High Expression Levels Research Articles

Multiomics sequencing and immune microenvironment characteristics define three subtypes of small cell neuroendocrine carcinoma of the cervix.

Small cell cervical carcinoma (SCCC) is the most common neuroendocrine tumor in the female genital tract, with an unfavorable prognosis and lacking an evidence-based therapeutic approach. Until now, the distinct subtypes and immune characteristics of SCCC combined with genome and transcriptome have not been described. We performed genomic (n = 18), HPV integration (n = 18), and transcriptomic sequencing (n = 19) of SCCC samples. We assessed differences in immune characteristics between SCCC and conventional cervical cancer, and other small cell neuroendocrine carcinomas, through bioinformatics analysis and immunohistochemical assays. We stratified SCCC patients through non-negative matrix factorization and described the characteristics of these distinct types. We further validated it using multiplex immunofluorescence (n = 77) and investigated its clinical prognostic effect. We confirmed a high frequency of PIK3CA and TP53 alterations and HPV18 integrations in SCCC. SCCC and other small cell carcinoma had similar expression signatures and immune cell infiltration patterns. Comparing patients with SCCC to those with conventional cervical cancer, the former presented immune excluded or 'desert' infiltration. The number of CD8+ cells in the invasion margin of SCCC patients predicted favorable clinical outcomes. We identified three transcriptome subtypes: an inflamed phenotype with high-level expression of genes related to the MHC-II complex (CD74) and IFN-α/β (SCCC-I), and two neuroendocrine subtypes with high-level expression of ASCL1 or NEUROD1, respectively. Combined with multiple technologies, we found that the neuroendocrine groups had more TP53 mutations and SCCC-I had more PIK3CA mutations. Multiplex immunofluorescence validated these subtypes and SCCC-I was an independent prognostic factor of overall survival. These results provide insights into SCCC tumor heterogeneity and potential therapies. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Robot-Assistant Radical Cystectomy as a Modern Method of Personalized Treatment for Patients with Muscle-Invasive Bladder Cancer

Introduction. Radical cystectomy (RC) is the “gold standard” treatment for muscle-invasive bladder cancer (MIBC). Robot-assisted RC (RARC) can act as a safe and effective method of treating MIBC.The aim of the study was to analyze our own experience with the use of RARC with intraoperative indocyanine green fluorescence (ICG) and postoperative determination of tumor macrophage levels in the treatment of patients with MIBC.Materials and methods. A study was conducted at the Clinic of the Bashkir State Medical University with the participation of 202 patients aſter RARC. 30 patients underwent intraoperative ICG fluorescence testing. The levels of tumor macrophages in the removed macropreparations were determined in all patients. In the postoperative period, a survival analysis was performed.Results. The sensitivity of the ICG fluorescence method was 94.4 %, and the specificity was 83.3 %. High levels of expression of macrophage markers CD68 and CD163 significantly predominate in the group with the cN+ stage (according to the clinical TNM classification, there are signs of damage) (p = 0.027 and p = 0.018, respectively). ICG fluorescence of regional lymph nodes (100 %) was observed in all patients with high CD68 and CD163 levels. A significant decrease in survival was recorded in patients with high pathomorphological stage T (pT according to the TNM classification) (r = 0.952, p < 0.05) and statistically significant survival rates were obtained for pT1–pT4 groups (p < 0.05 for overall, cancer-specific and recurrence-free survival (OS, CSS, RFS, respectively)).Discussion. More and more studies are appearing in the world literature proving the effectiveness of RARC in the treatment of patients with MIBC. Compared with open and laparoscopic techniques, better surgical and comparable oncological results are demonstrated.Conclusion. The results of the analysis of the RARC’s own experience indicate the effectiveness of this method of treating MIBC. The use of ICG fluorescence and verification of tumor macrophages makesit possible to provide personalized care to oncourological patients.

Insights into phage-bacteria interaction in cold seep Gigantidas platifrons through metagenomics and transcriptome analyses

Viruses are crucial for regulating deep-sea microbial communities and biogeochemical cycles. However, their roles are still less characterized in deep-sea holobionts. Bathymodioline mussels are endemic species inhabiting cold seeps and harboring endosymbionts in gill epithelial cells for nutrition. This study unveiled a diverse array of viruses in the gill tissues of Gigantidas platifrons mussels and analyzed the viral metagenome and transcriptome from the gill tissues of Gigantidas platifrons mussels collected from a cold seep in the South Sea. The mussel gills contained various viruses including Baculoviridae, Rountreeviridae, Myoviridae and Siphovirdae, but the active viromes were Myoviridae, Siphoviridae, and Podoviridae belonging to the order Caudovirales. The overall viral community structure showed significant variation among environments with different methane concentrations. Transcriptome analysis indicated high expression of viral structural genes, integrase, and restriction endonuclease genes in a high methane concentration environment, suggesting frequent virus infection and replication. Furthermore, two viruses (GP-phage-contig14 and GP-phage-contig72) interacted with Gigantidas platifrons methanotrophic gill symbionts (bathymodiolin mussels host intracellular methanotrophic Gammaproteobacteria in their gills), showing high expression levels, and have huge different expression in different methane concentrations. Additionally, single-stranded DNA viruses may play a potential auxiliary role in the virus–host interaction using indirect bioinformatics methods. Moreover, the Cro and DNA methylase genes had phylogenetic similarity between the virus and Gigantidas platifrons methanotrophic gill symbionts. This study also explored a variety of viruses in the gill tissues of Gigantidas platifrons and revealed that bacteria interacted with the viruses during the symbiosis with Gigantidas platifrons. This study provides fundamental insights into the interplay of microorganisms within Gigantidas platifrons mussels in deep sea.

Potential limitations of micro-dystrophin gene therapy for Duchenne muscular dystrophy.

