Search

OBJECTIVE To evaluate the diagnostic, prognostic, and therapeutic utility of a cancer genomic diagnostic assay (SearchLight DNA; Vidium Animal Health) for diagnostically ambiguous cancer cases. ANIMALS 69 privately owned dogs with ambiguous cancer diagnoses and for which the genomic assay was performed. PROCEDURES Genomic assay reports generated between September 28, 2020, and July 31, 2022, for dogs with malignancy or suspected malignancy were reviewed to determine the assay’s clinical utility defined as providing diagnostic clarity, prognostic information, and/or therapeutic options. RESULTS Genomic analysis provided diagnostic clarity in 37 of 69 cases (54%; group 1) and therapeutic and/or prognostic information in 22 of the remaining 32 cases (69%; group 2) for which the diagnosis remained elusive. Overall, the genomic assay was clinically useful in 86% (59/69) of cases. CLINICAL RELEVANCE To our knowledge, this was the first study to evaluate the multifaceted clinical utility of a single cancer genomic test in veterinary medicine. Study findings supported the use of tumor genomic testing for dogs with cancer, particularly those that are diagnostically ambiguous and therefore inherently challenging to manage. This evidence-driven genomic assay provided diagnostic guidance, prognostic support, and therapeutic options for most patients with an unclear cancer diagnosis that would otherwise have an unsubstantiated clinical plan. Furthermore, 38% (26/69) of samples were easily obtained aspirates. Sample factors (sample type, percentage of tumor cells, and number of mutations) did not influence diagnostic yield. Our study demonstrated the value of genomic testing for the management of canine cancer.

XLSX file - 23K, Mapping statistics for genome sequencing of tumor and germline DNA from mTNBC

XLSX file - 45K, Genes containing breakpoint from translocation in mTNBC

XLSX file - 273K, Somatic genic focal copy number changes in mTNBC

XLSX file - 598K, edgeR differential expression results in mTNBC for select cancer related genes

XLSX file - 607K, Outlier differential expression results in mTNBC for select cancer related genes

XLSX file - 61K, List of somatic point mutations in mTNBC detected by NGS sequencing analysis

XLSX file - 42K, Ingenuity Canonical Pathways results for mTNBC based on edgeR differential expression results

XLSX file - 40K, Characteristics of mTNBC study population

PDF file - 1635K, PET/CT images of mTNBC2 breast lesion before and after treatment on combination MEK and AKT inhibitors

XLSX file - 48K, Transcriptome sequencing (RNA-seq) run statistics for mTNBC

PDF file - 135K, Validation of RB1 39bp homozygous deletion in mTNBC1

Purpose:Naturally occurring primary canine lung cancers share clinicopathologic features with human lung cancers in never-smokers, but the genetic underpinnings of canine lung cancer are unknown. We have charted the genomic landscape of canine lung cancer and performed functional characterization of novel, recurrent HER2 (ERBB2) mutations occurring in canine pulmonary adenocarcinoma (cPAC).Experimental Design:We performed multiplatform genomic sequencing of 88 primary canine lung tumors or cell lines. Additionally, in cPAC cell lines, we performed functional characterization of HER2 signaling and evaluated mutation-dependent HER2 inhibitor drug dose-response.Results:We discovered somatic, coding HER2 point mutations in 38% of cPACs (28/74), but none in adenosquamous (cPASC, 0/11) or squamous cell (cPSCC, 0/3) carcinomas. The majority (93%) of HER2 mutations were hotspot V659E transmembrane domain (TMD) mutations comparable to activating mutations at this same site in human cancer. Other HER2 mutations were located in the extracellular domain and TMD. HER2V659E was detected in the plasma of 33% (2/6) of dogs with localized HER2V659E tumors. HER2V659E cPAC cell lines displayed constitutive phosphorylation of AKT and significantly higher sensitivity to the HER2 inhibitors lapatinib and neratinib relative to HER2-wild-type cell lines (IC50 < 200 nmol/L in HER2V659E vs. IC50 > 2,500 nmol/L in HER2WT).Conclusions:This study creates a foundation for molecular understanding of and drug development for canine lung cancer. These data also establish molecular contexts for comparative studies in dogs and humans of low mutation burden, never-smoker lung cancer, and mutant HER2 function and inhibition.

AKT S473 distribution based on ER expression measured by IHC for 31 lesions included of the discovery set. ER was measured in a binary scale (Panel A) and in continuous scale using the H-Score (Panel B).

Purpose: Little is known about the molecular signatures associated with specific metastatic sites in breast cancer. Using comprehensive multi-omic molecular profiling, we assessed whether alterations or activation of the PI3K–AKT–mTOR pathway is associated with specific sites of breast cancer metastasis.Experimental Design: Next-generation sequencing–based whole-exome sequencing was coupled with reverse-phase protein microarray (RPPA) functional signaling network analysis to explore the PI3K–AKT–mTOR axis in 32 pretreated breast cancer metastases. RPPA-based signaling data were further validated in an independent cohort of 154 metastatic lesions from breast cancer and 101 unmatched primary breast tumors. The proportion of cases with PI3K–AKT–mTOR genomic alterations or signaling network activation were compared between hepatic and nonhepatic lesions.Results: PIK3CA mutation and activation of AKT (S473) and p70S6K (T389) were detected more frequently among liver metastases than nonhepatic lesions (P < 0.01, P = 0.056, and P = 0.053, respectively). However, PIK3CA mutations alone were insufficient in predicting protein activation (P = 0.32 and P = 0.19 for activated AKT and p70S6K, respectively). RPPA analysis of an independent cohort of 154 tumors confirmed the relationship between pathway activation and hepatic metastasis [AKT (S473), mTOR (S2448), and 4EBP1 (S65); P < 0.01, P = 0.02, and P = 0.01, respectively]. Similar results were also seen between liver metastases and primary breast tumors [AKT (S473) P < 0.01, mTOR (S2448) P < 0.01, 4EBP1 (S65) P = 0.01]. This signature was lost when primary tumors were compared with all metastatic sites combined.Conclusions: Breast cancer patients with liver metastasis may represent a molecularly homogenized cohort with increased incidence of PIK3CA mutations and activation of the PI3K–AKT–mTOR signaling network. Clin Cancer Res; 23(16); 4919–28. ©2017 AACR.

