Archival Clinical Samples Research Articles

Challenges and Opportunities in the Clinical Translation of High-Resolution Spatial Transcriptomics.

Pathology has always been fueled by technological advances. Histology powered the study of tissue architecture at single-cell resolution and remains a cornerstone of clinical pathology today. In the last decade, next-generation sequencing has become informative for the targeted treatment of many diseases, demonstrating the importance of genome-scale molecular information for personalized medicine. Today, revolutionary developments in spatial transcriptomics technologies digitalize gene expression at subcellular resolution in intact tissue sections, enabling the computational analysis of cell types, cellular phenotypes, and cell-cell communication in routinely collected and archival clinical samples. Here we review how such molecular microscopes work, highlight their potential to identify disease mechanisms and guide personalized therapies, and provide guidance for clinical study design. Finally, we discuss remaining challenges to the swift translation of high-resolution spatial transcriptomics technologies and how integration of multimodal readouts and deep learning approaches is bringing us closer to a holistic understanding of tissue biology and pathology.

Positionally distinct interferon stimulated dermal immune acting fibroblasts promote neutrophil recruitment in Sweet's syndrome.

Sweet's syndrome is a poorly understood inflammatory skin disease characterized by neutrophil infiltration to the dermis. Single-nucleus and bulk transcriptomics of archival clinical samples of Sweet's syndrome revealed a prominent interferon signature in Sweet's syndrome skin that was reduced in tissue from other neutrophilic dermatoses. This signature was observed in different subsets of cells, including fibroblasts that expressed interferon-induced genes. Functionally, this response was supported by analysis of cultured primary human dermal fibroblasts that were observed to highly express neutrophil chemokines in response to activation by type I interferon. Furthermore, single-molecule resolution spatial transcriptomics of skin in Sweet's syndrome identified positionally distinct immune acting fibroblasts that included a CXCL1+ subset proximal to neutrophils and a CXCL12+ subset distal to the neutrophilic infiltrate. This study defines the cellular landscape of neutrophilic dermatoses and suggests dermal immune acting fibroblasts play a role in the pathogenesis of Sweet's syndrome through recognition of type I interferons.

Abstract 4195: Spatially resolved single cell transcriptomic profiling in formalin-fixed paraffin-embedded (FFPE) tissues

Abstract Formalin-fixed paraffin-embedded (FFPE) tissues are the most widely used clinical sample types in histology and molecular diagnosis, but these samples are often challenging for single-cell transcriptomic analysis due to RNA degradation and protein crosslinking. A spatial transcriptomics technique with high detection efficiency and single molecule resolution is required in order to accurately profile the gene expression in FFPE samples in situ. Vizgen’s MERSCOPE platform, built on multiplexed error robust in situ hybridization MERFISH technology, directly profiles intact tissue’s transcriptome with subcellular spatial resolution. Here, we demonstrate the FFPE MERSCOPE workflow in tissues from 10 mouse and human samples, including archival clinical samples. In each sample, hundreds of thousands of cells were captured with >100 million transcript counts, generating detailed spatial transcriptomic data for the profiled genes in each sample. A comparison of FFPE and matched fresh frozen samples indicated that the FFPE workflow performs similarly in detection efficiency as compared to the fresh frozen protocol. We further demonstrated the MERSCOPE FFPE workflow is compatible with protein imaging by performing simultaneous protein-based cell boundary staining with MERFISH to accurately profile gene expression and map cell types in archival clinical human samples. Finally, we constructed a spatially resolved single cell atlas across eight major tumor types, mapped and cataloged different cell types within the tumor microenvironment and systematically characterized the gene expression among cells. This study demonstrates the potential for spatially resolved transcriptomic profiling of FFPE samples at single cell level to contribute to a wide range of biomedical research areas, including many applications to study human diseases. Citation Format: Jiang He, Justin He, Timothy Wiggin, Rob Foreman, Renchao Chen, Nicolas Fernandez, George Emanuel. Spatially resolved single cell transcriptomic profiling in formalin-fixed paraffin-embedded (FFPE) tissues. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4195.

Abstract 3869: Short or long-term treatment with CDK4/6 inhibitors in patients with ER+ breast cancer: characterization and comparative analysis of resistance in seventeen XPDX models

Abstract Background: Mechanisms of resistance to CDK4/6 inhibitors (CDK4/6i) have been well studied and several alterations identified including RB loss and altered expression of related genes including CCNE1, E2F4 and CDK6. However, whether duration of clinical treatment might elicit specific mechanism(s) of CDK4/6i resistance is unclear. To better understand if duration of clinical treatment correlates with unique resistance mechanisms, we established, characterized, and compared a panel of ER+ breast XPDX models from patients who benefitted then progressed on a CDK4/6i. Patients were separated into two groups by time to progression (TTP): those who responded up to twelve months (RES12) and patients with clinical response greater than one year (RES13+). Methods: Seventeen breast cancer XPDX models were analyzed, including eight previously described (7xRES12; 1xRES13+: SABCS2021: T Hernandez et al). Nine new models were established from seven patients: six from fluid samples with three designated as ductal (ST3105B, ST3105C, STM001B) and three lobular carcinoma (STM182, STM229, STM229B); two from lymph node biopsies (ST5676, STM127) and one from a liver core biopsy (ST4887B), all reported as ductal carcinoma. STM182 was classified as RES12 and the remaining eight as RES13+. These models were passaged and challenged with CDK4/6i to confirm resistance. Receptor expression was determined by IHC and genomic analyses including WES and RNAseq, were performed to identify mechanisms of resistance. For in vivo studies, CDK4/6i were dosed PO once daily at 50 mg/kg; endpoints included tumor volume (TV) and time from treatment initiation (TTI) with %T/C values and tumor regression reported at study completion; a %T/C of ≤20 versus control was considered sensitive. Tumor regression (%T/C<0) versus Day 0 TV was also reported. Results: Clinical TTP for RES12 (n=8) was four to twelve months and RES13+ (n=9) from thirteen to forty-two months. All models retained ER expression in evaluated passages with similar histology compared with archival clinical samples. Sequencing identified several variants including RB1 truncations or deletions and increased gene expression in CCND1, CCNE1 and the PIK3CA/AKT pathway. Interestingly, 5/8 RES12 models reported ESR1 mutations or fusions versus 1/9 RES13+ and PIK3CA mutations were reported in 1/8 RES12 versus 5/9 RES13+. Several RES13+ models also reported variants and increased amplification in the RICTOR/TORC2 pathway versus RES12. Conclusion: We have established, characterized, and compared a panel of seventeen breast XPDX models from fourteen female patients representing early or late acquired resistance to CDK4/6i therapy and identified potential differences in each set. These models and resulting data are useful in developing novel therapies for CDK4/6i-resistant patients. Citation Format: Alyssa Simonson, Johnnie Flores, Morgan Lynch, Emily Carpenter, Justine Hruzek, Jim Lund, Natalia Baños Herraiz, Kyriakos Papadopoulos, Amy Vander Woude, Gladys Rodriguez, Sreenivasa Chandana, Thomas Gribbin, Nehal Lakhani, Tatiana Hernandez, Maria Jose de Miguel, Amy Lang, Michael J. Wick. Short or long-term treatment with CDK4/6 inhibitors in patients with ER+ breast cancer: characterization and comparative analysis of resistance in seventeen XPDX models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3869.

