Chia Jen Liu

Aldosterone-stimulating somatic gene mutations are common in normal adrenal glands

Significance Primary aldosteronism (PA) represents the most common adrenal disease and cause of secondary hypertension. However, little is known regarding adrenal cellular origins. Recently, subcapsular aldosterone-producing cell clusters (APCCs) were observed in normal adrenals. We hypothesize that APCCs are a contributor to PA. Here, we characterized the APCC transcriptome and show that CYP11B2 expression is increased compared with the rest of the adrenal cortex. We also show that many APCCs harbor known aldosterone-producing adenoma (APA)-related ion channels/pumps (ATPase, Na+/K+ transporting, α1-polypeptide and calcium channel, voltage-dependent, L-type, α1D-subunit) mutations that stimulate CYP11B2 expression and aldosterone production. Importantly, the mutation spectrum seen in APCCs differs from that reported for APA. These results provide molecular support for APCC as a precursor of PA. Primary aldosteronism (PA) represents the most common cause of secondary hypertension, but little is known regarding its adrenal cellular origins. Recently, aldosterone-producing cell clusters (APCCs) with high expression of aldosterone synthase (CYP11B2) were found in both normal and PA adrenal tissue. PA-causing aldosterone-producing adenomas (APAs) harbor mutations in genes encoding ion channels/pumps that alter intracellular calcium homeostasis and cause renin-independent aldosterone production through increased CYP11B2 expression. Herein, we hypothesized that APCCs have APA-related aldosterone-stimulating somatic gene mutations. APCCs were studied in 42 normal adrenals from kidney donors. To clarify APCC molecular characteristics, we used microarrays to compare the APCC transcriptome with conventional adrenocortical zones [zona glomerulosa (ZG), zona fasciculata, and zona reticularis]. The APCC transcriptome was most similar to ZG but with an enhanced capacity to produce aldosterone. To determine if APCCs harbored APA-related mutations, we performed targeted next generation sequencing of DNA from 23 APCCs and adjacent normal adrenal tissue isolated from both formalin-fixed, paraffin-embedded, and frozen tissues. Known aldosterone driver mutations were identified in 8 of 23 (35%) APCCs, including mutations in calcium channel, voltage-dependent, L-type, α1D-subunit (CACNA1D; 6 of 23 APCCs) and ATPase, Na+/K+ transporting, α1-polypeptide (ATP1A1; 2 of 23 APCCs), which were not observed in the adjacent normal adrenal tissue. Overall, we show three major findings: (i) APCCs are common in normal adrenals, (ii) APCCs harbor somatic mutations known to cause excess aldosterone production, and (iii) the mutation spectrum of aldosterone-driving mutations is different in APCCs from that seen in APA. These results provide molecular support for APCC as a precursor of PA.

Development and Validation of a Scalable Next-Generation Sequencing System for Assessing Relevant Somatic Variants in Solid Tumors

Next-generation sequencing (NGS) has enabled genome-wide personalized oncology efforts at centers and companies with the specialty expertise and infrastructure required to identify and prioritize actionable variants. Such approaches are not scalable, preventing widespread adoption. Likewise, most targeted NGS approaches fail to assess key relevant genomic alteration classes. To address these challenges, we predefined the catalog of relevant solid tumor somatic genome variants (gain-of-function or loss-of-function mutations, high-level copy number alterations, and gene fusions) through comprehensive bioinformatics analysis of >700,000 samples. To detect these variants, we developed the Oncomine Comprehensive Panel (OCP), an integrative NGS-based assay [compatible with < 20 ng of DNA/RNA from formalin-fixed paraffin-embedded (FFPE) tissues], coupled with an informatics pipeline to specifically identify relevant predefined variants and created a knowledge base of related potential treatments, current practice guidelines, and open clinical trials. We validated OCP using molecular standards and more than 300 FFPE tumor samples, achieving >95% accuracy for KRAS, epidermal growth factor receptor, and BRAF mutation detection as well as for ALK and TMPRSS2:ERG gene fusions. Associating positive variants with potential targeted treatments demonstrated that 6% to 42% of profiled samples (depending on cancer type) harbored alterations beyond routine molecular testing that were associated with approved or guideline-referenced therapies. As a translational research tool, OCP identified adaptive CTNNB1 amplifications/mutations in treated prostate cancers. Through predefining somatic variants in solid tumors and compiling associated potential treatment strategies, OCP represents a simplified, broadly applicable targeted NGS system with the potential to advance precision oncology efforts.