HER2 cellular location and function in primary canine lung cancer. (A) Canine papillary adenocarcinoma with intense, complete, circumferential membrane (white arrow) and lateral cytoplasmic membrane (black arrow) anti-HER2 antibody positive staining (brown) in a patient with wild-type HER2. (B) Canine papillary adenocarcinoma with moderate cytoplasmic (black arrow) anti-HER2 antibody positive staining (light brown) in a patient with wild-type HER2. x 40; bar 50 µm. (C) Anti-HER2 immunohistochemistry of a Grade 1 canine papillary adenocarcinoma wild type for HER2. x 20. (D) Segmentation mark-up of the tumor from adjacent normal lung. Tumor is identified by green, whereas red is area within tumor that contains no tissue, and yellow represents areas of non-tumor such as necrosis or tumor stroma. x20.

Supplementary material legend

Supplementary Legend

Concordance between PIK3CA, AKT, and PTEN mutation status and AKT (S473) and p70S6 (T389) activation.

Supplementary Table 1. Informatic tools utilized in primary canine lung cancer analysis Supplementary Table 2. Extended clinical and multiplatform annotation of primary canine lung cancer cases. Supplementary Table 3 : Sequencing metrics for primary canine lung cancer exome analysis. Supplementary Table 4. Somatic coding SNVs identified by exome sequencing of primary canine lung cancers. Supplementary Table 5. Somatic copy number variants identified by exome sequencing in primary canine lung cancers. Supplementary Table 6. Somatic coding structural variants identified by exome sequencing of primary canine lung cancers. Supplementary Table 7. Canine genomic regions covered by custom amplicon panel. Supplementary Table 8. Sequencing metrics for primary canine lung cancer amplicon analysis. Supplementary Table 9. Somatic coding SNVs identified by panel sequencing of primary canine lung cancers. Supplementary Table 10. Germline SNPs in COSMIC Tier 1 cancer genes identified by exome and panel sequencing in primary canine lung cancers. Supplementary Table 11. Multi-platform validation of HER2 mutations. Supplementary Table 12. Non-invasive detection of HER2V659E in the plasma of primary canine lung cancer patients. Supplementary Table 13. HER2 protein expression and quantification by immunohistochemistry.

The lack of preparedness for detecting the highly infectious SARS-CoV-2 pathogen, the pathogen responsible for the COVID-19 disease, has caused enormous harm to public health and the economy. It took ∼60 days for the first reverse transcription quantitative polymerase chain reaction (RT-qPCR) tests for SARS-CoV-2 infection developed by the United States Centers for Disease Control (CDC) to be made publicly available. It then took >270 days to deploy 800,000 of these tests at a time when the estimated actual testing needs required over 6 million tests per day. Testing was therefore limited to individuals with symptoms or in close contact with confirmed positive cases. Testing strategies deployed on a population scale at ‘Day Zero’ i.e., at the time of the first reported case, would be of significant value. Next Generation Sequencing (NGS) has such Day Zero capabilities with the potential for broad and large-scale testing. However, it has limited detection sensitivity for low copy numbers of pathogens which may be present. Here we demonstrate that by using CRISPR-Cas9 to remove abundant sequences that do not contribute to pathogen detection, NGS detection sensitivity of COVID-19 is comparable to RT-qPCR. In addition, we show that this assay can be used for variant strain typing, co-infection detection, and individual human host response assessment, all in a single workflow using existing open-source analysis pipelines. This NGS workflow is pathogen agnostic, and therefore has the potential to transform how both large-scale pandemic response and focused clinical infectious disease testing are pursued in the future.SIGNIFICANCE STATEMENTThe lack of preparedness for detecting infectious pathogens has had a devastating effect on the global economy and society. Thus, a ‘Day Zero’ testing strategy, that can be deployed at the first reported case and expanded to population scale, is required. Next generation sequencing enables Day Zero capabilities but is inadequate for detecting low levels of pathogen due to abundant sequences of little biological interest. By applying the CRISPR-Cas system to remove these sequences in vitro, we show sensitivity of pathogen detection equivalent to RT-qPCR. The workflow is pathogen agnostic, and enables detection of strain types, co-infections and human host response with a single workflow and open-source analysis tools. These results highlight the potential to transform future large-scale pandemic response.

Comparative studies of naturally occurring canine cancers have provided new insight into many areas of cancer research. Development and validation of circulating tumor DNA (ctDNA) analysis in pet dogs can help address diagnostic needs in veterinary as well as human oncology. Dogs have high incidence of naturally occurring spontaneous cancers, demonstrate molecular heterogeneity and clonal evolution during therapy, allow serial sampling of blood from the same individuals during the course of disease progression, and have relatively compressed intervals for disease progression amenable to longitudinal studies. Here, we present a feasibility study of ctDNA analysis performed in 48 dogs including healthy dogs and dogs with either benign splenic lesions or malignant splenic tumors (hemangiosarcoma) using shallow whole genome sequencing (sWGS) of cell-free DNA. To enable detection and quantification of ctDNA using sWGS, we adapted two informatic approaches and compared their performance for the canine genome. At the time of initial clinical presentation, mean ctDNA fraction in dogs with malignant splenic tumors was 11.2%, significantly higher than dogs with benign lesions (3.2%; p = 0.001). ctDNA fraction was 14.3% and 9.0% in dogs with metastatic and localized disease, respectively (p = 0.227). In dogs treated with surgical resection of malignant tumors, mean ctDNA fraction decreased from 11.0% prior to resection to 7.9% post-resection (p = 0.047 for comparison of paired samples). Our results demonstrate that ctDNA analysis is feasible in dogs with hemangiosarcoma using a cost-effective approach such as sWGS. Additional studies are needed to validate these findings, and determine the role of ctDNA to assess burden of disease and treatment response in dogs with cancer.

