare outcomes of patients with increased versus decreased expression. Kaplan-Meier curves were used to estimate OS and analyzed from the date of sample collection. Multivariate Cox regression models were created to adjust for clinical characteristics.Results A total of 50 patients were analyzed (25 TP53 MT and wildtype (WT), respectively) with a median age of 72 years and male predominance (58%). Overall 38% of patients (n=19) were treatment naïve while 40% were HMA refractory (n=20). TP53 MT patients had significantly higher percentage of PDL1+ hematopoietic stem cells (HSC; Lin-/CD34+/CD38-,15.5% v 5.7%; P=0.008) with a non-significant increase in progenitor cells (HPC; Lin-/CD34+/CD38+, 15.4% v 9.2%; P=0.3). Additionally, PD1+/CD71+ erythroid progenitors (6.5% v 28.1%; P <0.0001)) and granulocytic myeloid derived suppressor cells (MDSC-G (Lin-/CD33+/ CD38-/CD11b+/CD14-); 10 v 23.7%; P=0.001) were significantly decreased in TP53 MT versus WT patients. The percentage of PDL1+ HSC or HPC were similar in HMA-naïve versus HMA-failure or patients actively on HMA (P=0.61 and P=0.84, respectively). More importantly, TP53 MT patients were enriched for an immune suppressive/anergic profile with increased % of TIM3+ MDSC-G (4% v 2%, P=0.04) and decreased % of OX40 cytotoxic T-cells (2.2 v 2.9%; P=0.006). As TP53 MT strongly predicts for inferior OS (P <0.0001), survival was similarly predictive with checkpoint alterations associated with TP53 MT.Although there was no difference in % of T-regulatory cells (T-regs; CD3+/CD4+/ CD25+/CD127dim/-) in TP53 MT versus WT patients (P=0.75), there was a trend for increased ICOS+ T-regs in TP53 MT patients (16.9% v 13%; P=0.09). Increased ICOS+ T-regs was associated with inferior OS in the total cohort (5.7 v 13.3 months, P=0.003)) and TP53 WT cohort (5.5 months vs NR, P=0.01)). In multivariable analysis incorporating age, BMT status, HMA failure, and revised international prognostic scoring system (IPSS-R) category, increased ICOS+ T-regs remained an independent co-variate for inferior OS in both the total cohort (HR 2.28, 95% CI 1.13 to 4.62; P= 0.02) and TP53 WT cohort (HR 4.69, 95% CI 1.33 to 16.56; P= 0.02)).Conclusion In patients with MDS and sAML, immune checkpoint expression is heterogeneous in HSCs and immune cell subsets. TP53 mutation portends an immunologically distinct, immune evasive phenotype that may be the predominant driver of their poor prognosis. Increased % of tumor infiltrating ICOS+ T-regs is highly predictive of outcome, independent of TP53 mutation status. We conclude that comprehensive immune checkpoint profiling may offer rationale for targeted immunomodulatory therapeutic strategies. [Display omitted] DisclosuresSallman:Celgene: Research Funding. Komrokji:Celgene: Honoraria; Novartis: Honoraria, Speakers Bureau. Lancet:Boehringer Ingelheim: Consultancy; Celgene: Consultancy; Bio-Path Holdings: Consultancy; Janssen: Consultancy; Pfizer: Other: Institutional research funding, Research Funding; Erytech: Consultancy; Jazz Pharmaceuticals: Consultancy; BioSight: Consultancy; Novartis: Consultancy. Padron:Incyte: Honoraria, Research Funding.

Impact of STK11 mutation on first-line immune checkpoint inhibitor (ICI) outcomes in a real-world KRAS G12C mutant lung adenocarcinoma cohort.

