Loss Of Rb Research Articles

Predictors of response to CDK4/6i retrial after prior CDK4/6i failure in ER+ metastatic breast cancer

After disease progression on endocrine therapy (ET) plus a CDK4/6 inhibitor, there is no standardized sequence for subsequent treatment lines for estrogen receptor positive (ER+) metastatic breast cancer (MBC). CDK4/6i retrial as a treatment strategy is commonplace in modern clinical practice; however, the available prospective data investigating this strategy have had inconclusive results. To frame this data in a real-world context, we performed a retrospective analysis assessing the efficacy of CDK4/6is in 195 patients who had previous exposure to CDK4/6i in a prior treatment line at our institution. Among patients who had stopped a CDK4/6i due to toxicity, CDK4/6i retrial either immediately after with a different CDK4/6i or in a further treatment line with the same initial CDK4/6i was both safe and effective, with a median time to treatment failure (TTF) of 10.1 months (95%CI, 4.8–16.9). For patients whose disease progressed on a prior CDK4/6i, we demonstrated comparable median TTFs for patients rechallenged with the same CDK4/6i (4.3 months, 95%CI 3.2–5.5) and with a different CDK4/6i (4.7 months, 95%CI 3.7–6.0) when compared to the recent PACE, PALMIRA, and MAINTAIN trials. Exploratory genomic analysis suggested that the presence of mutations known to confer CDK4/6i resistance, such as TP53 mutations, CDK4 amplifications, and RB1 or FAT1 loss of function mutations may be molecular biomarkers predictive of CDK4/6i retrial failure.

The combination of p16 and Rb expression pattern is helpful to predict high-risk HPV infection and the primary site in lymph node metastases of squamous cell carcinoma

Identifying the primary site of metastatic squamous cell carcinoma in lymph nodes can be challenging. An immunohistochemistry (IHC) analysis recently revealed that high-risk human papillomavirus (HR-HPV)-associated oropharyngeal squamous cell carcinomas (OPSCCs) typically show overexpression of p16 protein and a partial loss pattern of Rb. Nevertheless, the status of these markers in metastatic lesions is still unclear. In this study, we examined p16 and Rb expression status by IHC and transcriptionally active HR-HPV infection by mRNA in situ hybridization in paired primary and metastatic SCC lesions. A total of 50 patients with OPSCCs (n=17), hypopharyngeal SCCs (n=16), laryngeal SCCs (n=6), or uterine cervical SCCs (n=11) were enrolled. HR-HPV and p16 were positive in 21/50 (42 %) and 23/50 (46 %) patients, respectively. Primary and metastatic lesions showed concordant results for those three markers in individual patients. Among the p16-positive patients (n=23), HPV-positive cases typically showed a partial loss of Rb (n=20) and, rarely, a complete loss of Rb (n=1), whereas HPV-negative cases showed preserved Rb expression (n=2). All 27 p16-negative cases lacked HPV infection, while preserved expression and complete loss of Rb were observed in 26 and 1 of the p16-negative cases, respectively. Compared to standalone p16, the combination of p16 overexpression and Rb-partial/complete loss showed equally excellent sensitivity and negative predictive value (each 100 %) as well as improved specificity (100 % versus 93.1 %) and positive predictive value (100 % versus 91.3 %). Our results suggest that combining p16 and Rb expression patterns may be helpful in screening for HR-HPV infection in metastatic lymph nodes and in estimating the primary site of SCC.

190 (PB178): BH3 mimetics targeting BCL-XL have efficacy in solid tumors with RB1 loss and replication stress

190 (PB178): BH3 mimetics targeting BCL-XL have efficacy in solid tumors with RB1 loss and replication stress

Integrated multi-omics assessment of lineage plasticity in a prostate cancer patient with brain and dural metastases

Metastases to the brain are rare in prostate cancer. Here, we describe a patient with two treatment-emergent metastatic lesions, one to the brain with neuroendocrine prostate cancer (NEPC) histology and one to the dural membrane of adenocarcinoma histology. We performed genomic, transcriptomic, and proteomic characterization of these lesions and the primary tumor to investigate molecular features promoting these metastases. The two metastatic lesions had high genomic similarity, including TP53 mutation and PTEN deletion, with the most striking difference being the additional loss of RB1 in the NEPC lesion. Interestingly, the dural lesion expressed both androgen receptor and neuroendocrine markers, suggesting amphicrine carcinoma (AMPC). When analyzing pioneer transcription factors, the AMPC lesion exhibited elevated FOXA1 activity while the brain NEPC lesion showed elevated HOXC10, NFYB, and OTX2 expression suggesting novel roles in NEPC formation or brain tropism. Our results highlight the utility of performing multi-omic characterization, especially in rare cancer subtypes.