Abstract 3855: Establishment and characterization of a panel of breast XPDX models representing innate or acquired resistance to trastuzumab deruxtecan (T-DXd)

Abstract Background: Trastuzumab deruxtecan (T-DXd) is an ADC consisting of an anti-HER2 antibody linked to a novel topoisomerase I inhibitor payload approved for T-DM1-refractory, HER2+ breast cancer (BC) and metastatic, recurrent HER2-low BC. While this therapy is effective in some patients, resistance often develops. To aid in developing new therapies for T-DXd-resistant BC and better understand potential resistance mechanisms, we established a panel of six breast XPDX models representing innate or acquired resistance to T-DXd. These models, designated ST4565C, ST4565D, STM148B, STM148C, STM148D, and ST4480B/EHR were developed and characterized for receptor expression, genomics, and drug sensitivities toward chemotherapies and targeted agents including T-DXd, T-DM1, and margetuximab. Methods: ST4565C/D was established from a patient with ER+/HER2+, metastatic BC who was T-DXd treatment naïve. ST4565C was collected post chemo- and HER2-targeted therapies; ST4565D was collected following eribulin/margetuximab. STM148B/C/D were established from a patient with ER-/HER2+ metastatic BC following trastuzumab, T-DM1, and eleven months of T-DXd. ST4480B/EHR was established by chronic in vivo T-DXd dosing to the parent ST4480B ER+/HER2+ model until resistance developed. Resulting models were passaged and receptor expression confirmed by IHC and genomic analyses, including WES and RNAseq, were performed to further characterize models. In vivo, models were tested with agents including: trastuzumab, T-DM1, and T-DXd; endpoints included tumor volume (TV) and time from treatment initiation (TTI) with %T/C values and tumor regression reported at study completion; a %T/C of ≤ 20 versus control was considered sensitive. Tumor regression (%T/C=<0) versus Day 0 TV was also reported. Results: Models retained receptor expression and similar histology comparable with archival clinical or model samples. In vivo, all ST4565 and STM148 models were refractory to trastuzumab, T-DM1, and T-DXd up to 10 mg/kg weekly with an average %T/C of 75%. ST4480B/EHR showed similar resistance to trastuzumab and T-DM1 at 10 mg/kg, and T-DXd at 3 mg/kg; T-DXd tested at 3 mg/kg in the ST4480B parent model resulted in tumor regressions. Sequencing of ST4565C/D identified a novel fusion, MTAP/CDKN2A/B deletions and elevated expression of FGFR1 and CCND1. In STM148 models an AKAP8L-NOTCH3 fusion and PIK3CA and CCNE1 mutations were reported. ST4480B/EHR reported PIK3CAE545K and an ESR1-CCDC170 fusion identical to the parent model, but differential expression was noted in several genes in the RICTOR/TORC2 pathway. Conclusion: We established and characterized six breast XPDX models representing innate or acquired resistance to T-DXd. These models are valuable tools in understanding resistance mechanisms and in developing novel therapies for T-DXd-resistant patients. Citation Format: Maci DeBoer, Albert Paez, Johnnie Flores, Robyn Baeza, Alyssa Simonson, Jim Lund, Kyriakos Papadopoulos, Thomas Gribbin, Amy Lang, Gladys Rodriguez, Lorena Mozas, Lorena Gonzalez, Manish Sharma, Emiliano Calvo, Tatiana Hernandez, Michael J. Wick. Establishment and characterization of a panel of breast XPDX models representing innate or acquired resistance to trastuzumab deruxtecan (T-DXd). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3855.

