High Mutational Load Research Articles

TERT Promoter Mutations and Telomerase in Melanoma.

Malignant melanoma is an extremely malignant tumor with a high mortality rate and an increasing incidence with a high mutation load. The frequency of mutations in the TERT promoter exceeds the frequency of any known noncoding mutations in melanoma. A growing number of recent studies suggest that the most common mutations in the TERT promoter (ATG start site −124C>T and −146C>T) are associated with increased TERT mRNA expression, telomerase activity, telomere length, and poor prognosis. Recently, it has been shown that TERT promoter mutations are more correlated with the occurrence, development, invasion, and metastasis of melanoma, as well as emerging approaches such as the therapeutic potential of chemical inhibition of TERT promoter mutations, direct telomerase inhibitors, combined targeted therapy, and immunotherapies. In this review, we describe the latest advances in the role of TERT promoter mutations and telomerase in promoting the occurrence, development, and poor prognosis of melanoma and discuss the clinical significance of the TERT promoter and telomerase in the treatment of melanoma.

Immune Checkpoint Blockade Therapy in Patients With Colorectal Cancer Harboring Microsatellite Instability/Mismatch Repair Deficiency in 2022.

Immune checkpoint inhibitors (ICIs) are shown to be effective among patients with metastatic colorectal cancer (mCRC) harboring high microsatellite instability (MSI-H) and/or mismatch repair deficiency (dMMR), with U.S. Food and Drug Administration approvals for all lines of therapy. In Europe, only pembrolizumab in the first line and the combination of nivolumab and ipilimumab beyond the first line are approved. Many questions remain about the clinical management of MSI-H/dMMR CRC. Biomarkers predictive of immune checkpoint inhibitor resistance among MSI-H/dMMR tumors are needed (1) to select the best treatment for patients with CRC (anti-PD-[L]1 monotherapy alone or combined with anti-CTLA-4 or chemotherapy) and (2) to develop new treatment strategies for patients whose disease progressed after immune checkpoint inhibitor monotherapy. The development of immune checkpoint inhibitors in the adjuvant and neoadjuvant settings is also of great interest for patients harboring MSI-H/dMMR, especially as a substantial proportion have Lynch syndrome or are at high risk of developing cancers in their lifetime and sporadic MSI-H/dMMR cancers occur most frequently in elderly and frail patients. Thus, CRC is not one, but two different diseases: (1) MSI-H/dMMR CRC (seen in 5% of mCRC and 15% of non-mCRC), which is genetically unstable with a high mutational load and many neoantigens, and for which immune checkpoint inhibitors radically changed clinical management, and (2) microsatellite stable CRC with chromosomal instability, for which immune checkpoint inhibitors are not proven efficient.

Иммунотерапия в комбинации с лучевой терапией при лечении пациентов с плоскоклеточным раком кожи

Squamous cell skin cancer (SCC) is the second most common malignant neoplasm. The incidence of SCC continues to grow. As a rule, the tumor is characterized by a benign clinical course and is cured surgically, but in some cases, the development of locally common (and/or metastatic) forms is observed. For a long period of time, there were no methods of drug therapy of patients with such forms of SCC that would lead not only to a local effect, but also to an increase in life expectancy. In most cases, SCC is characterized by a high mutational load, the presence of PD-L1 expression, which potentially suggests a possible sensitivity of the tumor to immunotherapy. This knowledge contributed to the conduct of clinical studies of anti-PD-1 immunotherapy in locally common inoperable and/or metastatic forms of SCC. In the USA, in 2018, the first drug for the treatment of patients with locally advanced inoperable and/or metastatic form of SCC, cemiplimab, was approved for the first time. In 2020 another anti-PD-1 drug, pembrolizumab, was approved, which allowed expanding the possibilities of drug therapy for this category of patients. Despite the high frequency of objective responses against the background of anti-PD-1 immunotherapy in the first line (about 50%), a number of patients have stabilization of the disease or progression of the process. Stabilization of the disease is not enough for most patients, taking into account the peculiarities of clinical manifestations characteristic of SCC, which requires the search for new approaches and the use of immunotherapy with other treatment options to improve immediate and long-term results. The article presents a clinical case of a patient with locally advanced right shin SCC who received anti-PD-1 immunotherapy (nivolumab) in combination with radiation therapy as part of first-line therapy. On the background of such therapy, it was possible to achieve some success.