Monoallelic loss of RB1 Enhances Osteogenic Differentiation and Delays DNA Repair Without Inducing Tumorigenicity

The Retinoblastoma (RB1) gene plays a pivotal role in osteogenic differentiation. Our previous study, employing temporal gene expression analysis using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), revealed the deregulation of osteogenic differentiation in patient-derived heterozygous RB1 mutant orbital adipose-derived mesenchymal stem cells (OAMSCs). The study revealed increased Alizarin Red staining, suggesting heightened mineralization without a corresponding increase in osteogenic lineage-specific gene expression. In this study, we performed high-throughput RNA sequencing on RB1+/+ and RB1+/- patient-derived OAMSCs differentiated towards the osteogenic lineage to investigate the pathways and molecular mechanisms. The pathway analysis revealed significant differences in cell proliferation, DNA repair, osteoblast differentiation, and cancer-related pathways in RB1+/- OAMSC-derived osteocytes. These findings were subsequently validated through functional assays. The study revealed that osteogenic differentiation is increased in RB1+/- cells, along with enhanced proliferation of the osteocytes. There were delayed but persistent DNA repair mechanisms in RB1+/- osteocytes, which were sufficient to maintain genomic integrity, thereby preventing or delaying the onset of tumors. This contrasts with our earlier observation of increased mineralization without corresponding gene expression changes, emphasizing the importance of high-throughput analysis over preselected gene set analysis in comprehending functional assay results.

Evaluating RB1 and p53 as diagnostic markers in treatment-related neuroendocrine prostate cancer through immunohistochemistry and genomic analysis of RB1 and TP53.

The diagnosis of treatment-related neuroendocrine prostate cancer (t-NEPC) often involves a pathological assessment and immunohistochemistry (IHC) for neuroendocrine markers. Genomic alterations in RB1 and TP53 are frequently observed in NEPC and are believed to play a crucial role in the transformation of adenocarcinoma to NEPC. In this study, we examined the clinicopathologic, immunohistochemical, and genetic features of patients with t-NEPC to better understand their prognosis and diagnostic utility. This retrospective study reviewed the records of patients diagnosed with t-NEPC at Kobe University Hospital between October 2018 and December 2022. Clinical data, including age, serum neuroendocrine marker levels, and treatment history, were collected. IHC was performed for conventional neuroendocrine markers (synaptophysin, chromogranin A, and CD56) and RB1 and p53 expression. Next-generation sequencing (NGS) was conducted using FoundationOne® CDx to identify mutations in RB1 and TP53. This study included 20 patients with t-NEPC. The median time from ADT initiation to development was 42.8 months. IHC revealed RB1 loss in 75% of cases and p53 abnormalities in 75% of cases. NGS identified RB1 mutations in 55% and TP53 mutations in 75% of cases. The concordance between NGS and IHC results was high, with 70% (14/20) agreement for RB1/RB1 and 80% (16/20) for p53/TP53. The immunostaining and genomic analysis of RB1/RB1 and p53/TP53 showed abnormal findings for the four negative cases for conventional neuroendocrine markers. This study indicated high concordance between IHC and NGS findings for RB1/RB1 and p53/TP53 in t-NEPC. We provide a comprehensive benchmark of NGS performance compared with IHC, and these findings may help increase the diagnostic sensitivity of t-NEPC.

Neuroendocrine Differentiation in Prostate Cancer Requires ASCL1.

Most patients with prostate adenocarcinoma develop resistance to therapies targeting the androgen receptor (AR). Consequently, a portion of these patients develop AR-independent neuroendocrine prostate cancer (NEPC), a rapidly progressing cancer with limited therapies and poor survival outcomes. Current research to understand the progression to NEPC suggests a model of lineage plasticity whereby AR-dependent luminal-like tumors progress towards an AR-independent NEPC state. Genetic analysis of human NEPC identified frequent loss of RB1 and TP53, and the loss of both genes in experimental models mediates the transition to a neuroendocrine lineage. Transcriptomics studies have shown that lineage transcription factors ASCL1 and NEUROD1 are present in NEPC. In this study, we modeled the progression of prostate adenocarcinoma to NEPC by establishing prostate organoids and subsequently generating subcutaneous allograft tumors from genetically-engineered mouse models harboring Cre-induced loss of Rb1 and Trp53 with Myc overexpression (RPM). These tumors were heterogeneous and displayed adenocarcinoma, squamous, and neuroendocrine features. ASCL1 and NEUROD1 were expressed within neuroendocrine-defined regions, with ASCL1 being predominant. Genetic loss of Ascl1 in this model did not decrease tumor incidence, growth, or metastasis; however, there was a notable decrease in neuroendocrine identity and an increase in basal-like identity. This study provides an in vivo model to study progression to NEPC and establishes the requirement for ASCL1 in driving neuroendocrine differentiation in prostate cancer.