Next-Gen Sequencing Exposes Frequent MED12 Mutations and Actionable Therapeutic Targets in Phyllodes Tumors

Phyllodes tumors are rare fibroepithelial tumors with variable clinical behavior accounting for a small subset of all breast neoplasms, yet little is known about the genetic alterations that drive tumor initiation and/or progression. Here, targeted next-generation sequencing (NGS) was used to identify somatic alterations in formalin-fixed paraffin-embedded (FFPE) patient specimens from malignant, borderline, and benign cases. NGS revealed mutations in mediator complex subunit 12 (MED12) affecting the G44 hotspot residue in the majority (67%) of cases spanning all three histologic grades. In addition, loss-of-function mutations in p53 (TP53) as well as deleterious mutations in the tumor suppressors retinoblastoma (RB1) and neurofibromin 1 (NF1) were identified exclusively in malignant tumors. High-level copy-number alterations (CNA) were nearly exclusively confined to malignant tumors, including potentially clinically actionable gene amplifications in IGF1R and EGFR. Taken together, this study defines the genomic landscape underlying phyllodes tumor development, suggests potential molecular correlates to histologic grade, expands the spectrum of human tumors with frequent recurrent MED12 mutations, and identifies IGF1R and EGFR as potential therapeutic targets in malignant cases. Implications: Integrated genomic sequencing and mutational profiling provides insight into the molecular origin of phyllodes tumors and indicates potential druggable targets in malignant disease. Visual Overview: http://mcr.aacrjournals.org/content/early/2015/04/02/1541-7786.MCR-14-0578/F1.large.jpg. Mol Cancer Res; 13(4); 613–9. ©2015 AACR. Visual Overview

Next-Generation Sequencing of Tubal Intraepithelial Carcinomas

IMPORTANCE High-grade serous carcinoma (HGSC) is the most prevalent and lethal form of ovarian cancer. HGSCs frequently arise in the distal fallopian tubes rather than the ovary, developing from small precursor lesions called serous tubal intraepithelial carcinomas (TICs, or more specifically, STICs). While STICs have been reported to harbor TP53 mutations, detailed molecular characterizations of these lesions are lacking. OBSERVATIONS We performed targeted next-generation sequencing (NGS) on formalin-fixed, paraffin-embedded tissue from 4 women, 2 with HGSC and 2 with uterine endometrioid carcinoma (UEC) who were diagnosed as having synchronous STICs. We detected concordant mutations in both HGSCs with synchronous STICs, including TP53 mutations as well as assumed germline BRCA1/2 alterations, confirming a clonal association between these lesions. Next-generation sequencing confirmed the presence of a STIC clonally unrelated to 1 case of UEC, and NGS of the other tubal lesion diagnosed as a STIC unexpectedly supported the lesion as a micrometastasis from the associated UEC. CONCLUSIONS AND RELEVANCE We demonstrate that targeted NGS can identify genetic alterations in minute lesions, such as TICs, and confirm TP53 mutations as early driving events for HGSC. Next-generation sequencing also demonstrated unexpected associations between presumed STICs and synchronous carcinomas, providing evidence that some TICs are actually metastases rather than HGSC precursors.

Tumor evolution and progression in multifocal and paired non-invasive/invasive urothelial carcinoma

Although multifocal tumors and non-invasive/invasive components are commonly encountered in surgical pathology, their genetic relationship is often poorly characterized. We used next-generation sequencing (NGS) to characterize somatic alterations in a patient with five spatially distinct, high-grade papillary urothelial carcinomas (UCs), with one tumor harboring an underlying invasive component. NGS of 409 cancer-related genes was performed on DNA isolated from formalin-fixed paraffin-embedded (FFPE) blocks representing each papillary tumor (n = 5), the invasive component of one tumor, and matched normal tissue. We identified nine unique non-synonymous somatic mutations across the six UC samples, including five present in each carcinoma sample, consistent with clonal origin and limited intertumoral heterogeneity. Copy number and loss of heterogeneity (LOH) profiles were similar in all six carcinomas; however, the invasive carcinoma component uniquely showed focal CDKN2A loss and chromosome 9 LOH and did not harbor gains of chromosomes 5p or X that were present in the other tumor samples. Phylogenetic analysis supported the invasive component arising from a shared progenitor prior to the outgrowth of cells in the non-invasive tumors. Results were extended to three additional cases of upper tract UC with paired non-invasive/invasive components, which identified driving alterations exclusive to both non-invasive and invasive components. Lastly, we performed targeted RNA sequencing (RNAseq) using a custom bladder cancer panel, which confirmed gene expression signature differences between paired non-invasive/invasive components. The results and approaches presented here may be useful in understanding the clonal relationships in multifocal cancers or paired non-invasive/invasive components from routine FFPE specimens.