This prospective phase II clinical trial (Side Out 2) explored the clinical benefits of treatment selection informed by multi‐omic molecular profiling (MoMP) in refractory metastatic breast cancers (MBCs). Core needle biopsies were collected from 32 patients with MBC at trial enrollment. Patients had received an average of 3.94 previous lines of treatment in the metastatic setting before enrollment in this study. Samples underwent MoMP, including exome sequencing, RNA sequencing (RNA‐Seq), immunohistochemistry, and quantitative protein pathway activation mapping by Reverse Phase Protein Microarray (RPPA). Clinical benefit was assessed using the previously published growth modulation index (GMI) under the hypothesis that MoMP‐selected therapy would warrant further investigation for GMI ≥ 1.3 in ≥ 35% of the patients. Of the 32 patients enrolled, 29 received treatment based on their MoMP and 25 met the follow‐up criteria established by the trial protocol. Molecular information was delivered to the tumor board in a median time frame of 14 days (11–22 days), and targetable alterations for commercially available agents were found in 23/25 patients (92%). Of the 25 patients, 14 (56%) reached GMI ≥ 1.3. A high level of DNA topoisomerase I (TOPO1) led to the selection of irinotecan‐based treatments in 48% (12/25) of the patients. A pooled analysis suggested clinical benefit in patients with high TOPO1 expression receiving irinotecan‐based regimens (GMI ≥ 1.3 in 66.7% of cases). These results confirmed previous observations that MoMP increases the frequency of identifiable actionable alterations (92% of patients). The MoMP proposed allows the identification of biomarkers that are frequently expressed in MBCs and the evaluation of their role as predictors of response to commercially available agents. Lastly, this study confirmed the role of MoMP for informing treatment selection in refractory MBC patients: more than half of the enrolled patients reached a GMI ≥ 1.3 even after multiple lines of previous therapies for metastatic disease., This prospective trial assessed the role of multi‐omic molecular profiling in informing treatment selection for metastatic breast cancer patients. Molecular profiles were collected in 14 business days and targetable alterations for commercial agents were identified in 92% of patients; 56% of patients reached a growth modulation index ≥ 1.3 after multiple lines of previous therapies. This work confirms the unique role for multi‐omic molecular profiling in guiding treatment selection for metastatic patients.

Cancer genomic heterogeneity presents significant challenges for understanding oncogenic processes and for cancer’s clinical management. Variation in driver mutation frequency between patients with the same tumor type as well as within individual patients’ cancers can limit the power of mutations to serve as diagnostic, prognostic, and predictive biomarkers. We have characterized genomic heterogeneity between and within patients in canine splenic hemangiosarcoma (HSA), a common naturally occurring cancer in pet dogs that is similar to human angiosarcoma (AS). HSA is a clinically, physiologically, and genomically complex canine cancer that may serve as a valuable model for understanding the origin and clinical impact of cancer heterogeneity. We conducted a prospective collection of 52 splenic masses from 44 dogs (28 HSA, 15 benign masses, and 1 stromal sarcoma) presenting to emergency care with hemoperitoneum secondary to a ruptured splenic mass. Multi-platform genomic analysis included matched tumor/normal cancer gene panel and exome sequencing. We found candidate somatic cancer driver mutations in 14/28 (50%) HSAs. Among recurrent candidate driver mutations, TP53 was most commonly mutated (29%) followed by PIK3CA (14%), AKT1 (11%), and CDKN2AIP (11%). We also identified significant intratumoral genomic heterogeneity, consistent with a branched evolution model, through multi-region exome sequencing of three distinct tumor regions from selected primary splenic tumors. These data provide new perspective on the genomic landscape and comparative value of understanding HSA in pet dogs, particularly as a naturally occurring cancer bearing intratumoral heterogeneity.

Comparative studies of naturally-occurring canine cancers have provided new insight into many areas of cancer research. The inclusion of pet dogs in the development and validation of circulating tumor DNA (ctDNA) diagnostics may be uniquely informative for human translation for many reasons, including: high incidence of certain spontaneous cancers, repeated access to blood and tumor from the same individuals during the course of disease progression, and molecular heterogeneity of naturally-occurring cancers in dogs. Here, we present a feasibility study of ctDNA analysis performed in 9 healthy dogs and 39 dogs with either benign or malignant splenic tumors (hemangiosarcoma) using shallow whole genome sequencing (sWGS) of cell-free DNA. To enable detection and quantification of ctDNA using sWGS, we adapted two informatic approaches and compared their performance for the canine genome. At presentation, mean ctDNA tumor fraction in dogs with malignant splenic tumors was 11.2%, significantly higher than dogs with benign lesions (3.2%; p 0.001), achieving an AUC of 0.84. ctDNA tumor fraction was 14.3% and 9.0% in dogs with metastatic and localized disease, respectively although this difference was not statistically significant (p 0.227). In paired analysis, ctDNA fraction decreased from 11.0% to 7.9% after resection of malignant tumors (p 0.047). Our results demonstrate that ctDNA analysis is feasible in dogs with hemangiosarcoma using a cost-effective approach such as sWGS. Future studies are underway to validate these findings, and further evaluate the role of ctDNA to assess burden of disease and treatment response during drug development.