Abstract P1-13-03: Establishment and characterization of two T-DM1-resistant, ER+/HER2+ breast XPDX models developed sequentially from the same patient with differential in vivo sensitivity to trastuzumab deruxtecan (DS-8201a)

Abstract Background: Trastuzumab deruxtecan (DS-8201a) is an antibody-drug conjugate (ADC), consisting of a humanized anti-HER2 (human epidermal growth factor receptor 2) monoclonal antibody linked to a topoisomerase I inhibitor payload using a cleavable tetrapeptide-based linker, approved for the treatment of HER2+ metastatic breast cancer patients refractory to anti-HER2 therapy including T-DM1. While some mechanisms for clinical T-DM1 resistance have been identified, less is known about innate or acquired resistance to DS-8201a. We established two XenoSTART Patient-Derived Xenograft (XPDX) models from tissue samples collected two years apart from a patient with ER+/HER2+ breast cancer before and after HER2 directed therapies. These models designated ST4565 and ST4565C were developed and characterized for receptor expression, genomic alterations, and in vivo drug sensitivities toward multiple chemotherapies and targeted agents, including T-DM1 and DS-8201a. Methods: Models ST4565 and ST4565C were established from breast samples collected from a Caucasian female with ER+/HER2+ metastatic breast cancer; ST4565 was collected at age 35 prior to therapy and ST4564C at age 37 following several treatment regimens including 5-FU/doxorubicin/cyclophosphamide, docetaxel/trastuzumab/pertuzumab, and T-DM1/anastrozole. Both were grown subcutaneously in female athymic nude mice supplemented with exogenous estradiol. The resulting models were passaged, and receptor expression confirmed immunohistochemically; genomic analysis, including WES and RNAseq, was performed to further characterize the models. For in vivo studies, both models were evaluated using several chemotherapy and targeted agents alone and in combination including: trastuzumab, pertuzumab, T-DM1, DS-8201a, neratinib, tucatinib, fulvestrant, alpelisib, sacituzumab, and irinotecan. In vivo study endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a T/C of ≤ 20% versus control was considered sensitive. Tumor regression (%T/C=< 0) versus Day 0 tumor volume was also reported. Results: ST4565 and ST4565C retained comparable receptor expression (ER=2+/HER2=3+) over tested passages with similar histology compared to archival clinical samples. DNA/RNA sequencing identified several conserved variants including loss of CDKN2A/B and MTAP and a CNV=14 for CCND1. In vivo, ST4565 and ST4565C were found resistant to T-DM1 at 3 mg/kg weekly with a T/C of 100% in both models. However, DS-8201a treatment at 3 mg/kg weekly resulted in partial tumor regressions in ST4565 (T/C=-51%) while ST4565C was found resistant (T/C=49%). Both models were found resistant to all tested chemotherapies and all other targeted therapies but reported similar sensitivity to fulvestrant (T/C=~40%). Conclusion: We established two XPDX models representing T-DM1-resistant, ER+/HER2+ breast cancer from breast samples collected two years apart from the same patient that were found differentially responsive to DS-8201a. These models can be utilized as a valuable tool in better understanding innate resistance to T-DM1 and acquired resistance to DS-8201a. Citation Format: George Plasko, Johnnie Flores, Alyssa Simonson, Peter Forofontov, Ashwin Varma, Amy Lang, Gladys Rodriguez, Kyriakos P. Papadopoulos, Drew Rasco, Amita Patnaik, Bruce Conway, Joe Johnston, Michael Wick. Establishment and characterization of two T-DM1-resistant, ER+/HER2+ breast XPDX models developed sequentially from the same patient with differential in vivo sensitivity to trastuzumab deruxtecan (DS-8201a) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-03.

Abstract P1-13-22: Establishment and characterization of two ER+/HER2- XPDX models developed sequentially before and after acquired resistance to the CDK4/6 inhibitor palbociclib from a patient with metastatic breast cancer

Abstract Background: Several CDK4/6 inhibitors have recently been approved in combination with letrozole or fulvestrant in hormone receptor-positive breast cancer. Although this combination therapy has been found effective in some patients, resistance often develops. To aid in developing new therapies for CDK4/6i-resistant breast cancer and better understand potential resistance mechanisms, we established two XenoSTART Patient-Derived Xenograft (XPDX) models representing ER+/HER2- breast cancer from tissue samples collected seventeen months apart from the same patient before and after palbociclib therapy. These models designated ST4887 and ST4887B were developed and characterized for receptor expression, genomic alterations, and in vivo drug sensitivities toward multiple chemotherapies and targeted agents, including CDK4/6i and fulvestrant. Methods: Models ST4887 and ST4887B were established from metastatic samples collected from a Caucasian female with ER+/HER2- metastatic breast cancer; ST4887 was collected at age 38 from a femur mass biopsy following several treatment regimens including paclitaxel/doxorubicin/cyclophosphamide, radiation and tamoxifen. ST4887B was collected at age 39 from a liver biopsy following treatment with palbociclib/letrozole then palbociclib/fulvestrant, and finally ixabepilone/capecitabine. Both were grown subcutaneously in female athymic nude mice supplemented with exogenous estradiol. The resulting models were passaged, and receptor expression confirmed immunohistochemically; genomic analysis, including WES and RNAseq, was performed to further characterize models. For in vivo studies, both models were evaluated using several chemotherapy and targeted agents alone and in combination including cisplatin, docetaxel, CDK4/6i, fulvestrant, letrozole, olaparib, niraparib, and sacituzumab. In vivo study endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a T/C of ≤ 20% versus control was considered sensitive. Tumor regression (%T/C=< 0) versus Day 0 tumor volume was also reported. Results: ST4887 and ST4887B retained comparable receptor expression (ER=3+/HER2=1+) over tested passages with similar histology compared to archival clinical samples. DNA/RNA sequencing identified several conserved variants including a somatic BRCA2 truncation (BRCA2Y2660*); transcriptomic analysis revealed upregulation of several related genes but no notable fusions. In vivo, both models were insensitive to cisplatin or docetaxel, however ST4887 but not ST4887B was sensitive to fulvestrant or CDK4/6i therapies, although abemaciclib demonstrated some activity toward ST4887B. PARP inhibitors were active toward ST4887 and to a lesser extent ST4887B, while sacituzumab did not have a significant effect on either model. Conclusion: We established and characterized two XPDX models from the same patient before and after acquired resistance to the CDK4/6i palbociclib. Both models were found to retain receptor status and drug sensitivities similar to the patient at the time of sample collection. These models can be utilized as a valuable tool in better understanding acquired resistance to palbociclib. Citation Format: Ashwin Varma, Johnnie Flores, Alyssa Simonson, Anna Stackpole, Kyriakos P. Papadopoulos, Amita Patnaik, Drew Rasco, Muralidhar Beeram, Marisa Sandera, Michael Wick. Establishment and characterization of two ER+/HER2- XPDX models developed sequentially before and after acquired resistance to the CDK4/6 inhibitor palbociclib from a patient with metastatic breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-22.