The expression of YAP1 and other transcription factors contributes to lineage plasticity in combined small cell lung carcinoma.

Lineage plasticity in small cell lung carcinoma (SCLC) causes therapeutic difficulties. This study aimed to investigate the pathological findings of plasticity in SCLC, focusing on combined SCLC, and elucidate the involvement of YAP1 and other transcription factors. We analysed 100 surgically resected SCLCs through detailed morphological observations and immunohistochemistry for YAP1 and other transcription factors. Component-by-component next-generation sequencing (n = 15 pairs) and immunohistochemistry (n = 35 pairs) were performed on the combined SCLCs. Compared with pure SCLCs (n = 65), combined SCLCs (n = 35) showed a significantly larger size, higher expression of NEUROD1, and higher frequency of double-positive transcription factors (p = 0.0009, 0.04, and 0.019, respectively). Notably, 34% of the combined SCLCs showed morphological mosaic patterns with unclear boundaries between the SCLC and its partner. Combined SCLCs not only had unique histotypes as partners but also represented different lineage plasticity within the partner. NEUROD1-dominant combined SCLCs had a significantly higher proportion of adenocarcinomas as partners, whereas POU2F3-dominant combined SCLCs had a significantly higher proportion of squamous cell carcinomas as partners (p = 0.006 and p = 0.0006, respectively). YAP1 expression in SCLC components was found in 80% of combined SCLCs and 62% of pure SCLCs, often showing mosaic-like expression. Among the combined SCLCs with component-specific analysis, the identical TP53 mutation was found in 10 pairs, and the identical Rb1 abnormality was found in 2 pairs. On immunohistochemistry, the same abnormal p53 pattern was found in 34 pairs, and Rb1 loss was found in 24 pairs. In conclusion, combined SCLC shows a variety of pathological plasticity. Although combined SCLC is more plastic than pure SCLC, pure SCLC is also a phenotypically plastic tumour. The morphological mosaic pattern and YAP1 mosaic-like expression may represent ongoing lineage plasticity. This study also identified the relationship between transcription factors and partners in combined SCLC. Transcription factors may be involved in differentiating specific cell lineages beyond just 'neuroendocrine'.

Revisiting the formation of lunar anorthosites via the Rb[sbnd]Sr isotope systematics

The incorporation of KREEP (potassium, rare-earth element, and phosphorus), mantle-derived mafic melts and trapped liquid into the lunar ferroan anorthosite (FAN) suite plays a pivotal role in generating their geochemical and isotopic variations. Nonetheless, the specific involvement and distinct roles of these different components remain controversial. This study presents in-situ Sr isotopic data for 28 anorthositic clasts found within lunar feldspathic meteorites to trace their sources and post-modification processes. We find that these plagioclases exhibit substantial variations in their measured 87Sr/86Sr values (0.69978–0.70357), in contrast to the relatively narrow range observed in Apollo 16 FANs, thereby likely reflecting a diverse chemical composition of lunar crustal rocks. In contrast, the analyzed plagioclases have consistently low 87Rb/86Sr ratios (0.00185–0.03962), similar to those of Apollo samples, reflecting impact-induced loss of Rb. Detailed investigations indicate that certain elevated 87Sr/86Sr ratios are probably not caused by terrestrial contamination or instrumental analysis, but most likely result from the decay of 87Rb from sources with initial 87Rb/86Sr higher than 0.0119–0.1380. However, such elevated 87Rb/86Sr values cannot solely result from crystallization of the lunar magma ocean (LMO) and likely involve KREEP components. Combined with trace element data, we estimate the maximum proportion of KREEP melt in the formation of lunar anorthosites. Future analyses of lunar anorthosites collected by China's Chang'e-5 and Chang'e-6 missions will be crucial for validating the observed Sr isotopic heterogeneity.

Identification and characterization of early human photoreceptor states and cell-state-specific retinoblastoma-related features.