Abstract 5767: Landscape of concomitant driver mutations in EGFR-mutated NSCLCs

Abstract Background While most oncogenic driver mutations are mutually exclusive, next-generation sequencing (NGS) has led to the identification of concomitant driver alterations in non-small cell lung cancer (NSCLC). However, the landscape of concomitant drivers and the clinical relevance remain to be explored. Methods: We profiled concomitant driver alterations, including EGFR, KRAS, ALK, RET, ROS1, MET, BRAF and ERBB2 from baseline tumor tissues of 22,610 NSCLC patients by using targeted NGS. The associated genomic and clinical features were analyzed and validated in a TCGA dataset of 136 EGFR+ NSCLC patients. Results: The overall frequency of concomitant driver mutations in EGFR+ patients was 3.09% (334/10,801). EGFR activating mutations most commonly co-occurred with KRAS mutations (2.42%), followed by ERBB2 (1.09%), MET (0.75%) and BRAF (0.15%). A total of six EGFR+ patients were identified with co-occurring ALK (n=2), RET (n=1), or ROS1 (n=3) fusions. Patients with dual EGFR/KRAS mutations were more likely to carry the p.G13D (6.1% vs. 2.9%, P<.05), p.G12S (6.1% vs. 2.3%, P<.01), p.A146T (8.3% vs. 0.9%, P<.0001), p.A146V (3.9% vs. 0.5%, P<.001)variants in KRAS, whereas those with single KRAS were enriched with p.G12C (15.0% vs. 33.0%, P<.0001) and p.G12A (3.9% vs. 8.5%, P<.05) variants. Patients with dual EGFR/MET alterations had a higher frequency of MET amplification (71.4% vs. 43.3%) and lower exon 14 skipping mutations (28.6% vs. 56.7%, P=.0001) than MET single drivers. Patients with dual EGFR/ERBB2 alterations had higher ERBB2 amplification (40.7% vs. 16.5%, P<.0001) and p.S310F/Y mutations (44.4% vs. 4.3%, P<.0001), and lower ERBB2 exon 20 insertions (9.9%, vs. 70.6%, P<.0001). In addition, dual drivers had higher frequencies of TP53 (P<.01), RB1 (P<.05), ARID2 (P<.05), NTRK1 (P<.01), TSC2 (P<.001), and BRIP1 (P<.01) alterations than single drivers. At the genomic level, dual drivers had higher mutational load than those with only EGFR mutations (median 4 vs. 2 muts, P<.01). In 75% of dual drivers, EGFR mutant allele frequency (MAF) was higher than that of the other concomitant driver. In patients with sufficient clinical follow-up, dual drivers were associated with unfavorable PFS outcomes compared with single drivers (HR=2.87, P=.04) following first-line first-generation EGFR TKIs, which was mainly attributed to the presence of MET amplification or PIK3CA mutations. In addition, in dual EGFR/KRAS and EGFR/BRAF drivers, a higher MAF of EGFR than that of the respective co-occurring driver was associated with a trend towards prolonged PFS (11.0 vs. 8.1m, HR= 0.21, P=.15), likely suggesting clonal advantage of EGFR sensitizing mutations in response to first-generation TKIs. Conclusion: In our large cohort study, we present the landscape of EGFR+ dual drivers and their associated clinical and genomic features and survival outcomes, which could aid in therapeutic decisions and facilitate the development of combination therapies. Citation Format: Xiaonan Wang, Junli Zhang, Chuqiao Liang, Jiani C. Yin, Yang Shao. Landscape of concomitant driver mutations in EGFR-mutated NSCLCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5767.

Abstract 2148: Pre-clinical models recapitulating the spectrum of response of BRCA associated pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease, with the majority of patients diagnosed at an advanced stage. Comprehensive genomic analysis identified the homologous recombination deficiency (HRD) subgroup which is predominantly enriched in patients harboring germline BRCA1/2 mutations (glBRCA) and presents ~7% and up to 15% in selected high-risk populations. Tumors with HRD are susceptible to DNA-damaging agents and PARP inhibition. However, not all patients demonstrate a similar response, and a spectrum is observed. We analyzed the clinical outcome of ninety-one glBRCA PDAC patients treated at Sheba Medical Center. We identify three main subgroups of response spanning from ~25% patients refractory to first line platinum agents and ~9% patients with durable long-term response with no evidence of disease for over three years. The majority of the patients display a prolonged response to platinum and PARPi maintenance (>24 months) followed by acquired resistance. We generated patient derived xenograft (PDX) models and an ex-vivo culture system (EVOC) from naïve to treatment tissue and at clinical resistance. In vivo efficacy to platinum agents and PARPi demonstrates that these models recapitulate the specific clinical spectrum of response. We demonstrate the utility of both pre-clinical models in their ability to predict response to platinum-based agents and PARPi, with EVOC having a potential to assist in medical decisions while treating the patient. Whole genome sequencing of the tumors revealed that BRCA monoallelic tumors are associated with innate resistance while biallelic tumors reflect sensitivity to platinum agents and PARPi. In the acquired resistance models, the main identified mechanism of resistance was due to reversion mutations in 4/6 of samples. Whole genome analysis demonstrates high mutational and neo-antigen load in majority of the glBRCA tumors. In a preliminary study, we show the efficacy of anti PD1 in a novel humanized glBRCA PDAC PDX model. This extensive preclinical and clinical collection of BRCA associated PDAC, enables further understanding and investigation of this unique subtype in aim to develop alternative treatments to overcome resistance. Citation Format: Talia Golan, Chani Stossel, Maria Raitses-Gurevitch, Dikla Atias, Tamar Beller, Steven Gallinger, Raanan Berger. Pre-clinical models recapitulating the spectrum of response of BRCA associated pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2148.