37 (PB027) - Establishment and characterization of HPV+ metastatic squamous cell anal carcinoma XPDX models in athymic nude mice

Background: Squamous cell carcinoma of the anus (SCCA) is a rare malignancy comprising less than 3% of all gastrointestinal cancers in the United States. While over 90% of SCCA have been found human papilloma virus positive (HPV+), a majority of cases present locally and can be cured with definitive therapy. However, metastatic SCCA is less treatable and poorly understood in part due to the lack of relevant preclinical models. To this end we have established four HPV+ XenoSTART ethnically and gender diverse Patient-Derived Xenograft (XPDX) models of metastatic SCCA designated ST4808, ST5678, ST5872 and ST6058. These models were established in athymic nude mice and characterized for receptor expression, genomic alterations, and in vivo drug sensitivity. Methods: ST4808 was established from biopsy collected from a 51-year-old Hispanic male with recurrent anal cancer following initial resection. ST5678 was established from a liver biopsy collected from a 55-year-old Hispanic male with newly-diagnosed SCCA. ST5872 was established from a liver biopsy collected from a 70-year-old Hispanic female with recurrent SCCA following prior treatment with 5-FU, mitomycin and radiation. ST6058 was established from a lung biopsy collected from a 55-year-old Caucasian female with recurrent SCCA following treatment with 5-FU/mitomycin/ radiation, carboplatin/gemcitabine, and investigational therapies. The resulting models were passaged and characterized using immunohistochemistry and genomic analysis including WES and RNAseq. In vivo studies were performed testing various chemotherapy and targeted agents; study endpoints included tumor volume and time from treatment initiation with % T/C values and tumor regression reported at study completion; a %T/C of £20 versus control was considered sensitive. Tumor regression (%T/C < 0) versus Day 0 tumor volume was also reported. Results: Immunohistochemistry analysis of each model confirmed disease similar to corresponding patient’s cancer using archival clinical samples. WES/RNAseq analysis identified EZH2 and STK11 variants in ST4808, ERBB2 and PIK3CA mutations in ST5678, an FGFR3-TACC3 fusion in ST5872 and KDM6A and CYLD variants in ST6058. In vivo, ST4808 demonstrated sensitivity to platinum chemotherapy while ST5678 was resistant to tucatinib, neratinib and alpelisib but not platinum. ST5872 reported modest sensitivity to erdafitinib and ST6058 was resistant to tested therapies. Conclusion: We have established and characterized four models of HPV+ metastatic SCCA cancer and benchmarked chemotherapy and relevant targeted therapies in each model. These models are valuable tools for evaluating potential therapies for metastatic anal cancer. No conflict of interest.

Abstract 353: Establishment and characterization of neuregulin-1 (NRG1) fusion driven XPDX models

Abstract Background: Neuregulin-1 (NRG1) rearrangements drive cancers by binding to ERBB3/ERBB2 heterodimers and activating downstream signaling. NRG1 fusion proteins have recently been identified in several cancer types including lung and ovary. To better understand the role of NRG1 fusions in cancer we established three XenoSTART patient-derived xenograft (XPDX) models driven by NRG1 rearrangements including two CD74-NRG1 lung models designated ST2891 and ST3204 and one SARAF-NRG1 ovary model designated ST2476. These models were established in athymic nude mice and characterized for receptor expression, genomic alterations, and in vivo drug sensitivity. Methods: ST2891 was established from an excision biopsy collected from a 68-year-old male with NSCLC following treatment with carboplatin/paclitaxel and carboplatin/pemetrexed. ST3204 was established from a lymph node biopsy collected from a 63-year-old female with NSCLC following treatment with cisplatin/pemetrexed. ST2476 was established from primary tissue collected from a chemo naïve 61-year-old female with ovarian cancer. The resulting models were passaged and characterized using immunohistochemical and genomic analysis including WES and RNAseq. In vivo studies were performed testing various chemotherapy and targeted agents; study endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a %T/C of ≤ 20 versus control was considered sensitive. Tumor regression (%T/C&amp;lt;0) versus Day 0 tumor volume was also reported. Results: All models reported ERBB2 and ERBB3 staining in evaluated passages with similar histology compared with archival clinical samples. WES analysis of the three models did not identify any variants in EGFR/ERBB2-4/RAS/RAF genes. RNA sequencing revealed CD74-NRG1 or SARAF-NRG1 gene fusions independently confirmed using molecular studies. In vivo, both lung models were resistant to weekly cisplatin or T-DM1 and daily palbociclib (50 mg/kg) or daily afatinib up to 10 mg/kg. ST3204 was found sensitive to weekly pertuzumab (1 mg/kg). Both lung models were resistant to trastuzumab (1 mg/kg). The ST2476 ovary XPDX was found resistant to endocrine therapies and reported some sensitivity to weekly cisplatin. Conclusion: We have established and characterized a panel of three XPDX models driven by NRG1 rearrangements including two CD74-NRG1 lung models and one SARAF-NRG1 ovary model. These models are valuable tools for further developing therapies targeting NRG1-driven cancers. Citation Format: Alyssa Simonson, Johnnie Flores, Crystal Moreno, Justine Hruzek, Kyriakos Papadopoulos, Drew Rasco, Amita Patnaik, Ronald Drengler, Allan White, Jun Ma, Michael J. Wick. Establishment and characterization of neuregulin-1 (NRG1) fusion driven XPDX models [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 353.