Triple-negative breast cancer (TNBC) is an aggressive subtype most prevalent among women of Western Sub-Saharan African ancestry. It accounts for 15-25% of African American (AA) breast cancers (BC) and up to 80% of Ghanaian breast cancers, thus contributing to outcome disparities in BC for black women. The aggressive biology of TNBC has been shown to be regulated partially by breast cancer stem cells (BCSC) which mediate tumor recurrence and metastasis and are more abundant in African breast tumors.We studied the biological differences between TNBC in women with African ancestry and those of Caucasian women by comparing the gene expression of the BCSC. From low-passage patient derived xenografts (PDX) from Ghanaian (GH), AA, and Caucasian American (CA) TNBCs, we sorted for and sequenced the stem cell populations and analyzed for differential gene enrichment.In our cohort of TNBC tumors, we observed that the ALDH expressing stem cells display distinct ethnic specific gene expression patterns, with the largest difference existing between the GH and AA ALDH+ cells. Furthermore, the tumors from the women of African ancestry [GH/AA] had ALDH stem cell (SC) enrichment for expression of immune related genes and processes. Among the significantly upregulated genes were CD274 (PD-L1), CXCR9, CXCR10 and IFI27, which could serve as potential drug targets.Further exploration of the role of immune regulated genes and biological processes in BCSC may offer insight into developing novel approaches to treating TNBC to help ameliorate survival disparities in women with African ancestry.

Background: While genomic alterations are central players in tumor progression, proteins are the targets for precision therapy. The degree by which “actionable” genomic alterations translate into activated/altered proteins and pathway is still under investigation. Using a multi-OMIC approach from the SideOut 2 metastatic breast cancer (MBC) trial, this study explored the concordance between selected “actionable” genomic alterations and protein expression/activation. Methods: Snap frozen biopsies from 29 MBC patients enrolled in a prospective phase II trial were used for this analysis. Exome WES and RNASeq data was processed using an in-house developed pipeline and identified amplification of CCND1 (6/29), FGFR1 (4/29), and FGF 3, 4, 5, and 19 (4/29) as some of most frequent “actionable” genomic alterations in our MBC cohort. Signaling analysis of the 29 cases was performed using Reverse Phase Protein Microarray coupled with Laser Capture Microdissection. Protein expression/phosphorylation was measured in a continuous scale and classified based on quartile distribution. Concordance between CCND1 amplification and Cyclin D1 expression, along with the activation of FOXM1 T600 and Rb S780, was explored. Amplification of the FGFR1 locus or its ligands was correlated with the level of activation/phosphorylation of FGFR1 Y653/654. Results: While Cyclin D1 protein expression was greater than the population mean for 4/6 (67%) patients with CCND1 amplification, only 2/6 (33%) patients with CCND1 amplification had Cyclin D1 level within the top quartile of the population (n=29). FOXM1 T600 activation was independent from CCND1 amplification, with high levels of FOXM1 T600 predominantly in the CCND1 wild-type population. Only 1/6 (17%) patients with CCND1 amplification had FOXM1 T600 level similar to the top quartile of the population while a second patient was above the population median. Activation of Rb S780 was above the population median, but below the top quartile, in 2/6 (33%) CCND1 amplified patients. Similarly, none of the patients with activation of FGFR Y653/654 equal to the top quartile harbored an FGFR1 amplification. Only 1/4 (25%) patients carrying an FGFR1 amplification had an activation of FGFR Y653/654 above the population median. Similarly, 1/4 (25%) patients with FGF ligand amplification showed FGFR Y653/654 level within the top quartile while three patients had FGFR Y653/654 activation below the population median. No significant results were found between proteomic (below/above the median) and genomic characteristics by Fisher test (p>0.05). Conclusion: Molecular genotyping of “actionable” cancer targets alone may be insufficient in predicting whether the actual drug target protein is expressed and/or activated in any given patient's tumor. Although these results need further validation, the combination of genomic and proteomic data may represent a more informative approach for identifying real molecular drivers of individual lesions as well as “actionable” protein/phosphoprotein targets in the absence of genomic events. Multi-OMIC approaches may lead to more effective stratification in precision medicine trials. Citation Format: Pierobon M, Wong S, Reeded A, Anthony S, Robert N, Northfelt DW, Jahanzeb M, Vocila L, Wulfkuhle J, Dunetz B, Aldrich J, Byron S, Craig D, Liotta L, Carpten J, Petricoin EF. A multi-OMIC analysis to explore the impact of “actionable” genomic alterations on protein pathway activation: Clinical implication for precision medicine in metastatic breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-07-09.

Purpose: Naturally occurring primary canine lung cancers share clinicopathologic features with human lung cancers in never-smokers, but the genetic underpinnings of canine lung cancer are unknown. We have charted the genomic landscape of canine lung cancer and performed functional characterization of novel, recurrent HER2 (ERBB2) mutations occurring in canine pulmonary adenocarcinoma (cPAC). Experimental Design: We performed multiplatform genomic sequencing of 88 primary canine lung tumors or cell lines. Additionally, in cPAC cell lines, we performed functional characterization of HER2 signaling and evaluated mutation-dependent HER2 inhibitor drug dose-response. Results: We discovered somatic, coding HER2 point mutations in 38% of cPACs (28/74), but none in adenosquamous (cPASC, 0/11) or squamous cell (cPSCC, 0/3) carcinomas. The majority (93%) of HER2 mutations were hotspot V659E transmembrane domain (TMD) mutations comparable to activating mutations at this same site in human cancer. Other HER2 mutations were located in the extracellular domain and TMD. HER2V659E was detected in the plasma of 33% (2/6) of dogs with localized HER2V659E tumors. HER2V659E cPAC cell lines displayed constitutive phosphorylation of AKT and significantly higher sensitivity to the HER2 inhibitors lapatinib and neratinib relative to HER2-wild-type cell lines (IC50 < 200 nmol/L in HER2V659E vs. IC50 > 2,500 nmol/L in HER2WT). Conclusions: This study creates a foundation for molecular understanding of and drug development for canine lung cancer. These data also establish molecular contexts for comparative studies in dogs and humans of low mutation burden, never-smoker lung cancer, and mutant HER2 function and inhibition.