Abstract 5742: The landscape of genetic alteration in Chinese lung adenosquamous carcinoma patients

Abstract Background: Lung adenosquamous carcinoma (LASC) is a mixed histologic component tumor type that contains both squamous cell carcinoma and adenocarcinoma, with each component accounting for at least 10% of tumors. The incidence of LASC is very low, only comprising 0.4-4% of all lung cancers. LASC yields a more aggressive clinical course, and its prognosis is generally worse than that of adenocarcinoma and squamous cell carcinoma of the lung. Due to the rarity of LASC, currently, there is no standard treatment. Also, to date, limited genomic data have been performed. In this study, we analyzed the genomic alterations and clinical signatures of LASC. Materials and Methods: Formalin-fixed, paraffin-embedded (FFPE) tumor tissues and matched blood samples were collected for a targeted, next-generation sequencing (NGS) assay at OrigiMed (Shanghai, China), a College of American Pathologists (CAP) accredited and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. Genomic alterations including single base substitution, indel, copy number variations, and gene fusion and rearrangement were assessed. Results: A total of 124 LASC patients were enrolled in our study, including 77 (62.1%) males and 47 (37.9%) females, with a median age of 61 years old (range 27-82). Forty-three (43, 34.68%) patients had a smoking history and half of patients possessed a Stage III/IV disease. The most commonly altered genes were TP53 (66.9%), EGFR (54.8%), CDKN2A (21%), TERT (21%), and LRP1B (18.5%). Surprisingly, 91.1% and 52.4% patients harbored at least one druggable alteration based on the solid cancer and NSCLC NCCN guides, respectively. The most frequent druggable mutation, EGFR, is comprised of 35 (51.47%) single mutations and 33 (48.53%) complexed mutations, with most containing a 19 exon deletion and EGFR amplification (12%). The median tumor mutational burden (TMB) of patients with LASC was 5.15 muts/Mb (range, 0-69.3 muts/Mb), and 33 (26.6%) patients were TMB-H (TMB ≥ 10 Muts/Mb). Patients with an ARID2, BRCA1, or KEAP1 mutation had a significantly higher value of TMB than wild type (median TMB 16.1 vs. 5 muts/Mb, 29.7 vs. 5 muts/Mb, 14.6 vs. 4.85 muts/Mb, respectively, P < 0.05). Patients with HRR or DDR mutations also displayed a higher TMB value than for wild type. We additionally determined that mutation signature 1 (the clock-like signature), signature 2 (the APOBEC signature), and signature 3 (the BRCA mutational signature) exhibited a higher mutational load for the entire cohort. All patients with signature 2 or 3 displayed a HLA-B heterozygous subtype. Conclusions: Our study revealed genomic profiling for the largest sample size of Chinese LASC patients to date. Profiling obtained from this study demonstrates that LASC patients have the same chance for targeted therapy and immune checkpoint inhibitors as compared to lung adenocarcinoma and lung squamous cell carcinoma, respectively. Citation Format: Hongbiao Wang, Yingcheng Lin, Jun Liu, Yuyin Cai, Xiaofang Qi, Lujia Huang. The landscape of genetic alteration in Chinese lung adenosquamous carcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5742.

Which angiosarcoma subtypes may benefit from immunotherapy?