Human cone photoreceptors differ from rods and serve as the retinoblastoma cell-of-origin, yet the developmental basis for their distinct behaviors is poorly understood. Here, we used deep full-length single-cell RNA-sequencing to distinguish post-mitotic cone and rod developmental states and identify cone-specific features that contribute to retinoblastomagenesis. The analyses revealed early post-mitotic cone- and rod-directed populations characterized by higher THRB or NRL regulon activities, an immature photoreceptor precursor population with concurrent cone and rod gene and regulon expression, and distinct early and late cone and rod maturation states distinguished by maturation-associated declines in RAX regulon activity. Unexpectedly, both L/M cone and rod precursors co-expressed NRL and THRB RNAs, yet they differentially expressed functionally antagonistic NRL and THRB isoforms and prematurely terminated THRB transcripts. Early L/M cone precursors exhibited successive expression of several lncRNAs along with MYCN , which composed the seventh most L/M-cone-specific regulon, and SYK , which contributed to the early cone precursors' proliferative response to RB1 loss. These findings reveal previously unrecognized photoreceptor precursor states and a role for early cone-precursor-intrinsic SYK expression in retinoblastoma initiation.

Wnt/β-Catenin–Activated Nonpilomatrical Carcinoma of the Skin: A Case Series

Among skin epithelial tumors, recurrent mutations in the APC/CTNNB1 genes resulting in activation of the Wnt/β-catenin pathway have been reported predominantly in neoplasms with matrical differentiation. In the present study, we describe the morphologic, immunohistochemical, and genetic features of 16 primary cutaneous carcinomas harboring mutations activating the Wnt/β-catenin pathway without evidence of matrical differentiation, as well as 4 combined tumors in which a similar Wnt/β-catenin–activated carcinoma component was associated with Merkel cell carcinoma (MCC) or pilomatrical carcinoma. Among the pure tumor cases, 6 of 16 patients were women with a median age of 80 years (range, 58-98 years). Tumors were located on the head and neck (n = 7, 44%), upper limb (n = 4, 25%), trunk (n = 3, 18%), and leg (n = 2, 13%). Metastatic spread was observed in 4 cases resulting in death from disease in 1 patient. Microscopically, all cases were poorly differentiated neoplasms infiltrating the dermis and/or subcutaneous tissue. In 13 cases, solid “squamoid” areas were associated with a basophilic component characterized by rosette/pseudoglandular formation resulting in a biphasic appearance. Three specimens consisted only of poorly differentiated carcinoma lacking rosette formation. Immunohistochemical studies showed frequent expression of EMA (100%), BerEP4 (100%), cytokeratin 7 (94%), chromogranin A (44%), synaptophysin (82%), and cytokeratin 20 (69%). Complete loss of Rb expression was observed in all but 1 case. Nuclear β-catenin and CDX2 expressions were detected in all cases. Recurrent pathogenic somatic mutations were observed in APC (60%), CTNNB1 (40%), and RB1 (n = 47%). Global methylation analysis confirmed that cases with rosette formation constituted a homogeneous tumor group distinct from established skin tumor entities (pilomatrical carcinoma, MCC, and squamous cell carcinoma), although the 3 other cases lacking such morphologic features did not. In addition, we identified 4 combined neoplasms in which there was a component showing a similar poorly differentiated rosette-forming carcinoma demonstrating Rb loss and β-catenin activation associated with either MCC (n = 3) or pilomatrical carcinoma (n = 1). In conclusion, we describe a distinctive neoplasm, for which we propose the term “Wnt/β-catenin–activated rosette-forming carcinoma,” morphologically characterized by the association of rosette formation, squamous and/or neuroendocrine differentiation, diffuse CDX2 expression, Rb loss, and mutations in CTNNB1/APC genes.

Arid1a Loss Enhances Disease Progression in a Murine Model of Osteosarcoma.

Osteosarcoma is an aggressive bone malignancy, molecularly characterized by acquired genome complexity and frequent loss of TP53 and RB1. Obtaining a molecular understanding of the initiating mutations of osteosarcomagenesis has been challenged by the difficulty of parsing between passenger and driver mutations in genes. Here, a forward genetic screen in a genetic mouse model of osteosarcomagenesis initiated by Trp53 and Rb1 conditional loss in pre-osteoblasts identified that Arid1a loss contributes to OS progression. Arid1a is a member of the canonical BAF (SWI/SNF) complex and a known tumor suppressor gene in other cancers. We hypothesized that the loss of Arid1a increases the rate of tumor progression and metastasis. Phenotypic evaluation upon in vitro and in vivo deletion of Arid1a validated this hypothesis. Gene expression and pathway analysis revealed a correlation between Arid1a loss and genomic instability, and the subsequent dysregulation of genes involved in DNA DSB or SSB repair pathways. The most significant of these transcriptional changes was a concomitant decrease in DCLRE1C. Our findings suggest that Arid1a plays a role in genomic instability in aggressive osteosarcoma and a better understanding of this correlation can help with clinical prognoses and personalized patient care.