Naturally occurring primary canine lung cancers are aggressive malignancies that are increasingly common in pet dogs. They share clinicopathologic features with human lung cancers in never-smokers, but their genetic underpinnings are unknown. Through multi-platform sequencing of 88 primary canine lung tumors or cell lines, we discovered somatic, coding HER2 (ERRB2) point mutations in 38% of canine pulmonary adenocarcinomas (cPAC, 28/74), but none in adenosquamous (cPASC, 0/11) or squamous cell (cPSCC, 0/3) carcinomas. In cPASC, PTEN was the most frequently mutated gene (18%) while one case each bore likely pathogenic HRAS, KRAS, EGFR, MET, TP53, or VHL somatic mutations. In cPSCC, no recurrently mutated genes were identified, but individual somatic coding mutations were found in BRAF and PTPN11. In cPAC, we also identified recurrent somatic mutation of TP53 (13.5%), SMAD4 (5.4%), PTEN (4.1%), and VHL (2.7%). cPACs assessed by exome sequencing displayed a low somatic mutation burden (median 64 point mutations, 19 focal copy number variants, and 1 translocation). The majority (93%) of HER2 mutations were hotspot V659E transmembrane domain (TMD) mutations comparable to activating mutations at this same site in human cancer. Other HER2 mutations identified in this study were located in the extracellular domain and TMD. HER2V659E was detected in the plasma of 33% (2/6) of dogs with localized HER2V659E tumors. HER2V659E correlated with constitutive phosphorylation of AKT in cPAC cell lines and HER2V659E lines displayed hypersensitivity to the HER2 inhibitors lapatinib and neratinib relative to HER2-wild-type cell lines. These findings have translational and comparative relevance for lung cancer and HER2 inhibition.

Background: The identification of organ-specific targetable signatures may help design more effective treatment for patients with metastatic breast cancer (MBC). We took a multi-OMIC approach to assess whether the AKT-mTOR pathway is globally activated during metastatic progression or whether it represents an organ-specific target. Methods: Snap frozen biopsies from 25 MBC patients enrolled in a prospective phase II trial were used. Sites of metastasis were classified as liver (n=8) and others (n=17), the latter including cutaneous, lung, lymph nodes, and intra-abdominal lesions. Signaling analysis of the 25 cases was performed using Reverse Phase Protein Microarray (RPPA) coupled with Laser Capture Microdissection. Activation of the AKT-mTOR pathway was quantified as phosphorylation of AKT (S473) and the mTOR target p70S6 (T389). Matched exome (WES) and RNASeq data were available for 17 of 25 patients, five with liver metastases. Sequencing data was processed using an in-house developed pipeline to identify somatic events including coding mutations, copy number alterations, gene fusions, and differential expression. Activation of the AKT-mTOR pathway and sequencing data were compared between hepatic and non-hepatic lesions using an integrated RPPA and genomic approach. Results: Among liver metastases, AKT was activated in 4 of the 8 (50.0%) patients, while 6 of the 8 cases (75.0%) showed activation of p70S6. Sequencing data revealed mutation of PIK3CA in 4 of the 5 liver metastases (80.0%). Three of the PIK3CA mutated specimens with catalytic domain mutations (codons 1023 and 147) demonstrated co-activation of AKT and p70S6, while the fourth case, containing a helical domain mutation (E542K), had activation of p70S6 only. The PIK3CA wild-type liver metastasis demonstrated low activation of AKT and p70S6. For non-hepatic metastases AKT was activated in 2 of the 17 cases (11.8%) and p70S6 in 5 of the 17 patients (29.4%). Discussion: Although these results need further validation, activation of the AKT-mTOR pathway appears to be a hepatic specific signature in MBC and could be used for the selection of targeted agents for hepatic lesions. Citation Format: Pierobon M, Wong S, Reeder A, Anthony SP, Robert NJ, Northfelt DW, Jahanzeb M, Vocila L, Wulfkuhle J, Dunetz B, Aldrich J, Byron S, Craig D, Liotta L, Petricoin EF, Carpten J. The AKT-mTOR pathway as a potential organ-specific drug target signature of hepatic metastases from breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-21.

Purpose: Prostate cancer is the most common cancer in males, with a ~1.5-2-fold higher incidence in African American men when compared with whites. Epidemiologic evidence supports a large heritable contribution to prostate cancer, with over 100 susceptibility loci identified to date that can explain ~33% of the familial risk. A portion of the undefined risk may be due to rare susceptibility variants. The African American Hereditary Prostate Cancer (AAHPC) Study, established in 1997, enrolled 77 African American families from seven clinical sites across the United States. The aim of this study is to identify rare, predictive, deleterious variants through exome sequencing of 99 cases from families selected from the AAHPC families and three 1000 Genome controls. Methods: To explore the contribution of rare variation in coding regions to prostate cancer risk, we sequenced the exomes of 99 AAHPC cases at a mean coverage of 30x. Post-variant calling quality control (QC) was implemented using Golden Helix SVS 8 software with filters set for removal of variants with Read Depth >10, Quality Score >10, and Quality Score: Read Depth Ratio > 0.5. Mendelian inconsistency was checked using PLINK. Prioritization of all candidate genes/variants was evaluated using online databases 1000 Genome and bioinformatics tool ANNOVAR for non-reference allele frequency and predictions of functional impact. Conclusions: Through exome sequencing of 99 AAHPC cases and three 1000 Genome controls, we identified 37 nonsynonymous single-nucleotide variants that are considered damaging by at least one predictive scoring tool in our candidate genes. Interesting candidate variants were found in known cancer susceptibility loci BRCA2, MSR1, PCNT, STAT3, WRN and ZFHX3. Future results are pending additional QC and analyses to determine which variants are shared by related individuals within each family compared to those not seen in the controls. Citation Format: Deyana D. Lewis, Shukmei Wong, Angela S. Baker, Joan E. Bailey-Wilson, John D. Carpten, Cheryl D. Cropp. Deleterious coding variants in African American Hereditary Prostate Cancer Study (AAHPC) families [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C050.