9106 Background: The introduction of KRAS G12C inhibitors into clinical trials has demonstrated promise and may provide a new therapeutic option for patients (pts) harboring KRAS G12C mutations. Recent data has also indicated that immune checkpoint inhibitors (ICI) have shown benefit in KRAS G12C mutant lung adenocarcinoma (LUAD); however, data on the impact of co-occurring STK11 mutations on outcomes are conflicting. We utilized the Guardant INFORM real-world clinical-genomic database to assess the impact of co-occurring STK11 mutations on outcomes in pts with KRAS G12C mutant LUAD treated with a first-line ICI containing regimen. Methods: This retrospective matched cohort observational study was conducted in a nationally representative clinical-genomic database covering over 137,000 pts with comprehensive ctDNA results and associated clinical information. Adult pts with metastatic LUAD who received ≥ 1 dose of first-line anti-PD1/PD-L1 ± chemotherapy and had at least 90 days follow-up after first KRAS G12C detection were included. A cohort of pts without KRAS G12C, including KRAS wildtype pts and pts with other KRAS mutations, were matched 3:1 for age, gender, year of index and baseline comorbidity. Time to next treatment (TTNT), time to discontinuation (TTD), real-world overall survival (rwOS) were compared with vs. without STK11 mutations for both cohorts using cox proportional-hazards model. Results: Among 330 pts in the KRAS G12C cohort, 21% (n = 70) had an STK11 mutation. Among the matched cohort (n = 938), 754 pts were KRAS wildtype, of whom 6% (n = 49) had STK11 mutations. Within the KRAS G12C cohort, pts with STK11 mutations had statistically significant shorter TTNT (hazard ratio [HR] 2.7, 95% confidence internal [CI] 1.8-4.0, p &lt; 0.0001), TTD (HR 1.4, 95% CI 1.0-2.0, p &lt; 0.04) and rwOS (HR 3.2, 95% CI 2.0-5.1, p &lt; 0.0001) than pts without STK11 mutations. Within the matched KRAS wildtype cohort, the differences in TTD (HR 1.4, 95% CI = 1.0-2.0, p = 0.08) and rwOS (HR 1.4, 95% CI = 0.8-2.4, p = 0.3) in patients with vs. without STK11 mutation did not reach statistical significance (Table). Conclusions: This study provides real-world evidence that STK11 co-mutations are associated with worse outcomes among pts with KRAS G12C mutant LUAD treated with first-line ICI. These inferior outcomes indicate a high unmet medical need among LUAD pts harboring co-occurring KRAS G12C and STK11 mutations and demonstrate the need for effective targeted and/or combination therapies in this patient population.[Table: see text]

MRI Radiomics Signature as a Potential Biomarker for Predicting KRAS Status in Locally Advanced Rectal Cancer Patients.

Background and PurposeLocally advanced rectal cancer (LARC) is a heterogeneous disease with little information about KRAS status and image features. The purpose of this study was to analyze the association between T2 magnetic resonance imaging (MRI) radiomics features and KRAS status in LARC patients.Material and MethodsEighty-three patients with KRAS status information and T2 MRI images between 2012.05 and 2019.09 were included. Least absolute shrinkage and selection operator (LASSO) regression was performed to assess the associations between features and gene status. The patients were divided 7:3 into training and validation sets. The C-index and the average area under the receiver operator characteristic curve (AUC) were used for performance evaluation.ResultsThe clinical characteristics of 83 patients in the KRAS mutant and wild-type cohorts were balanced. Forty-two (50.6%) patients had KRAS mutations, and 41 (49.4%) patients had wild-type KRAS. A total of 253 radiomics features were extracted from the T2-MRI images of LARC patients. One radiomic feature named X.LL_scaled_std, a standard deviation value of scaled wavelet-transformed low-pass channel filter, was selected from 253 features (P=0.019). The radiomics-based C-index values were 0.801 (95% CI: 0.772-0.830) and 0.703 (95% CI: 0.620-0.786) in the training and validation sets, respectively.ConclusionRadiomics features could differentiate KRAS status in LARC patients based on T2-MRI images. Further validation in a larger dataset is necessary in the future.