Organocatalyst treatment improves variant calling and mutant detection in archival clinical samples

Formalin fixation of biological specimens damages nucleic acids and limits their use in genomic analyses. Previously, we showed that RNA isolation with an organocatalyst (2-amino-5-methylphenyl phosphonic acid, used to speed up reversal of formalin-induced adducts) and extended heated incubation (ORGΔ) improved RNA-sequencing data from formalin-fixed paraffin-embedded (FFPE) tissue samples. The primary goal of this study was to evaluate whether ORGΔ treatment improves DNA-sequencing data from clinical FFPE samples. We isolated RNA and DNA ± ORGΔ from paired FFPE and frozen human renal and ovarian carcinoma specimens collected as part of the National Cancer Institute Biospecimen Pre-analytical Variables program. Tumor types were microscopically confirmed from adjacent tissue sections. Following extraction, DNA was fragmented and sequenced and differences were compared between frozen and FFPE sample pairs. Treatment with ORGΔ improved concurrent SNP calls in FFPE DNA compared to non-ORGΔ FFPE samples and enhanced confidence in SNP calls for all FFPE DNA samples, beyond that of matched frozen samples. In general, the concordant SNPs identified in paired frozen and FFPE DNA samples agreed for both genotype and homozygosity vs. heterozygosity of calls regardless of ORGΔ treatment. The increased confidence in ORGΔ FFPE DNA variant calls relative to the matched frozen DNA suggests a novel application of this method. With further optimization, this method may improve quality of DNA-sequencing data in FFPE as well as frozen tissue samples.

Development of a rapid, internally controlled, two target, real-time RT-PCR for detection of rubella virus

Rubella surveillance in elimination setting relies on rapid molecular detection of the virus. In this study a multiplex real-time RT-PCR assay for the detection of rubella virus was validated. The assay includes three independent probes with unique reporter dyes for the simultaneous detection of the rubella viral coding regions for envelope glycoprotein E1 and non-structural p150 protein, and an endogenous control (human RNaseP). Using dilution series of synthetic RNAs, the limits of detection were determined to be at least 50 copies of rubella RNA. The assay is reproducible with low intra-assay and inter-assay coefficients of variation for both the E1 and the p150 targets. After testing 62 confirmed rubella positive and 165 rubella negative archival clinical samples, the sensitivity and specificity of the multiplex assay were 98.4 and 100 %, respectively. No cross reactivity was identified with clinical specimens positive for eleven other viruses. This multiplex assay successfully detected nine viral genotypes including the predominant genotypes 1E, 1G, 1J, and 2B as well as the 1a vaccine genotype.

Abstract P5-01-11: Nonclinical activity of fulvestrant in a panel of ER+ breast XPDX models representing clinically acquired and innate resistance to endocrine therapies

Abstract Background: Fulvestrant is a selective estrogen receptor modulator (SERM) approved as a single agent for estrogen receptor positive, HER2 negative breast cancer patients at various stages of disease and in combination with CDK4/6 inhibitors following endocrine therapy failure. Although this agent requires intramuscular injection, it has demonstrated superior activity and fewer side effects versus some oral endocrine treatments however, resistance to fulvestrant often develops. To better understand fulvestrant resistance and its utility in patients who have failed other endocrine therapies, we evaluated the agent in a panel of ER+ breast models established from patients at various stages of disease representing endocrine-sensitive and -resistant disease. Methods: Sixty-five previously developed ER+ breast XPDX models were evaluated in this study. Models were grown subcutaneously in female athymic nude mice supplemented with estradiol in drinking water when necessary. All models were characterized at early and late passages for estrogen receptor expression by immunohistochemistry and profiled using WES and RNAseq. For in vivo studies, fulvestrant was administered by subcutaneous injection at 2.5 or 5 mg per dose once weekly until study completion. In vivo study endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a %T/C of ≤ 20 versus control was considered sensitive. Tumor regression (%T/C&amp;lt;0) versus Day 0 tumor volume was also reported. Models were grouped by patient clinical treatment prior to sample collection and model establishment including chemo-naïve, endocrine therapies other than fulvestrant, non-endocrine therapies, and fulvestrant. Results: All models retained ER expression in evaluated passages with similar histology compared with archival clinical samples. DNA/RNA sequencing identified several model-specific variants including ESR1 point mutations and fusions. In this study, 22% of models represented chemo-naïve patients, 24% were from patients treated with non-endocrine therapies, 20% were from patients treated with fulvestrant and the remaining 34% from patients treated with endocrine therapies other than fulvestrant. In vivo, 40% of the chemo-naïve group reported sensitivity to fulvestrant including several models with tumor regressions. Activity of fulvestrant was reported in 25% of models in the non-endocrine therapies group and in 20% of models from patients treated with either fulvestrant or endocrine therapies other than fulvestrant. Several models resistant to fulvestrant from chemo-naïve or patients treated with non-endocrine therapies harbored driver mutations including ESR1, PIK3CA, and AKT1 variants or were HER2+. In models from patients pretreated with fulvestrant, those who had also received a CDK4/6 inhibitor were particularly resistant. Conclusion: We evaluated fulvestrant in a panel of ER+ breast XPDX models representing endocrine-sensitive and -resistant disease from both chemo-naïve and pretreated patients, and identified models responsive and resistant to fulvestrant therapy. This panel can be utilized as a valuable tool in better understanding fulvestrant and endocrine therapy resistance and in developing novel therapies for patients resistant to currently available hormonal therapies. Citation Format: April Cabang, Crystal Moreno, Johnnie R Flores, Jenna Boedeker, Alyssa Simonson, Jun Ma, Amy Lang, Gladys Rodriguez, Arthur Rosenthal, Kyriakos Papadopoulos, Amita Patnaik, Drew Rasco, Lon Smith, Murali Beeram, Ronald Drengler, Luis Rodriguez, Steven Abbate, Scott Ulmer, Michael J Wick. Nonclinical activity of fulvestrant in a panel of ER+ breast XPDX models representing clinically acquired and innate resistance to endocrine therapies [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-01-11.