Concurrent RB1 Loss and BRCA Deficiency Predicts Enhanced Immunologic Response and Long-term Survival in Tubo-ovarian High-grade Serous Carcinoma.

The purpose of this study was to evaluate RB1 expression and survival across ovarian carcinoma histotypes and how co-occurrence of BRCA1 or BRCA2 (BRCA) alterations and RB1 loss influences survival in tubo-ovarian high-grade serous carcinoma (HGSC). RB1 protein expression was classified by immunohistochemistry in ovarian carcinomas of 7,436 patients from the Ovarian Tumor Tissue Analysis consortium. We examined RB1 expression and germline BRCA status in a subset of 1,134 HGSC, and related genotype to overall survival (OS), tumor-infiltrating CD8+ lymphocytes, and transcriptomic subtypes. Using CRISPR-Cas9, we deleted RB1 in HGSC cells with and without BRCA1 alterations to model co-loss with treatment response. We performed whole-genome and transcriptome data analyses on 126 patients with primary HGSC to characterize tumors with concurrent BRCA deficiency and RB1 loss. RB1 loss was associated with longer OS in HGSC but with poorer prognosis in endometrioid ovarian carcinoma. Patients with HGSC harboring both RB1 loss and pathogenic germline BRCA variants had superior OS compared with patients with either alteration alone, and their median OS was three times longer than those without pathogenic BRCA variants and retained RB1 expression (9.3 vs. 3.1 years). Enhanced sensitivity to cisplatin and paclitaxel was seen in BRCA1-altered cells with RB1 knockout. Combined RB1 loss and BRCA deficiency correlated with transcriptional markers of enhanced IFN response, cell-cycle deregulation, and reduced epithelial-mesenchymal transition. CD8+ lymphocytes were most prevalent in BRCA-deficient HGSC with co-loss of RB1. Co-occurrence of RB1 loss and BRCA deficiency was associated with exceptionally long survival in patients with HGSC, potentially due to better treatment response and immune stimulation.

STEM-06. THE ROLE OF SHH SIGNALING PATHWAY IN RETINOBLASTOMA INITIATION AND PROGRESSION

Abstract BACKGROUND Retinoblastoma is a rare childhood cancer of the retina that shows the biallelic loss of RB1 in nearly 95% of cases. It was reported that SHH pathway proteins, such as SHH, GLI1, and GLI2, are aberrantly high in human retinoblastoma, and high GLI2 expression is associated with aggressive clinicopathological features. Thus, we assessed whether activation of SHH pathway by overexpression of Gli2 or SMO induces the initiation of retinoblastoma. METHODS We generated a Cre-activated, Doxycycline (Dox)-controlled transgenic model to induce SMO or Gli2 expression in developing retina. In this transgenic mouse, expression of Smo or Gli2A is under the control of TRE, which permits temporal and spatial expression of Smo or Gli2A by administering/withdrawing doxycycline (DOX). RESULTS We fed the triple transgenic mice (Chx10-Cre/R26-LSL-rtTA/tetO-Smo or Chx10-Cre/R26-LSL-rtTA/tetO-Gli2A starting from E0 or P5. Our studies demonstrated that control littermates (Cre-) did not show any abnormalities, while mice with triple transgenes developed tumors in the bilateral eyes with 100% morbidity. The animals showed symptoms of leukocoria at P0-P3 or P25-p30 if the oncogenes were initiated at E0 or P5 respectively. The tumor cells were highly proliferative and expressed retina progenitor cell specific marker of Sox2, showing aberrant expression of pRB, CDK4/6, and CCND1. CONCLUSIONS Our results demonstrate that overexpressing Smo or Gli2 in Chx10+ cells induce rapid tumor formation in mouse retina. In future study, we plan to (i) investigate the impact of Shh pathway activation in RPCs on RB tumorigenesis within the context of Rb1 loss; (ii) define the molecular mechanisms underlying tumor initiation and progression; and (iii) evaluate whether targeting SHH pathway prevents or delays tumor initiation and progression using our Gli2-driven mouse model and patient-derived xenografts.