Background Polymerase chain reaction for antigen receptor rearrangement (PARR) is a molecular diagnostic tool used for discrimination of lymphoid malignancies in dogs from benign processes. Assay variations have been described and are commercially available, but performance metrics are not uniformly reported. Objectives To describe performance (accuracy, sensitivity, specificity) and rigorous benchmarking of a PARR protocol (ePARR) in clinically relevant samples. Animals One hundred eighty-one client-owned dogs. Methods Lymphoma and benign tissues representative of the clinical spectrum with gold standard histopathologic and immunohistochemical diagnoses were collected. Assay development and benchmarking were performed on fresh frozen (FF) tissue, formalin-fixed paraffin-embedded (FFPE) tissue, flow cytometry pellets, and air-dried fine-needle aspirates (FNA). Assay performance was determined for FFPE from 56 dogs (18 B-cell lymphoma, 24 T-cell lymphoma, and 14 non-lymphoma), 80 frozen flow cytometry pellets (66 B-cell lymphoma, 14 T-cell lymphoma, 0 non-lymphoma), and 41 air-dried FNA slides (23 lymphoma, 18 non-lymphoma). Results For discrimination of lymphoma versus non-lymphoma, ePARR had 92% and 92% sensitivity and specificity on FFPE with 92% accuracy, 85% sensitivity from flow cytometry pellets (non-lymphoma was not evaluated to calculate specificity) with 85% accuracy, and 100% and 100% sensitivity and specificity for FNA with 100% accuracy. Stringent quality control criteria decreased assay success rate without significant performance improvement. Performance metrics were lower in most cases for discrimination of B- or T-cell versus non-B- or non-T-cell samples than for lymphoma versus non-lymphoma. Conclusions and clinical importance These benchmarking data facilitate effective interpretation and application of PARR assays in multiple sample types.

Purpose: Little is known about the molecular signatures associated with specific metastatic sites in breast cancer. Using comprehensive multi-omic molecular profiling, we assessed whether alterations or activation of the PI3K–AKT–mTOR pathway is associated with specific sites of breast cancer metastasis. Experimental Design: Next-generation sequencing–based whole-exome sequencing was coupled with reverse-phase protein microarray (RPPA) functional signaling network analysis to explore the PI3K–AKT–mTOR axis in 32 pretreated breast cancer metastases. RPPA-based signaling data were further validated in an independent cohort of 154 metastatic lesions from breast cancer and 101 unmatched primary breast tumors. The proportion of cases with PI3K–AKT–mTOR genomic alterations or signaling network activation were compared between hepatic and nonhepatic lesions. Results: PIK3CA mutation and activation of AKT (S473) and p70S6K (T389) were detected more frequently among liver metastases than nonhepatic lesions (P < 0.01, P = 0.056, and P = 0.053, respectively). However, PIK3CA mutations alone were insufficient in predicting protein activation (P = 0.32 and P = 0.19 for activated AKT and p70S6K, respectively). RPPA analysis of an independent cohort of 154 tumors confirmed the relationship between pathway activation and hepatic metastasis [AKT (S473), mTOR (S2448), and 4EBP1 (S65); P < 0.01, P = 0.02, and P = 0.01, respectively]. Similar results were also seen between liver metastases and primary breast tumors [AKT (S473) P < 0.01, mTOR (S2448) P < 0.01, 4EBP1 (S65) P = 0.01]. This signature was lost when primary tumors were compared with all metastatic sites combined. Conclusions: Breast cancer patients with liver metastasis may represent a molecularly homogenized cohort with increased incidence of PIK3CA mutations and activation of the PI3K–AKT–mTOR signaling network. Clin Cancer Res; 23(16); 4919–28. ©2017 AACR.

Background: Despite a growing understanding of the somatic landscape of breast tumors from BRCA1 and BRCA2 mutation carriers, less is known about breast tumors from carriers of germline mutations in other homologous recombination and DNA repair pathway genes such as ATM, CHEK2, and PALB2. Methods: We identified 44 clinically annotated breast cancer cases that included carriers of germline mutations in BRCA1 (n=9), BRCA2 (n=9), ATM (n=5), CHEK2 (n=7), and PALB2 (n=6) from the Hereditary Cancer Risk Program at BUMC. Sporadic breast cancers cases (n=8) were also collected. Genomic DNA and RNA were extracted from macro-dissected FFPE tumor sections, adjacent normal FFPE tissue, along with constitutional genomic DNA from blood. Expanded whole exome sequencing (WES) was performed on normal/tumor pairs and RNA-seq from tumors for each case. Bioinformatics analysis was performed using industry standard methods for somatic characterization. Results: All germline mutations were confirmed by WES. Somatic mutational analysis and copy number profiling from WES revealed the greatest similarities among BRCA1 and CHEK2 carriers. As expected, TP53 mutations were found in 8 of 9 BRCA1 carriers as all were triple negative subtype. We also detected somatic TP53 mutations in tumors from 4 of 7 CHEK2 carriers. Somatic TP53 mutations were found in only 1 of 7 BRCA2 tumors and 1 of 4 PALB2 tumors tested. Furthermore, BRCA1 and CHEK2 tumors showed trends of having higher mutation burden. Analysis of copy number BRCAness demonstrated stronger similarities between BRCA1, ATM, CHEK2, and PALB2 tumors. BRCA2 tumors were unique with fewer events and characterized by specific amplifications including 11q23 (CCND1) and 17q23 (BRIP1). Hierarchical clustering of RNA-seq data revealed strong clustering of BRCA1 tumors compared to all other tumors, predominantly attributed to breast cancer subtype. Furthermore, pathway analysis of genes that distinguish BRCA1 mutation positive versus non-BRCA mutated tumors showed strong correlation to pro-inflammatory and immune pathway signatures. Conclusions: Molecular analysis of 44 breast cancers from individuals with inherited predisposition to breast cancer via BRCA1, BRCA2, ATM, CHEK2, and PALB2 germline mutations demonstrated strongest somatic similarities between BRCA1 and CHEK2 tumors although all BRCA1 were TNBC and all CHEK2 tumors were ER positive. Marked differential gene expression differences in RNA expression patterns were observed in BRCA1 mutation carriers compared with all other groups analyzed. Our study is among the first to interrogate the profile of non-BRCA mutated hereditary breast cancers. Citation Format: Blum JL, Wong S, Pearson EJ, Nair A, Snipes GJ, Briones N, Baker A, Cropp CD, Carpten JD. Molecular analysis of breast cancers from individuals with hereditary cancer syndromes secondary to mutations in BRCA1, BRCA2, ATM, CHEK2, and PALB2 [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-04-02.