Abstract P5-01-06: Establishment and characterization of luminal A breast XPDX models from patients with acquired resistance to CDK 4/6 inhibitors

Abstract Background: Several CDK 4/6 inhibitors have recently been approved in combination with letrozole or fulvestrant in hormone receptor-positive breast cancer. Although this combination therapy has been found effective in some patients, resistance often develops. To aid in developing new therapies for CDK4/6 inhibitor-resistant breast cancer and better understand potential resistance mechanisms, we established a panel of nine XPDX models from eight female patients with luminal A breast cancer at time of progression following acquired resistance to CDK4/6 inhibitor therapy. These models, designated ST940C, ST2056, ST3164B, ST3164B/PBR, ST3932, ST4316B, ST4378, STF160, and STM001B were developed in athymic nude mice and characterized for receptor expression, genomic alterations, and in vivo drug sensitivity. Methods: STF160 was established from a primary biopsy and ST3932 from a metastatic soft tissue lesion; the remaining models were established from malignant fluid samples collected at various stages of treatment post CDK4/6 inhibitor response and progression. The resulting models were passaged and challenged with CDK4/6 inhibitors to confirm resistance and fulvestrant to assess sensitivity. Receptor expression was determined immunohistochemically. Genomic analysis, including WES and RNAseq, were performed to characterize models and identify mechanisms of resistance. For in vivo studies, palbociclib and abemaciclib were dosed by oral administration once daily at 50 mg/kg and fulvestrant by subcutaneous administration once weekly at 2.5 mg. In vivo study endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a %T/C of ≤ 20 versus control was considered sensitive. Tumor regression (%T/C&amp;lt;0) versus Day 0 tumor volume was also reported. Results: All models retained ER expression in evaluated passages with similar histology compared with archival clinical samples; HER2 positivity was also confirmed in ST940C which was established from a clinically ER+/HER2+ patient. DNA/RNA sequencing identified several model-specific variants including the ESR1Y537S mutation in ST940C and ST2056 and the ESR1-CCDC170 fusion in ST3164B and ST3164B/PBR; BRCA2Q1089Sfs*10 was identified in ST4316B and PIK3CAE545G in ST4378. In vivo, seven models including ST3164B, ST3164B/PBR, ST3932, ST4316B, ST4378, STF160, and STM001B were found resistant (%T/C&amp;gt;20) to palbociclib or abemaciclib and single agent fulvestrant. ST940C and ST2056 were sensitive (%T/C≤20) to tested CDK4/6 inhibitors but resistant to fulvestrant. Conclusion: We have established and characterized a panel of nine XPDX models from eight female patients with luminal A breast cancer at time of progression following acquired resistance to CDK4/6 inhibitor therapy, seven of which were found resistant to single agent palbociclib and abemaciclib and all nine to fulvestrant. This panel can be utilized as a valuable tool in better understanding CDK4/6 inhibitor resistance and in developing novel therapies for CDK4/6 inhibitor-resistant patients. Citation Format: Tatiana Hernandez, Dustin Kneifel, Alyssa Simonson, Johnnie R Flores, Sarah Quick, April Cabang, Alexandra Ulmer, Kyriakos Papadopoulos, Amy Lang, Gladys Rodriguez, Murali Beeram, Drew Rasco, Amita Patnaik, Scott Ulmer, Michael J Wick. Establishment and characterization of luminal A breast XPDX models from patients with acquired resistance to CDK 4/6 inhibitors [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-01-06.

Abstract P5-01-09: Establishment and characterization of two simultaneously developed T-DM1-resistant, ER+/HER2+ XPDX models from the same patient with differential in vivo sensitivity to trastuzumab deruxtecan (DS-8201a)

Abstract Background: Trastuzumab deruxtecan (DS-8201a) is an antibody-drug conjugate (ADC) consisting of an anti-HER2 (human epidermal growth factor receptor 2) antibody linked to a topoisomerase I inhibitor payload using a cleavable tetrapeptide-based linker and was recently approved for unresectable or T-DM1-refractory HER2+ breast cancer. While some mechanisms for clinical T-DM1 resistance have been identified, less is known about acquired or innate resistance to DS-8201a. We established two XPDX models of ER+/HER2+ breast cancer from tissue and fluid samples collected simultaneously from the same patient. These models designated ST4480B and ST4480C were developed and characterized for receptor expression, genomic alterations, and in vivo drug sensitivities toward multiple chemotherapies and targeted agents including DS-8201a and T-DM1. Methods: ST4480B and ST4480C were established from a 70-year-old Caucasian female with ER+/HER2+ metastatic breast cancer pretreated with chemotherapy and targeted agents including T-DM1 for nine months followed by capecitabine/trastuzumab/tucatinib combination for one year prior to sample collections. ST4480B was established from a lymph node biopsy and ST4480C from a fluid sample collected the same day; both were grown subcutaneously in female athymic nude mice supplemented with estradiol. The resulting models were passaged and receptor expression confirmed immunohistochemically; genomic analysis, including WES and RNAseq, was performed to further characterize models. For in vivo studies, both models were evaluated with several chemotherapy and targeted agents alone and in combination including: trastuzumab, pertuzumab, T-DM1, DS-8201a, neratinib, tucatinib, alpelisib, everolimus, and irinotecan. In vivo study endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a %T/C of ≤ 20 versus control was considered sensitive. Tumor regression (%T/C=—&amp;lt;0) versus Day 0 tumor volume was also reported. Results: ST4480B and ST4480C retained comparable receptor expression (ER=2+/HER2=2+) over tested passages with similar histology compared with archival clinical samples. DNA/RNA sequencing identified several conserved variants including PIK3CAE545K and TP53Q192* mutations and an ESR1-CCDC170 fusion. However, two variants identified only in ST4480C sequences included PIK3CAG1007R and PTENS287L. In vivo, ST4480B and ST4480C were found resistant to T-DM1 up to 10 mg/kg weekly with an average %T/C of 46 and 100, respectively. However, DS-8201a treatment at 3 mg/kg weekly resulted in partial tumor regressions in ST4480B (%T/C=-68) while ST4480C was found resistant to the therapy up to 10 mg/kg weekly (%T/C=84%). Both models were found resistant to trastuzumab, pertuzumab, neratinib, tucatinib, and irinotecan but sensitive to both alpelisib and everolimus. Conclusion: We established two XPDX models representing T-DM1-resistant, ER+/HER2+ breast cancer from both tissue and fluid samples collected simultaneously from the same patient which were found differentially responsive to DS-8201a. These models can be utilized as a valuable tool in better understanding innate resistance to DS-8201a. Citation Format: Johnnie R Flores, Anna Stackpole, Abimael Garza, Alexandra Ulmer, Alyssa Simonson, Kyriakos Papadopoulos, April Cabang, Jun Ma, Amita Patnaik, Drew Rasco, Amy Lang, Gladys Rodriguez, Murali Beeram, Michael J Wick. Establishment and characterization of two simultaneously developed T-DM1-resistant, ER+/HER2+ XPDX models from the same patient with differential in vivo sensitivity to trastuzumab deruxtecan (DS-8201a) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-01-09.