Clinical trials delivering high doses of adeno-associated viruses (AAVs) expressing truncated dystrophin molecules (micro-dystrophins) are underway for individuals with Duchenne muscular dystrophy (DMD). We examined the efficiency and efficacy of this strategy with four micro-dystrophin constructs (three in clinical trials and a variant of the largest clinical construct), in a severe mouse model of DMD, using doses of AAV comparable to those used in the clinical trials. We achieved high levels of micro-dystrophin expression in striated muscle with cardiac expression ~10 fold higher than that observed in skeletal muscle. Significant, albeit incomplete, correction of the skeletal muscle disease was observed. Surprisingly, a lethal acceleration of cardiac disease progression occurred with two of the micro-dystrophins. The detrimental impact on the heart appears to be caused by the high levels of micro-dystrophin resulting in variable competition (dependent on the design of the micro-dystrophin) between micro-dystrophin and utrophin at the cardiomyocyte membrane. There may also be a contribution from an overloading of protein degradation. The significance of these observations for patients currently being treated with AAV-micro-dystrophin therapies is unclear since the levels of expression being achieved in the DMD hearts are unknown. However, it suggests that micro-dystrophin treatments need to avoid excessively high levels of expression in the heart and cardiac function should be carefully monitored in these patients.

Gene redundancy and gene compensation of insulin-like peptides in the oocyte development of bean beetle.

Bean beetle (Callosobruchus maculatus) exhibits clear phenotypic plasticity depending on population density; However, the underlying molecular mechanism remains unknown. Compared to low-density individuals, high-density individuals showed a faster terminal oocyte maturity rate. Four insulin-like peptide (ILP) genes were identified in the bean beetle, which had higher expression levels in the head than in the thorax and abdomen. The population density could regulate the expression levels of CmILP1-3, CmILP2-3, and CmILP1 as well as CmILP3 in the head, thorax, and abdomen, respectively. RNA interference results showed that each CmILP could regulate terminal oocyte maturity rate, indicating that there was functional redundancy among CmILPs. Silencing each CmILP could lead to down-regulation of some other CmILPs, however, CmILP3 was up-regulated in the abdomen after silencing CmILP1 or CmILP2. Compared to single gene silencing, silencing CmILP3 with CmILP1 or CmILP2 at the same time led to more serious retardation in oocyte development, suggesting CmILP3 could be up-regulated to functionally compensate for the down-regulation of CmILP1 and CmILP2. In conclusion, population density-dependent plasticity in terminal oocyte maturity rate of bean beetle was regulated by CmILPs, which exhibited gene redundancy and gene compensation.

Genome-wide identification and expression pattern analysis of the GRF transcription factor family in Astragalus mongholicus.

Astragalus membranaceus is a plant of the Astragalus genus, which is used as a traditional Chinese herbal medicine with extremely high medicinal and edible value. Astragalus mongholicus, as one of the representative medicinal materials with the same origin of medicine and food, has a rising market demand for its raw materials, but the quality is different in different production areas. Growth-regulating factors (GRF) are transcription factors unique to plants that play important roles in plant growth and development. Up to now, there is no report about GRF in A. mongholicus. This study conducted a genome-wide analysis of the AmGRF gene family, identifying a total of nine AmGRF genes that were classified into subfamily V based on phylogenetic relationships. In the promoter region of the AmGRF gene, we successfully predicted cis-elements that respond to abiotic stress, growth, development, and hormone production in plants. Based on transcriptomic data and real-time quantitative polymerase chain reaction (qPCR) validation, the results showed that AmGRFs were expressed in the roots, stems, and leaves, with overall higher expression in leaves, higher expression of AmGRF1 and AmGRF8 in roots, and high expression levels of AmGRF1 and AmGRF9 in stems. The results of this study provide a theoretical basis for the further exploration of the functions of AmGRFs in plant growth and development.

Highly conserved and extremely variable: The paradoxical pattern of toxin expression revealed by comparative venom-gland transcriptomics of Phalotris (Serpentes: Dipsadidae)

Although non-front fanged snakes account for almost two-thirds of snake diversity, most studies on venom composition and evolution focus exclusively on front-fanged species, which comprise most of the clinically relevant accidents. Comprehensive reports on venom composition of non-front fanged snakes are still scarce for several groups. In this study, we address such shortage of knowledge by providing new insights about the venom composition among species of Phalotris, a poorly studied Neotropical dipsadid genus. Phalotris are known for their specialized venom delivery system and toxic venoms, which can cause life-threatening accidents in humans. We evaluate the venom-gland transcriptome of Phalotris, comparing the following three South American species: P. reticulatus for the Araucaria Pine forests, P. lemniscatus for the Pampa grasslands, and P. mertensi for the Brazilian Cerrado. Our results indicate similar venom profiles, in which they share a high expression level of Kunitz-type inhibitors (KUNZ). On the other hand, comparative analyses revealed substantial differences in the expression levels of C-type lectins (CTL) and snake venom metalloproteinases (SVMP). The diverse set of SVMP and CTL isoforms shows signals of positive selection, and we also identified truncated forms of type III SVMPs, which resemble type II and type I SVMPs of viperids. Additionally, we identified a CNP precursor hosting a proline-rich region containing a BPP motif resembling those commonly detected in viperid venoms with hypotensive activity. Altogether, our results suggest an evolutionary history favoring high expression levels of few KUNZ isoforms in Phalotris venoms, contrasting with a highly diverse set of SVMP and CTL isoforms. Such diversity can be comparable with the venom variability observed in some viperids. Our findings highlight the extreme phenotypic diversity of non-front fanged snakes and the importance to allocate greater effort to study neglected groups of Colubroidea.

CXCL5/CXCL8 induces neutrophilic inflammation in peri-implantitis.