11572 Background: Angiosarcomas (AS) are aggressive mesenchymal tumors arising from cells with endothelial properties. They include de novo primary AS (pAS), and secondary AS (sAS) due to prior radiotherapy, UV exposure or chronic lymphedema. Treatment options are limited and their prognosis is poor. Development of new treatment strategies is difficult due to the heterogeneity and rarity of AS. We hypothesize that immunological and genomic profiles are significantly different between pAS and sAS and may result in different immune checkpoint inhibition (ICI) based treatment strategies. Methods: Tumor samples from AS patients were retrospectively collected. Patients were categorized as pAS or sAS. Lymphocytes were analyzed using multiplex immunohistochemistry on tissue microarrays. Genomic profiling was performed in a selected subgroup with “TruSight Oncology 500”, a Next Generation Sequencing panel containing 523 cancer related genes. Results: Immunological data were analyzed from 257 AS patients. The cohort comprised 80 pAS patients and 177 sAS patients. The median density of CD3+ T cells was 250 cells/mm2 in pAS vs 452 cells/mm2 in sAS (p< 0.001). Median CD4+ T helper cell density was 128 cells/mm2 in pAS vs 246 cells/mm2 in secondary AS (p< 0.001). The median density of CD8+ cytotoxic T cells was 85 cells/mm2 in pAS vs 111 cells/mm2 in sAS ( p= 0.057). Density of FoxP3+ T regulatory cells was higher in sAS (median 42 cells/mm2) compared to pAS (median 23 cells/mm2) (p< 0.001). The median count of CD20+ B cells in pAS was 24 cells/mm2 compared to 32 cells/mm2 in sAS ( p= 0.533). Genomic analysis was performed on tumor DNA from 51 patients (25 pAS and 26 sAS). Median tumor mutational burden (TMB) was 3.2 (range 0.8-11.9) mutations per megabase (mut/Mb) in pAS vs 3.9 (range 0.0-99.6) in sAS ( p= 0.485). No microsatellite instability was detected. A pathogenic mutation, gene amplification or gene loss was identified in 82% of all patients (n = 42, 70% of pAS vs 100% of sAS ( p< 0.01)). In 36 patients (71%) at least one (likely) pathogenic mutation was detected (54% pAS vs 88% sAS, ( p= 0.013)). In 20 patients (39%) mutations in the DNA damage response (DDR) pathway were detected (12% pAS vs 68% sAS ( p< 0.01)). The most frequently found mutated genes were TP53 (10%), BRAF (6%), ERCC4 (6%), PTPRD (6%), WETD2 (6%) and SETD2 (6%). Amplifications were found in 49% (n = 25) of all patients (15% pAS vs 84% sAS, ( p< 0,01)). MYC amplifications were most common and were detected in 15% of pAS and 68% of sAS. Immune profiles of the 51 genomically characterized patients are currently under further investigation. Conclusions: We showed a clear distinction in immunological and genomic profiles between pAS and sAS. The potential benefit of ICI seems to be most promising in sAS with a T cell inflamed tumor microenvironment, frequent MYC amplifications, DDR mutations, and high mutational load, while in pAS boosting strategies to enhance susceptibility to ICI might be interesting for further investigation.

Phase I results of the INFORM2 combination study of nivolumab and entinostat in children and adolescents: INFORM2 NivEnt.

10034 Background: Pediatric patients with relapsed or refractory high-risk solid and CNS tumors have dismal survival. To date treatment with immune checkpoint inhibitors in this population has been disappointing. This study exploits the immune enhancing effects of entinostat on nivolumab in biomarker enriched subpopulations. The study aims to determine the pediatric recommended phase II dose (pRP2D) and to evaluate activity and safety. Methods: This is an exploratory non-randomized, open-label, multinational seamless phase I/II trial in children and adolescents with relapsed / refractory or progressive high-risk solid and CNS tumors. The phase I is divided in 2 age cohorts: 12–21 years (y) and 6–11y and follows a 3 + 3 design with two dose levels for entinostat (dose level 1: 2 mg/m2 and dose level 2: 4 mg/m2 once per week) and fixed dose nivolumab (3 mg/kg every 2 weeks). Patients entering the trial on pRP2D can seamlessly enter phase II which consists of a biomarker defined four group basket trial: high mutational load (group A), high PD-L1 mRNA expression (group B), focal MYC(N) amplification (group C), low mutational load and low PD-L1 mRNA expression and no MYC(N) amplification (group D). Results: The first patient was enrolled in May 2020 and at the time of the data cut (21-JAN-2022), 19 patients were treated. The median age at enrollment was 14 y. In the 12 – 21y cohort 15 patients were enrolled and four patients in the 6 – 11y cohort. The most frequent treatment-related AEs to date were thrombocytopenia in six (32%), nausea and vomiting both in four (21%), and neutropenia in three patients (16%). Five patients (26%) experienced grade 3/4 mostly reversible treatment-related AEs, e.g. neutropenia/leukopenia. No treatment related deaths were reported. In the 6 – 11y cohort dose escalation is ongoing. In the 12 – 21y cohort, one DLT (CTCAE grade 3 thrombocytopenia) was observed in six patients on dose level two, which was determined as the pRP2D of the combination. At the time of the data cut, 10 patients (six in arm D and four patients in which the biomarker group was not yet determined) had received at least one RECIST/RANO response evaluation by central review in phase II. One patient (17%) in arm D with metastatic relapsed renal cell carcinoma (RCC) harboring a typical PRCC-TFE3 fusion showed a PR after two cycles and finally achieved an ongoing CR. Extensive explorative analyses of immune signatures derived from INFORM RNA-Seq and WES data revealed that both the primary diagnosis and the current relapse samples harbored a remarkable high immune cell infiltration, especially CD8+ T-cells. Conclusions: The first and ongoing global INFORM2 trial has identified the pRP2D for the nivolumab and entinostat combination in the older age cohort with good tolerability. A patient with metastasized relapsed RCC experienced a CR. The role of immune infiltration as a potential predictive biomarker is currently being explored. Clinical trial information: NCT03838042.

Establishment of a novel BRCAness score that predicts response to PARP inhibitors.