Development of a rapid, internally controlled, two target, real-time RT-PCR for detection of measles virus

Vaccination has greatly reduced global measles incidence, however measles remains endemic in many regions worldwide. Measles surveillance relies on high performance molecular detection of the virus. We have developed and validated a multiplex rRT-PCR assay for the detection of measles virus. The assay includes three independent probes with unique reporter dyes for the simultaneous detection of the measles hemagglutinin gene, nucleoprotein gene and endogenous RNaseP control. Using dilution series of synthetic RNAs the limits of detection were determined to be approximately 20 copies of measles RNA. The assay is extremely reproducible with very low intra-assay and inter-assay coefficients of varation for both the N and the H targets. After testing 68 confirmed measles positive and 86 measles negative archival clinical samples our data shows the multiplex assay has a sensitivity and specificity of 100 %, and a 100 % concordance with the expected results. No cross reactivity was identified with clinical specimens positive for six other viruses. According to the WHO, currently only the B3, D4, D8, H1 measles genotypes of the 24 recognized genotypes continue to circulate and this new multiplex assay successfully detected all four of those genotypes as well as six other genotypes.

Abstract 943: Insulin-like growth factor-2 a potential target for screening and treatment in patients with triple negative breast cancer

Abstract Triple-negative breast cancer (TNBC) affects only about 10-15% of women with breast cancer (BC) but accounts for almost 50% of all BC deaths. At this time, development of targeted treatments for TNBC are urgently needed. Insulin-like growth factor-2 (IGF2) is highly expressed in TNBCs and mediates its proliferative effects via two hormone receptors, insulin-like growth factor -1 receptor (IGF1R) and insulin receptor (IR). Using a TNBC tissue microarray (TMA) consisting of 159 archival clinical samples, we confirm that IGF2 is highly expressed in tumor tissue as compared to nearby normal tissue (P&amp;lt;0.001). In addition, evaluation of a second breast cancer TMA that included archival TNBC samples from African American and Latino women, IGF2 expression was similarly higher in these samples than in samples from Caucasian women (P&amp;lt;0.02). Since enrichment of IGF1R and IR are detected in TNBC and are known to allow signaling crosstalk, the effects of BMS-754807, a dual IGF1R/IR antagonist, were assessed on TNBC progression in vitro and in vivo with human and murine models. To determine the effects of BMS-754807 in vitro, we used cell viability assays with TNBC cell lines, MDA-MB-231 and HCC1806. BMS-754807 significantly reduced cell viability after 48 and 72 hours (P&amp;lt; 0.01), and a similar effect was observed in the murine TNBC cell line 4T1 (P&amp;lt; 0.01). In vivo, BMS-754807 inhibited tumor growth of human TNBC as well as mouse 4T1 xenografts (P&amp;lt; 0.05). Further, in studies of immune cell populations using syngeneic 4T1 tumor xenografts, BMS-754807 significantly decreased myeloid derived suppressor cells (P &amp;lt;0.05) in the tumor microenvironment (TME) and in the spleen of BALC/c mice. Additionally, there was an increase in T effector CD8+ and CD4+ tumor infiltrating lymphocytes (P&amp;lt; 0.05), particularly of spleen T central memory cells (P&amp;lt;0.01). Notably, conversion of CD8+ from T central memory to a T effector memory phenotype that is fully active against tumors can confer long term immunity against recurrence. This data suggest that IGF2 is a potential biological marker for breast cancer disparities and that targeting receptors of IGF2 with drugs such as BMS-754807 could provide additional treatment options in combination with current therapies, thus reducing TNBC progression and ultimately improving patient outcomes. [Funded by NIH U54 143931 Cancer Center Partnership and CBCRP] Citation Format: Nalo M. Hamilton, Diana Marquez-Garban, David Austin, Neda Moatamed, Lorena P. Burton, David Elashoff, Begonya Comin-Anduix, Tristan Grogan, Madhuri Wadehra, Helena Chang, Adrienne Martinez, Mary-Lynn Brecht, Isabel Jabara, Richard Pietras, Yahya Elshimali, Jaydutt Vadgama, Michael Jung. Insulin-like growth factor-2 a potential target for screening and treatment in patients with triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 943.