This research aimed to examine the role of C-X-C motif chemokine ligand 5 (CXCL5) and C-X-C motif chemokine ligand 8 (CXCL8; also known as IL-8) in neutrophilic inflammation triggered by peri-implantitis and to shed light on the underlying mechanisms that link them to the development of this condition. This study included 40 patients who visited the Department of Periodontology at Kyungpook University Dental Hospital. They were divided into two groups based on their condition: healthy implant (HI) group (n = 20) and peri-implantitis (PI) group (n = 20). Biopsy samples of PI tissue were collected from the patients under local anesthesia. HI tissue was obtained using the same method during the second implant surgery. To construct libraries for control and test RNAs, the QuantSeq 3' mRNA-Seq Library Prep Kit (Lexogen, Inc., Austria) was used according to the manufacturer's instructions. Samples were pooled based on representative cytokines obtained from RNA sequencing results and subjected to Reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Hematoxylin and eosin staining, and immunohistochemistry (IHC) analysis were performed to visually assess expression levels and analyze tissue histology. Student's t-test was employed to conduct statistical analyses. Initially, heatmaps were used to examine gene expression variations between the HI and PI groups based on the results of RNA sequencing. Notably, among various cytokines, CXCL5 and CXCL8 had the highest expression levels in the PI group compared with the HI group, and they are known to be associated with inflammatory responses. In the gingival tissues, the expression of genes encoding cytokines such as interleukin (IL)-1β, tumor necrosis factor-alpha (TNF)-α, interleukin (IL)-6, and CXCL5/CXCL8 was assessed via RT-qPCR. The mRNA expression level of CXCL5/CXCL8 significantly increased in the PI group compared with the HI group (p < .045). Contrarily, the mRNA expression level of interleukin 36 receptor antagonist (IL36RN) significantly decreased (p < .008). IHC enabled examination of the distribution and intensity of CXCL5/CXCL8 protein expression within the tissue samples. Specifically, increased levels of CXCL5/CXCL8 promote inflammatory responses, cellular proliferation, migration, and invasion within the peri-implant tissues. These effects are mediated through the activation of the PI3K/Akt/NF-κB signaling pathway. This study found that the PI sites had higher gene expression level of CXCL8/CXCL5 in the soft tissue than HI sites, which could help achieve more accurate diagnosis and treatment planning.

VBP1 promotes tumor proliferation as a part of the hypoxia-related signature in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a common malignant tumor in East Asia. Hypoxia, a hallmark of solid tumors, significantly alters redox homeostasis inside tumor microenvironment. This alteration drives tumor proliferation, invasion, and metastasis, leading to poor prognostic outcomes. However, the role of hypoxia-related genes in ESCC remains poorly understood. We employed RNA sequencing to identify differentially expressed genes in ESCC. Clinical data, transcriptome profiles, and a hypoxia-related gene set were extracted from open-source databases. A prognostic model was constructed using least absolute shrinkage and selection operator (LASSO) regression, which was then validated through Cox regression analysis. Within this prognostic model, we pinpointed and investigated a key hypoxia-related gene affecting prognosis. The gene's expression was validated using real-time PCR and immunohistochemistry in both esophageal carcinoma and normal tissues. Tumor proliferation was examined through in vitro and in vivo assays, including the Cell Counting Kit-8, EdU, colony formation, and subcutaneous tumor models. A robust four-gene prognostic model (VBP1, BGN, CDKN1A, and PPFIA1) was successfully constructed and validated. Among these, VBP1 emerged as a key gene, exhibiting high expression levels that correlated with poor prognosis in ESCC. Functional experiments confirmed that VBP1 significantly accelerated tumor proliferation both in vitro and in vivo. VBP1 is identified as a pivotal gene within the hypoxia-related prognostic signature, and it significantly promotes tumor proliferation in ESCC.

A novel SLC3A2-targeting antibody-drug conjugate exerts potent antitumor efficacy in head and neck squamous cell cancer

The development of innovative therapeutic strategies for head and neck squamous cell carcinoma (HNSCC) is a critical medical requirement. Antibody-drug conjugates (ADC) targeting tumor-specific surface antigens have demonstrated clinical effectiveness in treating hematologic and solid malignancies. Our investigation revealed high expression levels of SLC3A2 in HNSCC tissue and cell lines. This study aimed to develop a novel anti-SLC3A2 ADC and assess its antitumor effects on HNSCC both in vitro and in vivo. This study developed a potent anti-SLC3A2 ADC (19G4-MMAE) and systematically investigated its drug delivery potential and antitumor efficacy in preclinical models. This study revealed that 19G4-MMAE exhibited specific binding to SLC3A2 and effectively targeted lysosomes. Moreover, 19G4-MMAE induced a significant accumulation of reactive oxygen species (ROS) and apoptosis in SLC3A2-positive HNSCC cells. The compound demonstrated potent antitumor effects derived from MMAE against SLC3A2-expressing HNSCC in preclinical models, displaying a favorable safety profile. These findings suggest that targeting SLC3A2 with an anti-SLC3A2 ADC could be a promising therapeutic approach for treating HNSCC patients.

Abstract PO2-03-07: Immune Landscape in PD-L1-positive and TROP2-positive Triple-Negative Breast Cancer