549 Background: BRCAness is a generic term used to describe characteristic features of homologous recombination deficiency (HRD) mimicking mutations in BRCA genes. Although clinical genetic testing has increased the detection of mutations in BRCA1 and BRCA2, we hypothesized that a measure to quantify BRCAness will identify the responders to PARP inhibitors that cause synthetic lethality in BRCA mutation tumors. Methods: The BRCAness score was established by using gene set variation analysis (GSVA) algorithm on 34 BRCA-mutation related genes. We investigated the clinical relevance of the score by performing silico analyses of 6245 breast cancer patients using multiple independent large cohorts in this study. Results: A score to quantify BRCAness was generated using gene set variation analysis algorithm on 34 BRCA1-mutation related genes selected by high AUC levels in ROC curve between BRCA1 mutation and wildtype breast cancer. The score was significantly associated with BRCA1 mutation, high overall mutation load and intratumoral heterogeneity as well as high HRD, DNA repair and MKI67 expression regardless of mutation in BRCA gene. High score tumor enriched not only DNA repair, but also five cell proliferation-related gene sets (E2F targets, G2M checkpoint, MYC targets v1 and v2, and MITOTIC signaling) in Hallmark collection (all false discovery rate < 0.10). Breast cancers with high score were significantly associated with higher infiltration of anti-cancerous immune cells and higher cytolytic activity. Not all breast cancer cell lines with BRCA-mutation showed high score and the other cells in human breast cancer tumor microenvironment were contributing to the score. We found that the BRCAness score was the highest in triple-negative among subtypes consistently in all cohorts (all p < 0.001). Finally, BRCAness was associated with response to chemotherapy and correlated strongly with response to PARP inhibitor in both triple-negative (AUC = 0.815) and ER-positive/HER2-negative breast cancer (AUC = 0.715). Conclusions: We established a novel BRCAness score using mRNA expression of BRCA-mutation-related genes and found that it associates with DNA repair and response to PARP inhibitor regardless of BRCA mutation.

Neoadjuvant PD-1 blockade in patients with resectable desmoplastic melanoma (SWOG 1512).

9502 Background: Desmoplastic melanoma (DM) is a rare cancer defined by a dense fibrous collagen matrix. It is associated with high UV exposure leading to a high mutational load. When locally advanced, the standard of care is wide excision and radiation therapy due to its propensity for local relapses. Metastatic DM has a high response rate (RR) to PD-1 blockade therapy (Eroglu et al. Nature 2018). We hypothesized that neoadjuvant treatment with anti-PD-1 monotherapy may induce pathologically confirmed regressions in a high percentage of cases, potentially allowing for less extensive local treatment. Methods: Patients > 18 years old with histologically confirmed resectable (primary, recurrent, or regional lymph node metastasis) DM with clinical evidence of residual disease received pembrolizumab 200 mg q3 weeks (wk) 3 followed by excision. No adjuvant therapy was administered. Primary endpoint: pathological complete response (pCR), with the assumption that pCR of 25% would be considered a positive result worthy of future study. To test this hypothesis, a single arm trial with 25 eligible patients would have a 3.4% probability of a positive result with a true pCR of 5%, and a power of 90% of a positive result if the true pCR is 25%. Secondary endpoints: clinical RR by imaging and clinical exam, median overall survival (OS), and evaluation of safety/tolerability of neoadjuvant pembrolizumab. Adverse events were assessed q3 wk. Disease assessments occurred at baseline and q9 wk. NCT02775851. Results: We enrolled-29 eligible patients with resectable DM. One patient refused treatment and was omitted from further analysis. Median age was 75, 79% were male, primary sites of disease were 72% H&N, 10% torso, 14% extremities, 3% unknown. No patients received prior systemic therapy. Mean time from C1D1 treatment to surgery was 84.2 (range: 52-135) days, mean number of cycles received 3.3 (range: 2-4). 26/27 (93%) of patients underwent wide excision of the resectable disease, of which 14 (54%) underwent sentinel lymph node biopsy. One patient underwent resection of a nodal recurrence thus did not require wide excision. pCR was noted in 15/27 (56%) of patients (95% CI: 35%-75%). One patient without a pCR had a major pathologic response with 0.2 mm residual melanoma. In addition, one patient with a clinical CR did not undergo resection by choice. None became inoperable. Clinical RR was 52% (95% CI: 32%-71%). Median OS has not been reached, with two nontreatment related deaths (acute hypoxic respiratory failure; unknown). No > grade 2 related adverse events were observed. Conclusions: Neoadjuvant pembrolizumab in resectable DM results in a high pCR rate with excellent tolerance, which supports consideration of PD-1 blockade therapy prior to surgery. Funding: U10CA180888 and U10CA180819; and in part by Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA. Clinical trial information: NCT02775851.