RB1 Genetic Alterations in Estrogen Receptor–Positive Breast Carcinomas: Correlation With Neuroendocrine Differentiation

Genetic alterations in the retinoblastoma susceptibility gene (RB1) are present in up to 40% of triple-negative breast cancers (BCs) and frequent in tumors with neuroendocrine differentiation, including small cell neuroendocrine carcinoma. Data on RB1 genetic alterations in estrogen receptor (ER)–positive BCs are scarce. In this study, we sought to define the morphologic, immunohistochemical, and genetic features of ER-positive BCs harboring somatic alterations in RB1, with emphasis on neuroendocrine differentiation. ER-positive BCs with pathogenic RB1 genetic alterations were identified in <1% of cases (N = 55) from a cohort of 6026 BCs previously subjected to targeted next-generation sequencing, including 23 primary BCs (pBCs) and 32 recurrent/metastatic BCs (mBCs). In cases where loss of heterozygosity of the wild-type RB1 allele could be assessed (93%, 51/55), most pBCs (82%, 18/22) and mBCs (90%, 26/29) exhibited biallelic RB1 inactivation, primarily through loss-of-function mutation and loss of heterozygosity (98%, 43/44). Upon histologic review, a subset of RB1-altered tumors exhibited neuroendocrine morphology (13%, 7/55), which correlated with expression of neuroendocrine markers (39%, 9/23) in both pBCs (27%, 3/11) and mBCs (50%, 6/12). Loss of Rb protein expression was observed in BCs with biallelic RB1 loss only, with similar frequency in pBCs (82%, 9/11) and mBCs (75%, 9/12). All cases with neuroendocrine marker expression (n = 9) and/or neuroendocrine morphology (n = 7) harbored biallelic genetic inactivation of RB1 and exhibited Rb loss of expression. TP53 (53%, 29/55) and PIK3CA (45%, 25/55) were the most frequently comutated genes across the cohort. Overall, these findings suggest that ER-positive BCs with biallelic RB1 genetic alterations frequently exhibit Rb protein loss, which correlates with neuroendocrine differentiation in select BCs. This study provides insights into the molecular and phenotypic heterogeneity of BCs with RB1 genetic inactivation, underscoring the need for further research into the potential clinical implications associated with these tumors.

TOPOLOGY: A phase II study to evaluate the efficacy and toxicities of PLX038, in patients with locally advanced or metastatic triple-negative breast cancer.

TPS1142 Background: Sacituzumab govitecan (SG) is an anti-TROP2 antibody-drug conjugate (ADC) with an SN-38 payload (topoisomerase I inhibitor) that was recently approved for use in advanced triple-negative breast cancer (TNBC). The rapid spontaneous linker hydrolysis in sacituzumab-govitecan releases a very large amount of SN-38 cargo systemically, which may in part contribute to efficacy and also in part be responsible for systemic side effects (1). PLX038 is a 40 kDa 4-arm PEG-SN38 conjugate with a half-life of 5 days. It has a controlled, slow-release rate which allows high SN-38 exposure over an extended period of time with a low Cmax, and very low levels of SN-38-glucuronide. The PLX038 nanomolecule also penetrates large pores of tumor vasculature, and accumulates and is retained in the tumor for long periods. The prolonged exposure of tumors to SN-38 may result in increased efficacy without resulting in severe gastrointestinal toxicity. We hypothesize that PLX038 may be effective in patients with advanced TNBC who have been previously treated with standard chemotherapy regimens. Methods: This is an open label, multi-centric phase II study designed to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics and efficacy of PLX038 in locally advanced or metastatic TNBC previously treated with at least two prior cytotoxic chemotherapy regimens for locally advanced or metastatic breast cancer including an anthracycline, taxane and sacituzumab govitecan. Patients will be treated at a dose of 1730mg/m2 IV infusion on Day 1 of each cycle Q3W (every 21 days, 1 cycle = 1 injection) after at least a first cycle at 1300mg/m2. At the discretion of the treating physician, the PLX038 dose will be increased to 1730 mg/m2 for patients who tolerate at least 1 cycle. All included patients will receive PLX038 as single agent as long as study is ongoing or until progression of disease, unacceptable toxicity, patient withdrawal of consent, Investigator decision, lost to follow-up, death, patient non-compliance, or study termination by Sponsor. The primary endpoint is the best tumor response (defined as partial or complete response in the first 6 months of treatment) assessed by investigators per RECIST v1.1 criteria, with tumor assessments every 8 weeks (± 7 days). Secondary endpoints include i) efficacy criteria (time to response, duration of response, progression-free survival, overall survival), ii) toxicity criteria (AE/SAEs), iii) association between PLX038 efficacy and biomarkers (homologous recombination defect, replication stress-related biomarkers such as SFLN11 expression, RB1 loss), iv) PK/PD analysis. A Simon's two-stage optimum design will be used, and a maximum of N=44 patients will be included, 1. Santi et al., Biodrugs 2024. Clinical trial information: NCT06162351 .