Abstract Background: Triple-negative breast cancer (TNBC) tends to be more aggressive, and more likely to recur than other subtypes of breast cancer, and patients with metastatic TNBC have a poor prognosis. The recent approvals and data of pembrolizumab (anti-PD-1 inhibitor) in combination with chemotherapy in advanced-metastatic and early high-risk TNBC, and sacituzumab govitecan (an ADC that targets the tumor cell surface antigen TROP2 coupled with the irinotecan metabolite SN-38) in metastatic TNBC, has shed light on some of the immune and cancer signaling associated with TNBC. Although exciting progress has been made in the treatment of TNBC, there are still important gaps to fill to understand the biology of TNBC and identify additional therapies. Materials and Methods: We identified a total of 1500 patients whose tumors were negative for the expression of ER and HER2 receptors by gene array. PD-L1 and TROP2 expression data were available for 802 early TNBC patients, of whom 657 were treated with adjuvant chemotherapy. A comprehensive identification and analysis of immune-related genes (n= 1169) was conducted. Mann-Whitney test comparing the gene expression for all immune related-genes was evaluated in patients expressing PD-L1 and TROP2 at low and high expression levels. Immune gene modules resembling key immune gene signaling were analyzed in all subgroups identified. Fold changes and Mann-Whitney P values were calculated in each subgroup. Results: Patients were stratified based on high and low expression levels of PD-L1 and TROP-2 mRNA. PD-L1 highly expressing (PD-L1+) tumors showed a marked difference in immune-related genes as compared to PD-L1 low-level tumors (PD-L1-) with PD-L1+ tumors demonstrating a significantly higher proportion of correlated immune genes (CXCL9/10/11, IFN-γ, CCL5 and STAT1). TROP2 highly and low expressing tumors (TROP2+ and TROP2-, respectively) showed a relatively limited difference in terms of immune-related gene expression with key immune-related genes having statistical differential expression in the two subgroups. TROP2+ tumors showed a high presence of several Mucin family gene members (MUC1, -5AC, -4). In addition, CLDN 4, VTCN1, and MET were among the key immune genes significantly expressed in TROP2+ tumors (P = 5.73E-20; P = 6.2E-13, P = 2.1E-11). Overall, PD-L1+ tumors and TROP2+ tumors showed an immune suppressive genomic landscape but were potentially regulated by different signaling. Of note, tumors with very high expression of PD-L1 showed an inverse correlation with VTCN1 gene expression. Conclusions: This preliminary analysis confirmed that only a limited number of early TNBC highly expressed PD-L1 and that a different immune suppressive landscape for PD-L1± and TROP2± tumors exists. In addition, our data suggest that early TNBC cases lacking PD-L1 might escape immune surveillance by virtue of the upregulation of key alternate signaling. Ongoing efforts investigating the expression of such markers on stroma/epithelial tumor components will help to clarify their biological, clinical, and potential therapeutic role in TNBC. Citation Format: Balazs Gyorffy, Libero Santarpia. Immune Landscape in PD-L1-positive and TROP2-positive Triple-Negative Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-03-07.

Association analysis between Acetyl-Coenzyme A Acyltransferase-1 gene polymorphism and growth traits in Xiangsu pigs.

Acetyl-Coenzyme A Acyltransferase-1 (ACAA1) is a peroxisomal acyltransferase involved in fatty acid metabolism. Current evidence does not precisely reveal the effect of the ACAA1 gene on pig growth performance. The present study assessed the mRNA expression levels of the ACAA1 gene in the heart, liver, spleen, lung, kidney of 6-month-old Xiangsu pigs and in the longissimus dorsi muscle at different growth stages (newborn, 6months and 12months of age) using RT-qPCR. The relationship between single-nucleotide polymorphisms (SNPs) of ACAA1 gene and growth traits in 6-month-old and 12-month-old Xiangsu pigs was investigated on 184 healthy Xiangsu pigs using Sanger sequencing. The ACAA1 gene was expressed in heart, liver, spleen, lung, kidney, and longissimus dorsi muscle of 6-month-old pigs, with the highest level of expression in the liver. ACAA1 gene expression in the longissimus dorsi muscle decreased with age (p < 0.01). In addition, four SNPs were identified in the ACAA1 gene, including exon g.48810 A>G (rs343060194), intron g.51546 T>C (rs319197012), exon g.55035 T>C (rs333279910), and exon g.55088 C>T (rs322138947). Hardy-Weinberg equilibrium (p > 0.05) was found for the four SNPs, and linkage disequilibrium (LD) analysis revealed a strong LD between g.55035 T>C (rs333279910) and g.55088 C>T (rs322138947) (r 2 = 1.000). Association analysis showed that g.48810 A>G (rs343060194), g.51546 T>C (rs319197012), g.55035 T>C (rs333279910), and g.55088 C>T (rs322138947) varied in body weight, body length, body height, abdominal circumference, leg and hip circumference and living backfat thickness between 6-month-old and 12-month-old Xiangsu pigs. These findings strongly demonstrate that the ACAA1 gene can be exploited for marker-assisted selection to improve growth-related phenotypes in Xiangsu pigs and present new candidate genes for molecular pig breeding.

Abstract PO3-27-02: A novel PTK6 PROTAC degrader induces apoptosis of drug resistant breast cancer cells

Abstract Background. PTK6/Brk, a non-receptor tyrosine kinase, is an oncogenic driver for several tumor types, including breast, prostate, liver, and pancreatic cancers. PTK6 is expressed in all breast cancer subtypes and higher levels of expression are associated with worse patient outcomes. The optimal approach to targeting PTK6 for therapeutic benefit has not been established. Although several inhibitors of PTK6 kinase activity have been developed, none have thus far been successfully translated to clinical trials. This may be in part due to kinase activity-independent functions of PTK6, for which there is growing evidence, that are not effectively targeted by traditional small molecule kinase inhibitors. Downregulation of PTK6 using shRNA inhibits growth, invasion/migration, survival and metastasis of breast cancer cells, including those that are resistant to standard therapies. PTK6 kinase inhibitor treatment phenocopies effects of PTK6 downregulation with respect to some, but not all PTK6-driven oncogenic activities. Moreover, growth of PTK6 CRISPR-targeted cells can be rescued either with wild type or catalytically inactive PTK6, supporting kinase activity-independent activities of PTK6 in regulating growth and viability of breast cancer cells. Therefore, PROTAC degraders may be a better way to chemically target and downregulate PTK6 expression in cancer, thereby phenocopying the effects of PTK6 shRNA. Methods. We developed a first-in-class PTK6 degrader using PROTAC (proteolysis-targeting chimeric) technology to target PTK6 expression in breast cancer cells of diverse subtypes. Degrader controls were also synthesized to assess specificity of MS105’s effects. Results. PTK6 degrader, but not degrader controls, potently downregulates PTK6 expression in multiple breast cancer cell types. Proteomic and kinome profiling confirm the specificity of our PTK6 degrader. PTK6 PROTAC degrader inhibits viability and induces apoptosis of PTK6-expressing breast cancer cells, whereas treatment with parent kinase inhibitor or degrader controls that do not engage PTK6 or E3 ligase have minimal effect. Treatment with PTK6 degrader, but not kinase inhibitor or degrader controls, induces expression of pro-apoptotic Bim, phenocopying the effects observed with PTK6 shRNA. In contrast, PTK6 degrader and kinase inhibitor are comparably effective in inhibiting cell migration, supporting the differential kinase dependency of PTK6-dependent oncogenic functions. Therefore, our PTK6 PROTAC degrader effectively inhibits both kinase-dependent and independent oncogenic activities of PTK6. Our studies support the development of PTK6 PROTAC degraders as a preferred approach to clinically targeting PTK6 in cancer. Citation Format: Hanna Irie, Criseyda Martinez, Yan Xiong, Alison Bartkowski, Jessica Byerly, Jian Jin. A novel PTK6 PROTAC degrader induces apoptosis of drug resistant breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-27-02.