FAT1 mutations in colorectal cancer patients are associated with the therapeutic benefit of immunotherapy.

e15523 Background: Immunotherapy is one of the main treatment modalities for advanced colorectal cancer. Although microsatellite instability (MSI) is widely applied as an effective immune efficacy prediction biomarker in clinical practice, the majority of the colorectal patients are microsatellite stable (MSS). This study is designed to identify new biomarkers for immune efficacy in colorectal cancer. Methods: Targeted sequencing with a 425 gene panel was performed with the tumor tissues from a total of 161 colorectal cancer (CRC) patients (discovery cohort) for mutation analysis. The association between FAT1 mutations and immunotherapy markers MSI and TMB was investigated using an external database of 727 CRC patients from cBioportal (validation cohort). Results: The discovery cohort consisted of 95(59%) males and 66(41%) females with a median age of 59 years old. There were 2(1.2%) stage II, 5(3.1%) stage III, 24(14.9%) stage IV, and 130(80.8%) unknown stage patients. The majority of the discovery cohort was MSS (154, 95.7%), which was similar to the validation cohort (518, 71.2%). FAT1 mutations were present around similar ratios in both discovery cohort (12.4%) and validation cohort (11.6%). In both cohorts, the co-occurrence analysis revealed that MSI events were often co-occurred with FAT1 mutations and patients with FAT1 mutations had a higher mutational load compared to the FAT1 wild-type patients. Analysis with survival data from the validation cohort showed that OS was significantly higher in patients with FAT1-mutated tumors than in ones with FAT1 wild-type. Compared to the FAT1 wild-type tumors, the higher infiltration rate of immune cell identified in the colorectal tumors with FAT1 mutation may also explain the good immune efficacy of patients with FAT1 mutation. Conclusions: Colorectal cancer patients with FAT1 mutations may benefit from immunotherapy. The increased immunogenicity in patients with FAT1 mutations may be due to increased immune cell infiltration.

Relationship between lifespan and somatic mutation in D. melanogaster after treatment with chlorophyllin

Sodium copper chlorophyllin (SCC) has a genetic damage inhibitory capacity due to its antioxidant action. For this reason, it was considered to investigate its role in the life span of Drosophila melanogaster and its relationship with the frequency of somatic mutation induced by gamma rays. Results indicated that SCC alone prolonged the lifespan only in females, but in combination with 20 Gy of gamma rays, the aging delay in both sexes was significant. In addition to confirming that the porphyrin reduces the frequency of mutation, the individuals with the highest mutation load are the individuals who die more quickly, and once they are eliminated, the survivor individuals treated with 20 Gy or with SCC + 20 Gy, died at the same rate. The results together indicate that SCC not only inhibits induced genetic damage, but it also has beneficial effects that probably cause an aging delay of the treated population that need to be investigated.

Nonsense-mediated RNA decay: an emerging modulator of malignancy.

Nonsense-mediated RNA decay (NMD) is a highly conserved RNA turnover pathway that selectively degrades RNAs harbouring truncating mutations that prematurely terminate translation, including nonsense, frameshift and some splice-site mutations. Recent studies show that NMD shapes the mutational landscape of tumours by selecting for mutations that tend to downregulate the expression of tumour suppressor genes but not oncogenes. This suggests that NMD can benefit tumours, a notion further supported by the finding that mRNAs encoding immunogenic neoantigen peptides are typically targeted for decay by NMD. Together, this raises the possibility that NMD-inhibitory therapy could be of therapeutic benefit against many tumour types, including those with a high load of neoantigen-generating mutations. Complicating this scenario is the evidence that NMD can also be detrimental for many tumour types, and consequently tumours often have perturbed NMD. NMD may suppress tumour generation and progression by degrading subsets of specific normal mRNAs, including those encoding stress-response proteins, signalling factors and other proteins beneficial for tumours, as well as pro-tumour non-coding RNAs. Together, these findings suggest that NMD-modulatory therapy has the potential to provide widespread therapeutic benefit against diverse tumour types. However, whether NMD should be stimulated or repressed requires careful analysis of the tumour to be treated.

Abstract PR012: Genetic and immune landscape evolution defines subtypes of MMR deficient colorectal cancer