Alisertib in patients with extensive-stage small-cell lung cancer: The phase 2 ALISCA-Lung1 study.

TPS8128 Background: Treatment options are limited for patients (pts) with small-cell lung cancer (SCLC) whose disease has progressed on or after platinum-based chemotherapy. Therefore, there is an urgent need for evaluation of novel agents in this setting. Aurora kinase A (AURKA) is a key regulator of mitosis and AURKA expression is associated with worse prognosis in multiple solid tumor types. Alisertib is a highly selective, reversible, ATP-competitive, orally administered, small-molecule AURKA inhibitor under investigation for treating SCLC. Phase 1/2 clinical trials of alisertib as either monotherapy or in combination with paclitaxel for relapsed/refractory solid tumors (including SCLC) reported response rates of 21–22%. The most common treatment-related grade ≥3 AEs were neutropenia, febrile neutropenia, and leukopenia. Preclinically, greater alisertib sensitivity has been reported in models with high c-Myc expression and/or loss of RB1 function. In a clinical study of alisertib + paclitaxel vs placebo + paclitaxel in SCLC, either c-Myc expression or mutation in RB1, RBL1, RBL2, or CDK6 showed strong correlation with an improvement in both PFS and OS in the alisertib arm. Methods: ALISCA-Lung1(NCT06095505) is a phase 2 study to determine whether there is a biomarker-defined population of pts with extensive-stage SCLC that derives increased benefit from alisertib monotherapy. Key inclusion criteria: ≥18 years of age; progression on or after first-line treatment with platinum-based chemotherapy + anti-PD-L1 immunotherapy; ≥1 measurable lesion per RECIST v1.1; availability of tissue sample for retrospective biomarker evaluation. Key exclusion criteria: prior treatment with AURKA-specific or pan-Aurora-targeted agents; active infection; immunocompromise; unstable brain metastases; inability/unwillingness to swallow tablets. Primary objective: to determine whether any biomarker(s) correlate with increased benefit to alisertib monotherapy. Biomarkers will be assessed by next-generation sequencing, mRNA expression analysis, and immunohistochemistry. Candidate biomarkers include, but are not limited to, RB1 loss of function, c-Myc expression, TP53mutation, AURKA expression, and SCLC molecular subtype. Secondary objectives: to determine investigator-assessed efficacy, survival, safety, and population pharmacokinetics. Eligible pts will receive alisertib 50 mg orally BID d1−7 q21d (including primary prophylaxis with G-CSF) until disease progression, unacceptable toxicity, or withdrawal of consent. All pts will undergo sparse pharmacokinetic sampling. Recruitment is underway and up to 60 pts will be enrolled at ~20 centers in the USA. Findings are anticipated to identify a biomarker-defined pt population that derives the greatest clinical benefit from alisertib. Future development of alisertib in SCLC is anticipated to focus on this biomarker-defined population. Clinical trial information: NCT06095505 .

Osimertinib, platinum, etoposide as initial treatment for patients with EGFR mutant lung cancers with TP53 and RB1 alterations.