Abstract PO4-15-12: Personalized circulating tumor DNA monitoring to predict response to neoadjuvant therapy in patients with early-stage breast cancer

Abstract Introduction Longitudinal ctDNA testing offers a minimally invasive approach for monitoring treatment response in patients with breast cancer (BC). In this study, we evaluated whether serial ctDNA testing during neoadjuvant therapy (NAT) can provide an early indication of treatment response or resistance and disease progression. Methods In this real world study, longitudinal blood samples collected from 159 patients with stage I-III BC were analyzed using a personalized, tumor-informed ctDNA assay (SignateraTM bespoke mPCR-NGS assay). Of the 159 patients, 157 received standard of care NAT; the remaining 2 patients were treated with endocrine therapy only in the neoadjuvant setting. Blood samples were collected at the time of diagnosis from 71 patients (baseline) and longitudinally during treatment from 159 patients (median time between time points: 1.5 months). ctDNA status and dynamics were correlated with clinicopathological features and surgical outcomes available at the time of data cut off. Results At baseline, ctDNA was detected in 72% (51/71) of patients. Baseline ctDNA detection rates varied based on histologic subtypes: 82% (28/34) in patients with triple negative breast cancer, 88% (15/17) in patients with HER2+ disease and 40% (8/20) in patients with ER+HER2- BC. ctDNA was detectable at baseline in 72% (26/36) of patients with clinical T1-2 disease and 92% (11/12) of patients with T3-4 disease. ctDNA was detected in 64% (16/25) of patients with clinical N0, whereas among patients with at least one positive lymph node, ctDNA was detected in 92% (22/24). At baseline, 65% (31/48) of patients with low/intermediate-grade disease were ctDNA-positive, while 81% (25/31) of patients with high-grade disease were ctDNA-positive. Similarly, 83% (24/29) of patients with high levels (&amp;gt;20%) of Ki67 expression had detectable ctDNA prior to treatment initiation, while only 22% (2/9) of those with low level (&amp;lt; 20%) of Ki67 expression had ctDNA detected. Surgical outcomes were available for 22 patients of whom 16 had achieved pathologic complete response (pCR). While 3/16 pts were ctDNA negative at baseline, all of the remaining cases except one patient with baseline ctDNA detection and serial measurements (11/12) became ctDNA negative by the end of two months of treatment. Despite achieving pCR, one patient with inflammatory ER+HER2+ BC remained ctDNA positive before and after the surgery, which informed a change in the adjuvant treatment strategy. Among those with residual disease (pT1c-T3), 28% (7/25) remained ctDNA positive after two months of NAT. Association between ctDNA dynamics and surgical outcomes of the remaining 137 patients will be presented at the time of the conference. Conclusions This real world study demonstrates the prevalence of ctDNA detection and dynamics in patients with eBC treated with NAT. ctDNA monitoring during NAT can facilitate real-time assessment of treatment response and serve as an early indicator of subsequent pCR to NAT. Citation Format: Mridula George, Trishala Meghal, Coral Omene, Ekaterina Kalashnikova, Janie Fielder, Nisha Ohri, Shicha Kumar, Valeria Burkovskaya, Preena Patel, Ashley Young, Melanie Racenstein, Tinamarie Bauman, Wassim Mchayleh, Deborah Toppmeyer, Shridar Ganesan, Barry Rosen, Angel Rodriguez, Minetta Liu. Personalized circulating tumor DNA monitoring to predict response to neoadjuvant therapy in patients with early-stage breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-15-12.

Abstract PO1-24-08: Targeting of HER3 potentiates the antitumor activity of paclitaxel against triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is characterized by the absence of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) overexpression. This aggressive form of breast cancer carries a higher risk of recurrence compared to other subtypes, presenting a significant clinical challenge. Although chemotherapy is commonly used as the primary treatment for a substantial number of TNBC patients, it is often accompanied by drug resistance and frequent tumor recurrence. Urgent efforts are needed to identify novel molecular targets specific to TNBC and develop effective therapies to combat this aggressive disease. The ultimate objective is to discover treatments capable of overcoming drug resistance and improving the overall survival of patients with TNBC. Methods: Immunohistochemistry was performed to examine the expression of HER3 in TNBC samples. Western blots were used to assess protein expression and activation. Cell proliferation and viability were determined by cell growth (MTS) assays. TCGA databases were analyzed to correlate HER3 mRNA expression with the clinical outcomes of TNBC patients. Specific shRNA was used to knockdown HER3 expression. LIVE/DEAD Cell Imaging was to detect live and dead cells. Orthotopic tumor models were established in nude mice to determine the capability of TNBC cells forming tumors and to test if our newly developed anti-HER3 monoclonal antibody (mAb) 4A7 could potentiate the antitumor activity of paclitaxel in vivo. Results: The chemo-resistant subline HCC1806-TR was obtained by subjecting TNBC HCC1806 cells to prolonged treatment with paclitaxel. In comparison to the parental cell line HCC1806-P, HCC1806-TR exhibited enhanced HER3 expression and activation. It is noteworthy that approximately half of the tested TNBC specimens and cell lines displayed elevated expression of HER3. Analyzing TCGA databases revealed that TNBC patients with high levels of HER3 mRNA expression in their tumors had significantly poorer overall survival (OS) and relapse-free survival (RFS) compared to those with low HER3 expression. Specifically knockdown of HER3 markedly inhibited TNBC cell proliferation and mammosphere formation in vitro and tumor growth in vivo. Blockade of HER3 signaling with our mAb (4A7) in combination with paclitaxel exerted significant antitumor activity against TNBC in vitro and in vivo. Conclusions: Our data demonstrate that increased HER3 is an effective therapeutic target for TNBC and our anti-HER3 mAb (4A7) may enhance the efficacy of paclitaxel in TNBC. Keywords: HER3, Anbibody, Chemotherapy, Triple Negative Breast Cancer Citation Format: Hui Lyu, Sanbao Ruan, Congcong Tan, Ann Thor, Bolin Liu. Targeting of HER3 potentiates the antitumor activity of paclitaxel against triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-08.