Abstract Mismatch repair deficient (MMRd) CRCs have high mutation/neo-antigen loads, leading to high immunogenicity and good immunotherapy response rates. We reasoned that the MMRd hypermutator phenotype should also promote intratumor heterogeneity (ITH) and evolvability; and investigated the genetic and immunological co-evolution in 69 regions from 6 localized and 13 metastatic MMRd CRCs by multi-region DNA-, RNA-sequencing and immunohistochemistry. All tumors had high truncal mutation loads (median: 44 mutations in 191 sequenced genes; whole exome equivalent: 1870 mutations). A median of 16.1% mutations per region were heterogeneous, indicating pervasive ITH. Phylogenetic analyses showed that metastases had diverged before subclonal diversification of the primary tumor in 75% of assessable cases. Thus, the ability to metastasize was frequently acquired early during tumor evolution. Driver aberrations evolved with a clear hierarchy: those in the WNT and RTK-MAPK pathways and in TGFbR-family members were almost always truncal (87.0%, 86.4% and 83.7%), indicating a critical role for cancer initiation. In contrast, genetic aberrations that are known to confer immune evasion (IE) were predominantly subclonal (71.4%) and parallel evolution of IE drivers occurred in 4/6 tumors that harboured any subclonal IE driver. This substantiates immune selection pressure as the main driver of Darwinian evolution during tumor progression. These IE drivers are known to confer resistance to checkpoint-inhibitor immunotherapy. ITH therefore needs to be addressed for predictive biomarker development. We quantified CD8 T-cell infiltrates as a surrogate measure of tumor immunogenicity; distinguishing tumors with low CD8 T-cell infiltrates (mean: 3.9% T-cells of all nucleated cells) and those with high infiltrates (mean: 12.2%). T-cell infiltrates showed high ITH in the latter group. This suggested a tumor-intrinsic setpoint, accompanied by marked variability in tumors with dense infiltrates. T-cell densities did not correlate with truncal mutation loads or heterogeneity metrics, questioning how immunogenicity is regulated. Phylogenetic analysis defined three patterns of IE evolution: tumors with subclonal, with pan-tumor, or without any identifiable IE drivers. CD8 T-cell abundance was highest in tumors with subclonal IE, supporting selection pressure from high CD8 T-cell infiltrates as the proximate cause for IE evolution. Tumors with pan-tumor IE showed low CD8 T-cell infiltrates. Surprisingly, tumors without IE drivers had the lowest CD8 T-cell abundance, indicating an alternative mechanism of immune escape. Low densities of CD8 T-cells at the tumor margin and low expression of T-cell chemo-attractants suggested impaired T-cell recruitment in these. Together, we show that immune recognition is a major driver of Darwinian evolution in MMRd CRCs and that immune infiltrates and IE drivers co-evolve interdependently. Whether sensitivity to checkpoint-inhibitor immunotherapy differs between the three phylogenetic MMRd CRC subtypes needs to be assessed in clinical trials. Citation Format: Benjamin R. Challoner, Andrew Woolston, David Lau, Marta Buzzetti, Louise J. Barber, Tom Lund, Harold B. Sansano, Katharina von Loga, Héctor Lázare-Iglesias, Ruwaida Begum, Richard Crux, David Cunningham, Ian Chau, Naureen Starling, Juan Ruiz-Bañobre, Tony Dhillon, Marco Gerlinger. Genetic and immune landscape evolution defines subtypes of MMR deficient colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr PR012.

47P Immune tumor microenvironment and markers of apoptosis in breast cancer in carriers of hereditary mutations in the BRCA1 gene

Tumor immune microenvironment is currently recognized as an important participant in carcinogenesis, influencing the behavior of tumor cells and treatment efficacy. It is suggested that defects in BRCA1/2 genes contribute to a high mutation load and high immunogenicity, which modulates immune microenvironment. At the same time, it was shown that BRCA1/2-associated breast cancer tumors do not belong to the category of immunoactive ones. In addition, there is evidence of the additional evaluation of TP53 mutation in these tumors and disruption of the cell death process, which can also be a factor of resistance to therapy. 40 patients with breast cancer with BRCA1 mutations 185delAG, 4153delA, 5382insC, 3819DELGTAA, 3875delGTCT, 300T>G, 2080delA and BRCA2 (6174delT)) determined by real-time polymerase chain reaction (PCR) were included. Immunohistochemical study was performed using monoclonal antibodies to Ki67, p53, CD8, CD4, CD68, CD163, BCL2. In the presented study patients with BRCA1-associated breast cancer, had high low tumor CD4/CD8 ratio in 50% of cases, which characterizes an immunosuppressive microenvironment. Mutation BRCA1 5382insC is associated with high level of CD4+ T-lymphocytes (p˂0.05), G3 is associated with a low CD4/CD8 ratio (p=0.056) and a low levels of CD68+M1, CD163+M2-macrophages (p=0,022, p=0.002); T1 correlates with high levels of CD8+ TILs (p=0.038) and high levels of CD163 (p=0.033). High Ki-67 is associated with a lack of BCL2 expression (p=0.04). Negative expression of BCL2 occurred in 52.5% of cases. A high level of p53 expression is described as the main type of expression in these tumors, suggesting a combination of TB53 and BRCA1 mutations and a violation of the mechanism of cell death in these patients. The immunosuppressive type of microenvironment prevails in the tumors of breast cancer patients with hereditary mutations in the BRCA1 gene, where a violation of the mechanism of cell death in BRCA1-deficient breast cancer was revealed. The main directions of future therapy of these tumors may be modification of the immune microenvironment and activation of the mechanisms of cell death.

The complexity of cancer immunotherapy illustrated through skin tumors.

Skin tumours are among the cancer types most sensitive to immunotherapy, due to their unique immunogenic features including skin-associated lymphoid tissue, high mutational load, overexpression of tumour antigens, and high frequency of viral antigens. Despite this high immunotherapy response rate, however, ultimately most skin tumours develop similar treatment resistance to most other malignant tumours, which highlights the need for in-depth study of mechanisms of response and resistance to immunotherapy. A bibliographic review of the most recent publications regarding currently in use and emerging biomarkers on skin tumors has been done. Predictive biomarkers of treatment response, biomarkers that warn of possible resistance, and emerging markers, the majority of a systemic nature, are described. Including factors affecting not only genomics, but also the immune system, nervous system, microbiota, tumour microenvironment, metabolism and stress. For accurate diagnosis of tumour type, knowledge of its functional mechanisms and selection of a comprehensive therapeutic protocol, this inclusive view of biology, health and disease is fundamental. This field of study could also become a valuable source of practical information applicable to other areas of oncology and immunotherapy.