TRK Fusion Cancer: Patient Characteristics and Survival Analysis in the Real-World Setting.

BackgroundNeurotrophic tyrosine receptor kinase (NTRK) gene fusions are oncogenic drivers in various tumor types. While NTRK gene fusions are predictive of benefit from tropomyosin receptor kinase inhibitors regardless of tumor type, the prognostic significance of NTRK gene fusions in a pan-tumor setting remains unclear.ObjectiveThis study evaluated the characteristics and prognosis of tropomyosin receptor kinase fusion cancer in the real-world setting.Patients and MethodsThis retrospective study used a de-identified clinico-genomic database and included patients with cancer who had comprehensive genomic profiling between January 2011 and July 2018. Patients were classified as having cancer with NTRK gene fusions or NTRK wild-type genes. Patients were matched with a 1:4 ratio (NTRK fusion:NTRK wild-type) using the Mahalanobis distance method on demographic and clinical characteristics, including age and Eastern Cooperative Oncology Group performance status. Descriptive analysis of clinical and molecular characteristics was conducted. Kaplan–Meier estimator and Cox regression were used for overall survival analysis.ResultsMedian overall survival was 12.5 months (95% confidence interval 9.5–not estimable) and 16.5 months (95% confidence interval 12.5–22.5) in the NTRK gene fusion (n = 27) and NTRK wild-type cohorts (n = 107), respectively (hazard ratio 1.44; 95% confidence interval 0.61–3.37; p = 0.648). Co-occurrence of select targetable biomarkers including ALK, BRAF, ERBB2, EGFR, ROS1, and KRAS was lower in cancers with NTRK gene fusions than in NTRK wild-type cancers.ConclusionsAlthough the hazard ratio for overall survival suggested a higher risk of death for patients with NTRK gene fusions, the difference was not statistically significant. Co-occurrence of NTRK gene fusions and other actionable biomarkers was uncommon.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11523-021-00815-4.

Impact of splicing mutations in acute myeloid leukemia treated with hypomethylating agents combined with venetoclax

AbstractSpliceosome mutations (SRSF2, SF3B1, U2AF1, ZRSR2), are encountered in ∼50% of secondary acute myeloid leukemia cases (sAML) and define a molecular subgroup with outcomes similar to sAML in de novo AML patients treated with intensive chemotherapy. Outcomes in patients with spliceosome mutations treated with hypomethylating agents in combination with venetoclax (HMA+VEN) remains unknown. The primary objective was to compare outcomes in patients with spliceosome mutations vs wild-type patients treated with HMA+VEN. Secondary objectives included analysis of the mutational landscape of the spliceosome cohort and assessing the impact of co-occurring mutations. We performed a retrospective cohort analysis of patients treated with HMA+VEN–based regimens at The University of Texas MD Anderson Cancer Center. A total of 119 patients (spliceosome mutated n = 39 [SRSF2, n = 24; SF3B1, n = 8; U2AF1, n = 7]; wild-type, n = 80) were included. Similar responses were observed between spliceosome and wild-type cohorts for composite complete response (CRc; 79% vs 75%, P = .65), and measurable residual disease–negative CRc (48% vs 60%, P = .34). Median overall survival for spliceosome vs wild-type patients was 35 vs 14 months (P = .58), and was not reached; 35 months and 8 months for patients with SRSF2, SF3B1, and U2AF1 mutations, respectively. IDH2 mutations were enriched in patients with SRSF2 mutations and associated with favorable outcomes (1- and 2-year overall survival [OS] of 100% and 88%). RAS mutations were enriched in patients with U2AF1 mutations and associated with inferior outcomes (median OS, 8 months). Comparable outcomes were observed between patients with vs without spliceosome mutations treated with HMA+VEN regimens, with specific co-mutation pairs demonstrating favorable outcomes.