Abstract PO4-06-12: Distinction of basal-like and triple-negative basal-like breast cancers utilizing a novel comprehensive single-cell liquid biopsy-based test

Abstract Introduction: Breast cancer is a highly heterogeneous disease and is a leading cause of death for women worldwide. Triple-negative breast cancer (TNBC), the most aggressive form of the disease, is clinically defined by the lack of expression of estrogen receptor (ER) and progesterone receptor (PR), as well as lack of over-expression of human epidermal growth factor receptor-2 (HER2). Approximately 70% of all TNBC cases exhibit basal-like cells, characterized by high basal expression of keratins as well as extensive genomic instability with copy number gains and losses across most chromosomes. In this study we show that novel single cell genomic evaluation coupled with immunofluorescence (IF) protein analysis of CTCs can detect TNBC with basal-like genomic features as well as basal-like tumors with expression of ER and HER2. Materials and Methods: Epic Sciences’ DefineMBC™ liquid biopsy test characterizes CTC proteomics and genomics as well as ctDNA for the treatment of metastatic breast cancer. Blood samples from 813 metastatic breast cancer patients, with either newly diagnosed or progressing disease, with historical tissue subtype [HR(+)HER2(-), HR(+/-)HER2(+), and TNBC] supplied by the referring physician were collected and tested. Up to 5 CTCs were selected and isolated from each sample based on morphology and IF signal. DNA was extracted from individual cells, and whole genome amplified and sequenced at low coverage. Copy number analysis was performed to determine copy number gains and losses across chromosomes, as well as large scale-state transitions (LSTs) and ERBB2 amplification. CTCs characterized with high chromosomal instability (e.g., exhibiting &amp;gt;30 LSTs; 1Q firestorm and 8Q stair step) and high levels of cytokeratin expression (mean fluorescence intensity signal &amp;gt;10K) were categorized as basal-like. Results: By previous metastatic tissue biopsy, 77% were HR(+), 8% were HER2(+), and 12% were TNBC. CTCs categorized in DefineMBC as basal-like subtype (9.8 % of all samples tested) showed to be highly present in patients called as TNBC (79% basal-like). Additionally, 54% of HR(+) and 23% of HER2(+) had basal-like CTCS. Patients characterized with TNBC by metastatic tissue biopsy and by DefineMBC exhibited basal-like cells in 88% of the cases. Conclusion: The results show a novel clinical assay which utilizes CTC protein detection as well as single-cell genomic analysis to identify tumors driven by basal-like cells. Generally, TNBC is classified with negative biomarker expression. We show that CTCs in TNBC display genomically basal-like cells. CTCs from HR(+) and HER2(+) tumors may express basal-like features with preserved ER and HER2 expression. The comprehensive nature of DefineMBC™ utilizing genomic analysis of detected CTCs has provided a new tool to diagnose both basal-like TNBC and basal-like hormone receptor positive and HER2 over-expressing tumors. Citation Format: Alessandra Cunsolo, David Bourdon, Ernest Lam, Giuseppe Di Caro, Nilesh Dharajiya, Timothy Pluard, Lee Schwartzberg. Distinction of basal-like and triple-negative basal-like breast cancers utilizing a novel comprehensive single-cell liquid biopsy-based test [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-06-12.

Abstract PO5-24-11: An integrated approach for comprehensive molecular and tumor microenvironment characterization of invasive lobular carcinoma