This abstract was withdrawn by the authors.

Spontaneous primary canine lung cancers are aggressive malignancies that are increasingly common in dogs. They share clinicopathologic features with human lung cancers in never-smokers, but their genetic underpinnings are unknown. As never-smoker lung cancer incidence increases in humans, a need exists for improved biologic understanding and development of new models to fuel translational research. Here, we describe for the first time the detailed clinical and genetic features of primary canine lung cancers through case review and multi-platform sequencing of 90 primary canine lung tumors and cell lines. We performed multi-platform genomic profiling including whole exome sequencing (WES), amplicon panel-based sequencing of 250 regions of 51 cancer genes, and single nucleotide polymorphism (SNP) array-based analysis of tumors and matched constitutional DNA. We profiled 76 cPAC, 11 canine pulmonary adenosquamous carcinomas (cPASC) and three canine pulmonary squamous cell carcinomas (cPSCC). The median age at the time of diagnosis was 11 year old. The most common affected pure breed was the Labrador retriever (19%). Five cPACs assessed by WES displayed low mutation burden with a median of 64 somatic single nucleotide variants (SNVs), 19 somatic copy number variants (CNVs), and 1 structural variant (SV), and frequent C>T substitutions (80%) at NpCpG trinucleotides, a common mutation signature seen across human and canine cancers. Based on WES and amplicon sequencing, we discovered somatic, coding HER2 (ERRB2) point mutations in 37% of cPAC, but none in cPASC or cPSCC. Additional recurrently mutated genes in cPAC included CDKN2A/B (~40%), TP53 (~12%), K/HRAS (~7%), SMAD4 (~5%), and PTEN (~5%). In cPASC, KRAS and TP53 were the most frequently mutated genes (18% each). In cPSCC, no recurrently mutated genes were identified, but individual somatic coding mutations were found in BRAF and PTPN11. The majority (93%) of HER2 mutations were hotspot V659E, located on the transmembrane domain (TMD), and comparable to activating mutations at this same site in human cancer. We were able to detect this mutation not only in biopsied tumors, but also in the plasma of 33% (2/6) of dogs with localized V659E-positive tumors. Other somatic HER2 mutations identified were similarly located in the HER2 juxtamembrane domain and TMD including A664T and K676E. HER2 point mutations occurred in the absence of significant focal amplification (assessed by WES and SNP array) or overexpression (assessed by qRT-PCR and IHC). We also showed that HER2 mutation correlated with constitutive phosphorylation of Akt in cPAC cell lines. Furthermore, HER2 V659E-mutant lines displayed hypersensitivity to the HER2 kinase inhibitors neratinib (IC50, 23nM) and lapatinib (IC50, 168nM) relative to HER2 wild-type cell lines (IC50, >2500nM). These data bear implications for comparative understanding of HER2-mutant lung cancer across species. Citation Format: Gwendolen Lorch, Karthigayini Sivaprakasam, Victoria Zissman, Nieves Perdigones, Tania Contente-Cuomo, Alexandra Nazareno, Salvatore Facista, ShukMei Wong, Winnie Liang, Joseph M. Amann, Sara L. Sinicropi-Yao, Michael J. Koenig, Krista La Perle, Timothy G. Whitsett, Muhammed Murtaza, Jeffrey Trent, David P. Carbone, William P. Hendricks. Identification of frequent HER2 activating mutations in canine primary pulmonary adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3705.

Purpose: Prostate cancer is the most common cancer in males, but also exhibits a ∼1.5-2-fold higher incidence in African American men compared with whites. Epidemiologic evidence supports a large heritable contribution to prostate cancer, with over 100 susceptibility loci identified to date that can explain ∼33 % of the familial risk. A portion of the undefined risk may be due to rare susceptibility variants. The African American Hereditary Prostate Cancer (AAHPC) Study, established in 1997, enrolled 77 AA families from seven clinical sites across the United States (Ann Epidemiol, 2000). The aim of this study is to identify rare, predictive, deleterious variants through exome sequencing of 99 cases from 26 families selected from the AAHPC families (2 to 3 affected men sequenced per family) and three female 1000 Genome controls. Methods: To explore the contribution of rare variation in coding regions of 38 known cancer-causing genes to prostate cancer risk (PCa), we sequenced the exomes of 99 AAHPC cases at a mean coverage of 30x. Post-variant calling quality control (QC) was implemented using Golden Helix SVS 8 software with filters set for removal of variants with Read Depth >10, Quality Score >10, and Quality Score: Read Depth Ratio > 0.5. Mendelian inconsistency was checked using PLINK. Prioritization of all candidate genes/variants were evaluated using online databases including 1000 Genomes and ANNOVAR for non-reference allele frequency and predictions of functional impact. Conclusions. Through exome sequencing of 99 AAHPC cases and 3 female 1000 Genome controls, we identified 37 non-synonymous single nucleotide variants that are considered damaging by at least one predictive scoring tool in our 38 candidate genes. However, most of these variants were common and thus unlikely to be causal. We observed three rare variants that were considered damaging by three predictive scoring tools in two known PCa genes, PCNT and ZFHX3. These 3 variants were each observed in a single different family (in 1 of 3 affected individuals). Interesting rare candidate variants (MAF ≤ 0.01) rs190517526 & rs114692729 were found in known cancer susceptibility loci (CD82 and EZH2). The CD82 variant was observed in all 3 sequenced affecteds in one family and the EZH2 variant was observed in 2 of 3 sequenced affecteds in a different family. These predicted damaging variants in these four genes represent potentially novel causal candidates for some of the 26 AAHPC families sequenced here. Future work will carefully analyze the remainder of the genome in the other 21 sequenced families including planned sequencing of additional family members. Citation Format: Deyana D. Lewis, Shukmei Wong, Angela S. Baker, Isaac Powell, John D. Carpten, Joan E. Bailey-Wilson, Cheryl Cropp. Rare candidate variants shared among affected family members in the African American Hereditary Prostate Cancer Study families [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4240.