8565 Background: EGFR-mutant lung adenocarcinomas (EGFR+LC) transform to small cell lung cancer (SCLC) in up to 14% of cases, which creates an aggressive disease phenotype that is treated like de novo small lung cancer with platinum/etoposide chemotherapy. Transformed EGFR+LC molecularly mimic de novo small cell lung cancers and harbor TP53and RB1 loss. We hypothesized that if small cell directed chemotherapy is utilized prior to histologic transformation in EGFR/TP53/RB1 mutant LC, we may eradicate any pre-existing small cell clones, prevent small cell histologic transformation, and improve patient outcomes. Methods: We administered osimertinib, platinum, and etoposide in patients with newly diagnosed EGFR+LC with TP53and RB1alterations. Patients received 3 months of osimertinib induction, then 4 cycles of osimertinib, platinum and etoposide followed by osimertinib until progression. Objective response rate (ORR), progression-free survival (PFS), overall survival (OS), and safety were assessed. Patients were required to have an on-treatment biopsy prior to initiation of chemotherapy upon which single cell RNA sequencing (scRNAseq) was performed. All patients had pre-treatment and post-progression biopsies that underwent next-generation sequencing. Patients were evaluated for acquired resistance (classified as on-target, off-target, transformation, and unknown). Among patients that transformed, markers of SCLC (ASCL1, NEUROD1, POU2F3, YAP1, synaptophysin, chromogranin, and Ki-67) were analyzed. Results: Eleven patients were enrolled [median age 58 years old range (49-81), 45% female, 73% EGFRexon 19 deletions and 27% EGFR L858R]. Three patients received cisplatin/etoposide, six patients carboplatin/etoposide, and two patients progressed two months on induction osimertinib before they could start chemotherapy. The ORR was 82%, median PFS 15.6 months and median OS was 37.9 months. All patients retained EGFR, TP53, and RB1mutations on post-treatment biopsy. Acquired on-target resistance mechanisms were identified in 9% of patients ( EGFR C797S), off-target in 9% of patients ( CDKN2A), and small cell transformation in 46% of patients. Among the transformed patients, all had high Ki-67 (70-95%) and 80% were positive for synaptophysin and chromogranin. 40% of cases were ASCL1-dominant, 40% were NEUROD1-dominant, and 20% were unknown. Additionally, we utilized copy number inference and CytoTrace to identify subclones within pre-transformed EGFR+LC that potentially harbor features of early plasticity. Further scRNAseq and whole exome sequencing data are forthcoming. Conclusions: In patients with EGFR+LCs with concurrent TP53and RB1alterations, osimertinib plus platinum and etoposide demonstrated activity and no new safety signals were seen. Early addition of small cell directed chemotherapy did not prevent histologic transformation. Clinical trial information: NCT03567642 .

A synthetic lethal dependency on casein kinase 2 in response to replication-perturbing therapeutics in RB1-deficient cancer cells.

Resistance to therapy commonly develops in patients with high-grade serous ovarian carcinoma (HGSC) and triple-negative breast cancer (TNBC), urging the search for improved therapeutic combinations and their predictive biomarkers. Starting from a CRISPR knockout screen, we identified that loss of RB1 in TNBC or HGSC cells generates a synthetic lethal dependency on casein kinase 2 (CK2) for surviving the treatment with replication-perturbing therapeutics such as carboplatin, gemcitabine, or PARP inhibitors. CK2 inhibition in RB1-deficient cells resulted in the degradation of another RB family cell cycle regulator, p130, which led to S phase accumulation, micronuclei formation, and accelerated PARP inhibition-induced aneuploidy and mitotic cell death. CK2 inhibition was also effective in primary patient-derived cells. It selectively prevented the regrowth of RB1-deficient patient HGSC organoids after treatment with carboplatin or niraparib. As about 25% of HGSCs and 40% of TNBCs have lost RB1 expression, CK2 inhibition is a promising approach to overcome resistance to standard therapeutics in large strata of patients.

Comprehensive genomic and transcriptomic characterization of high-grade gastro-entero-pancreatic neoplasms

BackgroundHigh-grade gastro-entero-pancreatic neoplasms (HG GEP-NENs) can be stratified according to their morphology and Ki-67 values into three prognostic classes: neuroendocrine tumors grade 3 (NETs G3), neuroendocrine carcinomas with Ki-67 < 55% (NECs <55) and NECs with Ki-67 ≥ 55% (NECs ≥55).MethodsWe analyzed a cohort of 49 HG GEP-NENs by targeted Next-Generation Sequencing (TrueSight Oncology 500), RNA-seq, and immunohistochemistry for p53, Rb1, SSTR-2A, and PD-L1.ResultsFrequent genomic alterations affected TP53 (26%), APC (20%), KRAS and MEN1 (both 11%) genes. NET G3 were enriched in MEN1 (p = 0.02) mutations, while both NECs groups were enriched in TP53 (p = 0.001), APC (p = 0.002) and KRAS (p = 0.02) mutations and tumors with TMB ≥ 10 muts/Mb (p = 0.01). No differentially expressed (DE) gene was found between NECs <55% and NECs ≥55%, while 1129 DE genes were identified between NET G3 and NECs. A slight enrichment of CD4+ and CD8+ T cells in NECs and of cancer-associated fibroblasts and macrophages (M2-like) in NET G3. Multivariate analysis identified histologic type and Rb1 loss as independent prognostic factors for overall survival.ConclusionsThis study showed that GEP-NET G3 and GEP-NECs exhibit clear genomic and transcriptomic differences, differently from GEP-NECs <55% and GEP-NECs ≥55%, and provided molecular findings with prognostic and potentially predictive value.