Abstract Introduction: Although recent studies have shown that invasive lobular carcinoma (ILC) is associated with worse long-term outcomes compared to invasive breast cancer of no special type (NST), they are treated in a similar manner. Therefore, classification approaches to help understand the molecular characteristics associated with the different BC subtypes are needed to personalize therapy. Here, we describe an integrated analytical approach to comprehensively characterize ILC samples. Methods: We collected genomic and transcriptomic data for 16,087 ILC and NST samples, with clinical and pathological annotation, from 59 datasets and used an internally developed algorithm based on clustering performed after gene expression analysis to classify them into 5 intrinsic subtypes: Basal, Luminal A (LumA), Luminal B (LumB), HER2 high, and HER2 low. We performed gene set enrichment analyses and utilized the DESeq2 algorithm to perform differential expression analysis of RNA-seq data. We performed molecular grade subtyping using methods described by Antysheva et al. (Cancer Res., 2022). Samples that were listed as ILC or had an inactivated CDH1 gene by mutation, deletion or low expression (&amp;lt; −2.5 MAD after median scaling) were defined as having an ILC histomolecular subtype. Using methods described by Bagaev et. al. (Cancer Cell, 2021), 29 functional gene expression signatures were selected, and unsupervised dense Louvain clustering was performed to identify TME subtypes. The Mann-Whitney U test was used for statistical analysis. The tertiary lymphoid structures (TLS) gene signature was evaluated using methods described by Sautès-Fridman et al. (Nat. Rev. Cancer, 2019). Results: In the ILC samples (n=1,887), we identified LumA (46%), LumB (40%), Basal (8%,), HER2 high (3%), and HER2 low (3%) subtypes. In contrast, the NST samples were classified as LumB (41%), LumA (25%), Basal (19%), HER2 high (9%), and HER2 low (5%). Molecular grade subtyping revealed that 67% of ILC samples were low grade and 33% were high grade, compared to NST where 55% were high grade and 45% were low grade. Notably, among high-grade ILC, LumB (64%) and Basal (23%) subtypes were predominant. Key differentially expressed genes included downregulated CDH1 and upregulated MMP23B, IGF1, and TFAP2B across LumA and LumB ILC samples. Further analysis revealed downregulation of TACSTD2 and CDH3 in the Basal subtype for ILC samples compared to NST samples. Transcriptomic-based TME subtyping revealed distinct TME distributions across the BC subtypes and molecular grades for ILC samples (Table 1). In high-grade ILC LumB samples, 38% had a Desert (D; predictive of poor immunotherapy response) and 39% had an Immune-Enriched (IE; predictive of favorable immunotherapy response) TME. In low-grade ILC LumB samples we found that while 51% had a D TME, only 14% had an IE TME. We found an overrepresentation of the TLS signature [Solid TLS] in ILC compared to NST samples, and higher matrix remodeling and CAF signatures in LumA compared to the LumB subtype among low-grade ILC samples. Samples with high CD274 expression levels had higher percentages of T cells and M1 macrophages, and LumA samples had significantly higher (p &amp;lt; 0.001) CD274 expression compared to the other BC subtypes. Conclusion: These results provide in-depth molecular and tumor microenvironment characterization of ILC. Further optimization of this analytical approach could lead to the development of more effective therapeutic strategies. Table 1. ILC subtypes classification Citation Format: Jason Mouabbi, Konstantin Chernyshov, Oleg Baranov, Vladimir Kushnarev, Polina Turova, Anna Butusova, Sofia Menshikova, Jessica Brown, Nikita Kotlov, Patrick Clayton, Krystle Nomie, Nathan Fowler, Debu Tripathy. An integrated approach for comprehensive molecular and tumor microenvironment characterization of invasive lobular carcinoma [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-24-11.

Abstract PO5-04-04: Development of a method to detect very low levels of HER2 expression in Circulating Tumor Cells (CTCs) by liquid biopsy in patients with metastatic breast cancer

Abstract Introduction Blood-based liquid biopsies are a non-invasive diagnostic approach for detecting circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) that may provide clinically actionable information for treatment decisions for metastatic breast cancer (MBC) patients when a conventional tissue biopsy is not feasible. Recently, the novel anti-HER2 antibody-drug conjugate, Trastuzumab Deruxtecan (T-DXd), has shown effectiveness against MBC with low HER2 expression detected by tissue biopsy characterized as HER2-low (immunohistochemically 1+ or 2+ and ERBB2 amplification-negative via in situ hybridization) and is being explored in an ultra-low population. Here we report on the application of a liquid biopsy platform designed to sensitively detect low levels of HER2 protein expression in CTCs from MBC patients. Methods Blood samples from 704 metastatic breast cancer patients as well as control cells lines were collected for cell-based and cell-free DNA analysis. After plasma isolation, nucleated cells deposited on glass slides went through immunofluorescent staining and imaging. CTCs were identified using Epic Sciences’ digital imaging and machine learning algorithms, and a subset of CTCs were isolated and sequenced for genomic quantification of large scale-state transitions (LSTs) as well as copy number variants (CNVs). The HER2 status was based upon on ASCO-CAP guidelines and the previous tissue biopsy results from the ordering healthcare provider, as documented on the DefineMBC™ test requisition form. Results Within a DefineMBC early-access program (EAP), 93% of patients with MBC had detectable CTCs. Among ERBB2-non-amp patients tested with the DefineMBC assay, 25% of patients with MBC had findings that were reported as consistent with HER2-low in the CAP-CLIA setting. To explore the lower boundaries of HER2 protein expression, we created and validated an MCF-7 HER2-negative ERBB2 knock-out cell line and utilized its 95% confidence level threshold (271 MFI) as a negative fluorescence cutoff for very low HER2 expression. Based upon publicly available CCLE datasets, MDA-MB-453, MCF7, and MCF7 ERBB2 KO breast cancer cell lines were selected for their high and low HER2 RNA expression levels and were corroborated via observed HER2 immunofluorescence median levels of 15740, 381, and 188 MFI respectively. In an exploratory analysis, among ERBB2-non-amp patients, we retrospectively applied the 271 MFI HER2 cutoff to the EAP cohort and identified 38% of patients with very low HER2 expression. We examined the impact of the HER2 ultra-low cutoff in patients not eligible for T-DXd treatment. Among patients reported as IHC=0 or with unknown IHC status from the most recent tissue biopsy, 44% and 42%, respectively, may qualify for treatment with T-DXd. Conclusion Using our higher sensitivity assay that combines CTC immunofluorescence and single-cell genomic analysis, we examined the lower boundary of HER2 protein expression. Feasibility data support further characterization of a very low HER2 expression phenotype, for the eventual inclusion into our comprehensive cancer profiling solution. To date, studies with biomarker expression have been limited to tissue biopsy, which may not always yield contemporaneous sampling in the metastatic setting. These results offer a liquid biopsy test that might identify patients potentially responsive to HER2-directed therapies. Citation Format: Giuseppe Di Caro, David Bourdon, Andrew Kunihiro, Alessandra Cunsolo, Ernest Lam, Megan Slade, Martin Blankfard, Lee Schwartzberg. Development of a method to detect very low levels of HER2 expression in Circulating Tumor Cells (CTCs) by liquid biopsy in patients with metastatic breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-04-04.