Genomic Profiling of Penile Squamous Cell Carcinoma Reveals New Opportunities for Targeted Therapy

Penile squamous cell carcinoma (PeSCCA) is a rare malignancy for which there are limited treatment options due to a poor understanding of the molecular alterations underlying disease development and progression. Therefore, we performed comprehensive, targeted next-generation sequencing to identify relevant somatic genomic alterations in a retrospective cohort of 60 fixed tumor samples from 43 PeSCCA cases (including 14 matched primary/metastasis pairs). We identified a median of two relevant somatic mutations and one high-level copy-number alteration per sample (range, 0-5 and 0-6, respectively). Expression of HPV and p16 was detectable in 12% and 28% of patients, respectively. Furthermore, advanced clinical stage, lack of p16 expression, and MYC and CCND1 amplifications were significantly associated with shorter time to progression or PeSCCA-specific survival. Notably, four cases harbored EGFR amplifications and one demonstrated CDK4 amplification, genes for which approved and investigational targeted therapies are available. Importantly, although paired primary tumors and lymph node metastases were largely homogeneous for relevant somatic mutations, we identified heterogeneous EGFR amplification in primary tumor/lymph node metastases in 4 of 14 cases, despite uniform EGFR protein overexpression. Likewise, activating HRAS mutations occurred in 8 of 43 cases. Taken together, we provide the first comprehensive molecular PeSCCA analysis, which offers new insight into potential precision medicine approaches for this disease, including strategies targeting EGFR.

Comprehensive genomic profiling of orbital and ocular adnexal lymphomas identifies frequent alterations in MYD88 and chromatin modifiers: New routes to targeted therapies

Non-Hodgkin lymphoma of the orbit and ocular adnexa is the most common primary orbital malignancy. Treatments for low- (extra-nodal marginal zone and follicular lymphomas) and high-grade (diffuse large B-cell lymphoma) are associated with local and vision-threatening toxicities. High-grade lymphomas relapse frequently and exhibit poor survival rates. Despite advances in genomic profiling and precision medicine, orbital and ocular adnexal lymphomas remain poorly characterized molecularly. We performed targeted next-generation sequencing (NGS) profiling of 38 formalin-fixed, paraffin-embedded orbital and ocular adnexal lymphomas obtained from a single-center using a panel targeting near-term, clinically relevant genes. Potentially actionable mutations and copy number alterations were prioritized based on gain- and loss-of-function analyses, and catalogued, approved, and investigational therapies. Of 36 informative samples, including marginal zone lymphomas (n=20), follicular lymphomas (n=9), and diffuse large B-cell lymphomas (n=7), 53% harbored a prioritized alteration (median=1, range 0–5/sample). MYD88 was the most frequently altered gene in our cohort, with potentially clinically relevant hotspot gain-of-function mutations identified in 71% of diffuse large B-cell lymphomas and 25% of marginal zone lymphomas. Prioritized alterations in epigenetic modulators were common and included gain-of-function EZH2 and loss-of-function ARID1A mutations (14% of diffuse large B-cell lymphomas and 22% of follicular lymphomas contained alterations in each of these two genes). Single prioritized alterations were also identified in the histone methyltransferases KMT2B (follicular lymphoma) and KMT3B (diffuse large B-cell lymphoma). Loss-of-function mutations and copy number alterations in the tumor suppressors TP53 (diffuse large B-cell and follicular lymphoma), CDKN2A (diffuse large B-cell and marginal zone lymphoma), PTEN (diffuse large B-cell lymphoma), ATM (diffuse large B-cell lymphoma), and NF1 (diffuse large B-cell lymphoma), and gain-of-function mutations in the oncogenes HRAS (follicular lymphoma) and NRAS (diffuse large B-cell lymphoma) were also observed. Together, our study demonstrates that NGS can be used to profile routine formalin-fixed, paraffin-embedded orbital and ocular adnexal lymphomas for identification of somatic-driving alterations and nomination of potential therapeutic strategies.