Genetic and Clinical Characteristics of ARID1A Mutated Melanoma Reveal High Tumor Mutational Load without Implications on Patient Survival.

Simple SummaryMelanoma is a highly malignant skin cancer with the highest mortality of all cutaneous tumors. Relevant genetic events have been identified, which shape the tumor and also the response to treatment. Recurrent ARID1A mutations have been identified, which are associated with improved outcomes to immune checkpoint inhibition in various tumors. Not much was known about the role of ARID1A mutations in melanoma to date. We investigated the largest cohort of ARID1A mutated melanoma to date and were able to show that despite a high mutational load the described beneficial treatment response is not apparent in melanoma.(1) Background: Melanoma has the highest mortality of all cutaneous tumors, despite recent treatment advances. Many relevant genetic events have been identified in the last decade, including recurrent ARID1A mutations, which in various tumors have been associated with improved outcomes to immunotherapy. (2) Methods: Retrospective analysis of 116 melanoma samples harboring ARID1A mutations. Assessment of clinical and genetic characteristics was performed as well as correlations with treatment outcome applying Kaplan–Meier (log-rank test), Fisher’s exact and Chi-squared tests. (3) Results: The majority of ARID1A mutations were in cutaneous and occult melanoma. ARID1A mutated samples had a higher number of mutations than ARID1A wild-type samples and harbored UV-mutations. A male predominance was observed. Many samples also harbored NF1 mutations. No apparent differences were noted between samples harboring genetically inactivating (frame-shift or nonsense) mutations and samples with other mutations. No differences in survival or response to immunotherapy of patients with ARID1A mutant melanoma were observed. (4) Conclusions: ARID1A mutations primarily occur in cutaneous melanomas with a higher mutation burden. In contrast to findings in other tumors, our data does not support ARID1A mutations being a biomarker of favorable response to immunotherapies in melanoma. Larger prospective studies would still be warranted.

SCLC Treatment in the Immuno-Oncology Era: Current Evidence and Unmet Needs.

Small cell lung cancer (SCLC) represents about 13%–15% of all lung cancers. It has a particularly unfavorable prognosis and in about 70% of cases occurs in the advanced stage (extended disease). Three phase III studies tested the combination of immunotherapy (atezolizumab, durvalumab with or without tremelimumab, and pembrolizumab) with double platinum chemotherapy, with practice-changing results. However, despite the high tumor mutational load and the chronic pro-inflammatory state induced by prolonged exposure to cigarette smoke, the benefit observed with immunotherapy is very modest and most patients experience disease recurrence. Unfortunately, biological, clinical, or molecular factors that can predict this risk have not yet been identified. Thanks to these clinically meaningful steps forward, SCLC is no longer considered an “orphan” disease. Innovative treatment strategies and combinations are currently under investigation to further improve the expected prognosis of patients with SCLC. Following the recent therapeutic innovations, we have reviewed the available literature data about SCLC management, with a focus on current unmet needs and potential predictive factors. In detail, the role of radiotherapy; fragile populations, such as elderly or low-performance status patients (ECOG PS 2), usually excluded from randomized studies; predictive factors of response useful to optimize and guide therapeutic choices; and new molecular targets and future combinations have been explored and revised.

Intratumor Heterogeneity and Antitumor Immunity Shape One Another Bidirectionally.

Over the last decade, it has become clear that the genomic landscapes of tumors profoundly impact their immunogenicity and how tumor cells interact with immune cells. Whereas past discoveries mainly focused on the interplay between tumor immunogenicity and tumor mutational burden (TMB), under the assumption that a higher mutation load would give rise to a better patient response to immune checkpoint blockade therapies, we and others have underlined intratumor heterogeneity (ITH) as an important determinant of the magnitude of the antitumor response and the nature of the tumor microenvironment. In this review, we define TMB versus ITH and how the two factors are being inferred from data, examine key findings in the cancer immunogenomics literature deciphering the complex cross-talk between TMB, ITH, and antitumor immunity in human cancers and in vivo models, and discuss the mutual influence of ITH and immunity-how the antitumor response can give rise to tumors with higher ITH, and how higher ITH can put shackles on the antitumor response.

Arrhythmia prevalence and sudden death risk in adults with the m.3243A>G mitochondrial disorder

AimsTo define the prevalence of non-sustained tachyarrhythmias and bradyarrhythmias in patients with the m.3243A>G mitochondrial genotype and a previously defined, profile, associated with ‘high sudden-death risk’.Methods and resultsPatients at high...