Abstract PO5-16-03: RT LAMP assay for the subtyping of breast cancer

Abstract Introduction Our previous study showed that direct reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay can be employed as a substitute in order to detect tumor involvement of lymph nodes within breast cancer patients. In this research, we aimed to investigate the potential applicability of RT-LAMP for the classification of breast cancer subtype. Method Breast Cancer Cell line RNA extraction Total RNA from the cell lines was isolated from cultured cells using RNAiso Plus (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions; MCF10A, MCF7, T47D, BT474, SKBR3, MB468, HCC1806, MB231 Primer design For the Primer design, ER, PR, HER2, Ki 67, CK7 and GATA were targeted. LAMP primers targeted at ER were created between exons 5 and 6, PR were created between exons 6 and 7, HER2 were created between exons 17 and 18, KI67 were created between exons 2 and 3, CK7 were created between exons 5 and 6, and GATA3 were created between exons 2 and 3 utilizing Primer Explorer V4 (http://primereplorer.jp/elamp4.00/index.html). RT-LAMP assays RT-LAMP assays were carried out within a 25 µL reaction which included 12.5 µL of 2 × reaction buffer, 2µL of each primer, 1 µL of enzyme mixture (16U Bst DNA polymerase and 120U M-MLV reverse transcriptase) 8 µL of DEPC-treated water, and 2 µL of the template RNA. DEPC-treated water was also employed for purposes of a negative control. The reaction mixture was conducted within qRT-PCR. Results ER, PR, HER2, Ki-67, CK7, GATA3 expression level were checked for each breast cancer cell line using RT-LAMP. The luminal cell line exhibited elevated levels of ER and PR expression, while the her2 cell line demonstrated a high level of expression for her2. In the RT LAMP assay, there is a direct correlation between shorter detection time and higher expression levels of the substance. Specifically, in the luminal type, ER/PR was detected within a timeframe of 10 to 12 seconds, while HER2 was detected within a timeframe of 15 to 16 seconds. The expression levels of ER, PR, and HER2 were compared between MCF7 (luminal A) and MDA-MB-231 (TNBC) cell lines. RT-LAMP was performed to examine the RNA expression levels, while RT-PCR was applied to confirm the cDNA expression levels. MCF7 and MDA-MB-231 were mixed at 1:9, 5:5 and 9:1 ratios, respectively, and the difference in ER/PR/HE2 expression level was confirmed (Table1, Figure1). The presence of ER was detected in the MCF7 cell line at 14.44 minutes, and PR was observed at 18.84 minutes. HER2 was detected at a similar time (approximately 15 minutes) in both the MCF7 and MB 231 cell lines. Conclusion This study demonstrated that ER, PR, and HER2 can be quantified using RT-LAMP. Ongoing experiments are being conducted to classify breast cancer using the RT-LAMP method, aiming to enhance sensitivity in the quantification of ER, PR, and HER2. Additionally, new primers are being developed specifically for HER2 detection. RT-LAMP offers the advantages of being a point-of-care test that can be conducted on-site and reducing the discrepancies between pathologists. Citation Format: In Hee Lee, Byeongju Kang, Jeeyeon Lee, Jin Hyang Jung, Ho Yong Park, Nora Jee-Young Park, Ji-Young Park, Eun Ae Kim, Jieun Kang, Yee Soo Chae, Soo Jung Lee. RT LAMP assay for the subtyping of breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-16-03.

Investigating the novel role of sarcospan in modulating immune function in mice

Objective: The tetraspanin-like protein sarcospan (SSPN) has been shown to have an important in stabilizing muscle cell membranes and promoting cell adhesion. SSPN has documented expression in immune cells with the highest level of expression in B cells. Examination of SSPN-deficient (SSPN−/−) mice revealed alterations in important immune pathways, which remain uncharacterized. This led us to hypothesize that SSPN may play a role in modulating immune cell development and/or function. Methods: Flow cytometry was used to perform immunophenotyping of spleens obtained from SSPN−/− and WT mice. Cytokine analysis was performed using Luminex to assess cytokines that serve as important signals for immune cell maturation. Hematopoietic stem cells were isolated from red bone marrow and stimulated with phorbol myristate acetate (PMA), a mitogen and polyclonal activator of B cells, to assess whether SSPN deficiency affected their proliferative capacity. To determine whether SSPN plays a role in immunoglobulin isotype switching — antibody isotyping was performed. The presence of immune (antibody-antigen) complexes was investigated in kidney sections obtained from WT and SSPN−/− mice and visualized using biotinylated IgG and IgM antibodies. Autoantigen screening was also performed to assess whether SSPN−/− mice generated abnormal levels of autoantibodies that could be responsible for circulating antibody-antigen complexes. Results: The presence of SSPN on major immune cell types was confirmed using flow cytometry. In addition, flow cytometry analysis showed insignificant baseline differences in the percentage of B cells, T cells, plasmacytoid dendritic cells in SSPN−/− compared to WT spleens. One difference was noted in SSPN−/− females with increased counts of dendritic cells. Spleens in female SSPN−/− mice were found to be slightly larger compared to WT. Levels of B-cell activating factor (BAFF) and B-cell maturation antigen (BCMA) were elevated in SSPN−/− mice, therefore it is expected that SSPN−/− B cells received appropriate signals. Overall B cell proliferation was assessed by CD19+ and appeared unaffected by the absence of SSPN. Subtle increases in IgG1 and IgG2b were detected in SSPN−/− mice, however immunoglobulin isotypes were within normal ranges. SSPN−/− mice were also assessed for signs of dysregulated immune reactivity, evidenced by the presence of immune (antibody-antigen) complexes within filtration units of the kidney. Overall, male SSPN−/− mice were found to have a larger subset of increased IgG autoantibodies compared to male WT mice while female SSPN−/− mice exhibited a larger subset of increased IgM autoantibodies compared to female WT mice. Conclusions: This study examines potentially important functions of SSPN in the immune system. Overall, deletion of SSPN has not been found to affect maturation of major immune cell classes. However, SSPN does appear to influence formation of antigen-antibody complexes in mice, which may contribute to kidney dysfunction. These studies are ongoing and directed toward understanding the role SSPN plays in immune function. FSU Funding, Florida Department of Health, James and Esther King Biomedical Research Program #21K12. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.