Large-scale multiplexed identification of somatic alterations in cancer has become feasible with next generation sequencing (NGS). However, calibration of NGS somatic analysis tools has been hampered by a lack of tumor/normal reference standards. We thus performed paired PCR-free whole genome sequencing of a matched metastatic melanoma cell line (COLO829) and normal across three lineages and across separate institutions, with independent library preparations, sequencing, and analysis. We generated mean mapped coverages of 99X for COLO829 and 103X for the paired normal across three institutions. Results were combined with previously generated data allowing for comparison to a fourth lineage on earlier NGS technology. Aggregate variant detection led to the identification of consensus variants, including key events that represent hallmark mutation types including amplified BRAF V600E, a CDK2NA small deletion, a 12 kb PTEN deletion, and a dinucleotide TERT promoter substitution. Overall, common events include >35,000 point mutations, 446 small insertion/deletions, and >6,000 genes affected by copy number changes. We present this reference to the community as an initial standard for enabling quantitative evaluation of somatic mutation pipelines across institutions.

TNBC is the only subtype of breast cancer for which there are no approved targeted therapies. In the US, its incidence is highest in women with African ancestry (AA); in western sub-Saharan Africa, single-institution studies show that TNBC constitutes 40- 80% of all breast cancers. Given the Caucasian/AA survival disparity in breast cancer, there is an urgent need to find actionable targets in TNBC of all ethnicities, but especially in TNBC in AA, which are suspected to be more aggressive. Breast cancer stem cells, the small population of cells that have been shown to mediate breast tumor initiation, metastasis, and resistance to conventional therapy have also been reported to mediate the heterogeneity of TNBC and are especially abundant in TNBC in AA women. Here, we sought to better understand the biology of TNBC by finding genes and pathways that are differentially expressed in the stem cell population of patient derived xenografts (PDX) from TNBC from Ghanaian (G), AA and Caucasian (C) women and the effect of these differentially expressed genes on the stem cell phenotype in these primary tumors. We isolated the ALDH+ and the CD44+/CD24- stem cell populations from the bulk cells from 15 PDXs using flow cytometry. We performed RNA sequencing (Illumina HiSeq platform) on the isolated populations and bulk cells (45). Comprehensive bioinformatics analyses led to the identificationof highly significantly differentially expressed genes and pathways between the cell populations. By principal component analysis, the tumors were very heterogeneous. However, the ALDH+ cells separated out from the CD44+/CD24- and the bulk cells. We identified 14 genes that were simultaneously differentially expressed between the ALDH+ vs the CD44+/CD24- as well as ALDH+ VS bulk (p-value Citation Format: Evelyn M. Jiagge, Shukmei Wong, Rabia Gilani, Sean Mcdermott, Lisa Newman, Jessica Bensenhaver, Max Wicha, John Carpten, Sofia Merajver. Aldh expressing stem cells mediate tumor initiation and metastasis in triple negative breast cancers across different ethnicities [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 3371. doi:10.1158/1538-7445.AM2017-3371

A regional key comparison, APMP.L-K3 (calibration of angle standards) was held in 2005. Thirteen National Metrology Institutes (NMISA, NMIA, KRISS, NMIJ/ AIST, NMC/ A*STAR, NIMT, SCL, CMS/ ITRI, NPLI, RCM- LIPI, NIM, and NSCL) have participated in this comparison and the measurements were carried out during the period from April 2005 to December 2007. This report describes the measurement results of a 12-sided optical polygon with nominal angles of 30°, and four angle blocks with nominal angles of 5 arc seconds, 5 minutes, 30 minutes and 5 degrees, respectively. Main text To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCL, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

Triple-negative breast cancer (TNBC) is characterized by the absence of expression of estrogen receptor, progesterone receptor, and HER-2. Thirty percent of patients recur after first-line treatment, and metastatic TNBC (mTNBC) has a poor prognosis with median survival of one year. Here, we present initial analyses of whole genome and transcriptome sequencing data from 14 prospective mTNBC. We have cataloged the collection of somatic genomic alterations in these advanced tumors, particularly those that may inform targeted therapies. Genes mutated in multiple tumors included TP53, LRP1B, HERC1, CDH5, RB1, and NF1. Notable genes involved in focal structural events were CTNNA1, PTEN, FBXW7, BRCA2, WT1, FGFR1, KRAS, HRAS, ARAF, BRAF, and PGCP. Homozygous deletion of CTNNA1 was detected in 2 of 6 African Americans. RNA sequencing revealed consistent overexpression of the FOXM1 gene when tumor gene expression was compared with nonmalignant breast samples. Using an outlier analysis of gene expression comparing one cancer with all the others, we detected expression patterns unique to each patient's tumor. Integrative DNA/RNA analysis provided evidence for deregulation of mutated genes, including the monoallelic expression of TP53 mutations. Finally, molecular alterations in several cancers supported targeted therapeutic intervention on clinical trials with known inhibitors, particularly for alterations in the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways. In conclusion, whole genome and transcriptome profiling of mTNBC have provided insights into somatic events occurring in this difficult to treat cancer. These genomic data have guided patients to investigational treatment trials and provide hypotheses for future trials in this irremediable cancer. Mol Cancer Ther; 12(1); 104–16. ©2012 AACR.