Abstract 2708: A custom gene expression panel for consensus molecular subtype classification of archival primary and metastatic colorectal cancers

Abstract Stratification of Colorectal Cancer (CRC) into actionable molecular subtypes has tremendous clinical value. Recently, a consolidated classifier identified four molecularly distinct CRC subtypes (CMS1-4) that were associated with unique biology and clinical outcomes based on global transcriptional analysis of frozen tissues. Here, we developed and applied a novel CRC panel that is ideally suited for transcriptional classification of archival clinical samples. Findings from in silico analysis demonstrated that the 800 genes on our panel could accurately classify CRC samples from external public datasets into the correct CMS subtypes. We applied our panel in the analysis of a novel cohort of 312 formalin-fixed paraffin-embedded (FFPE) tissues from 205 patients, and were able to detect all 4 CMS subtypes in primary CRCs and in metastases. When we examined the CMS subtypes of primary tumors and matched metastases from 50 patients we found 70% of cases to be concordant, as were key biologies, such as WNT/MYC pathway activation in CMS2 and EMT features in tumors of the CMS4 subtypes. This was confirmed by in situ hybridization (ISH) using the markers ASCL2 for CMS2 and SPARC for CMS4, respectively. Discordance in the CMS subtypes between primary tumors and matched metastases were observed in 30% of cases and may reflect tumor heterogeneity. Our findings suggest that our CRC-focused panel many have clinical utility for CMS classification of FFPE samples, and point to potential risks of using CMS subtypes of primary tumors to inform clinical decision-making at the metastatic stage in a subset of patients. Citation Format: Ling-Yuh Huw, Robert Piskol, Felipe de Sosa e Melo, Doris Kim, Xueping Qu, Hartmut Koeppen, Mark Lackner, Garret Hampton, Omar Kabbarah, Rachel Tam. A custom gene expression panel for consensus molecular subtype classification of archival primary and metastatic colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2708. doi:10.1158/1538-7445.AM2017-2708

Development and Application of a Microfluidics-Based Panel in the Basal/Luminal Transcriptional Characterization of Archival Bladder Cancers.

In the age of personalized medicine stratifying tumors into molecularly defined subtypes associated with distinctive clinical behaviors and predictable responses to therapies holds tremendous value. Towards this end, we developed a custom microfluidics-based bladder cancer gene expression panel for characterization of archival clinical samples. In silico analysis indicated that the content of our panel was capable of accurately segregating bladder cancers from several public datasets into the clinically relevant basal and luminal subtypes. On a technical level, our bladder cancer panel yielded robust and reproducible results when analyzing formalin-fixed, paraffin-embedded (FFPE) tissues. We applied our panel in the analysis of a novel set of 204 FFPE samples that included non-muscle invasive bladder cancers (NMIBCs), muscle invasive disease (MIBCs), and bladder cancer metastases (METs). We found NMIBCs to be mostly luminal-like, MIBCs to include both luminal- and basal-like types, and METs to be predominantly of a basal-like transcriptional profile. Mutational analysis confirmed the expected enrichment of FGFR3 mutations in luminal samples, and, consistently, FGFR3 IHC showed high protein expression levels of the receptor in these tumors. Our bladder cancer panel enables basal/luminal characterization of FFPE tissues and with further development could be used for stratification of bladder cancer samples in the clinic.

Retrospective analysis of HPV 16/18-related disease burden using archival clinical samples.

Estimation of HPV-related disease burden lies at the core of effective disease management. HPV testing is heavily reliant on its retrospective detection in archival clinical cancer samples, especially in parts of the world where HPV screening is not routinely practiced. During the last decade, valuable insights were gained through regional reports based on occasional screening of cervical smears or biopsy sections for the presence of high-risk HPV. HPV 16 and 18 were found to be predominant high-risk HPV subtypes with some regional differences and incidences of co-infections, detected mostly through PCR-based methods. In cases of multiple infections, the presence of viral DNA may not signify its etiologic involvement. The current study, therefore, combines PCR-based detection method with the immunohistochemical (IHC) detection of early viral protein E6 expression, in order to obtain a reliable read out for the disease causing viral subtype, especially in cases of co-infections with oncogenic subtypes other than HPV 16 and 18. Immunohistochemistry (IHC) and PCR-based methods are routinely used laboratory techniques in local hospitals. The concordance between IHC and PCR-based analyses may be useful for determining effective method for the retrospective testing of HPV 16 and 18 disease-related burden. A total of 49 paraffin-embedded cervical cancer biopsy sections representing patients from the northwest region of the country were collected from the tertiary care hospital for this study. Genotyping for HPV 16 and 18 was carried out through PCR. The HPV 16/18 E6 protein expression was evaluated by IHC and was compared with the clinicopathological features of cervical cancer. Molecular analysis of 33 (67%), E6-expressing paraffin-embedded cervical cancer biopsy sections revealed the presence of HPV 16 (n=23; 47%), HPV 18 (n=6; 12%) and co-infection (n=4; 8%) in 49 tumors through PCR. Despite the PCR-based detection of viral DNA in 37 cervical cancer samples, IHC analysis of E6 expression revealed the etiological involvement of HPV 16/18 in 33 out of 37 cervical cancer samples. Overall, there was 85% concordance in the results of the two techniques. IHC analysis provides more conclusive evidence regarding the etiological involvement of the viral subtypes, especially in the presence of multiple infections. About two-thirds (67%) of cervical cancer samples were found to be caused due to HPV 16/18. Latent occurrence of HPV 16 and 18 is suggested in less than 10% cervical cancer samples which were found to harbor viral DNA without E6 expression. Furthermore, E6 expression was found to be significantly correlated with the tumor grade.

Evidence of broad-based family support for the use of archival childhood tumour samples in future research

ObjectivesThis study aimed to determine the ability to successfully contact past paediatric patients and their families to request participation in research, to assess familial views on the use of previously...