Comprehensive serial molecular profiling of an “N of 1” exceptional non-responder with metastatic prostate cancer progressing to small cell carcinoma on treatment

ImportanceSmall cell carcinoma/neuroendocrine prostate cancer (NePC) is a lethal, poorly understood prostate cancer (PCa) subtype. Controversy exists about the origin of NePC in this setting.ObjectiveTo molecularly profile archived biopsy specimens from a case of early-onset PCa that rapidly progressed to NePC to identify drivers of the aggressive course and mechanisms of NePC origin and progression.Design, setting, and participantsA 47-year-old patient presented with metastatic prostatic adenocarcinoma (Gleason score 9). After a 6-month response to androgen deprivation therapy, the patient developed jaundice and liver biopsy revealed exclusively NePC. Targeted next generation sequencing (NGS) from formalin-fixed paraffin-embedded (FFPE)-isolated DNA was performed from the diagnostic prostate biopsy and the liver biopsy at progression.InterventionAndrogen deprivation therapy for adenocarcinoma followed by multiagent chemotherapy for NePC.Main outcomes and measuresIdentification of the mutational landscape in primary adenocarcinoma and NePC liver metastasis. Whether the NePC arose independently or was derived from the primary adenocarcinoma was considered based on mutational profiles.ResultsA deleterious somatic SMAD4 L535fs variant was present in both prostate and liver specimens; however, a TP53 R282W mutation was exclusively enriched in the liver specimen. Copy number analysis identified concordant, low-level alterations in both specimens, with focal MYCL amplification and homozygous PTEN, RB1, and MAP2K4 losses identified exclusively in the NePC specimen. Integration with published genomic profiles identified MYCL as a recurrently amplified in NePC.Conclusions and relevanceNGS of routine biopsy samples from an exceptional non-responder identified SMAD4 as a driver of the aggressive course and supports derivation of NePC from primary adenocarcinoma (transdifferentiation).

Molecular Profiling to Determine Clonality of Serial Magnetic Resonance Imaging/Ultrasound Fusion Biopsies from Men on Active Surveillance for Low Risk Prostate Cancer

Purpose: To determine whether MRI/ultrasound (MRI/US) fusion biopsy facilitates longitudinal resampling of the same clonal focus of prostate cancer and to determine whether high-grade cancers can evolve from low-grade clones. Experimental Design: All men on active surveillance who underwent tracking MRI/US fusion biopsy of Gleason 6 prostate cancer, on at least two distinct occasions, between 2012 and 2014 were enrolled. MRI/US fusion was used to track and resample specific cancer foci. IHC for ERG and targeted RNA/DNA next-generation sequencing (NGS) were performed on formalin-fixed paraffin-embedded prostate biopsy specimens to assess clonality. Results: Thirty-one men with median age and PSA of 65 years and 4.6 ng/mL, respectively, were analyzed. The median sampling interval was 12 months (range, 5–35). Of the 26 evaluable men, ERG IHC concordance was found between initial and repeat biopsies in 25 (96%), indicating resampling of the same clonal focus over time. Targeted NGS supported ERG IHC results and identified unique and shared driving mutations, such as IDH1 and SPOP, in paired specimens. Of the nine men (34.6%) who were found to have Gleason ≥7 on repeat biopsy, all displayed temporal ERG concordance. Prioritized genetic alterations were detected in 50% (13/26) of paired samples. Oncogenic mutations were detected in 22% (2/9) of Gleason 6 cancers prior to progression and 44% (4/9) of Gleason ≥7 cancers when progression occurred. Conclusions: Precise tracking of prostate cancer foci via MRI/US fusion biopsy allowed subsequent resampling of the same clonal focus of cancer over time. Further research is needed to clarify the grade progression potential of Gleason 6 prostate cancer. Clin Cancer Res; 23(4); 985–91. ©2016 AACR.

Rapid, ultra low coverage copy number profiling of cell-free DNA as a precision oncology screening strategy

Current cell-free DNA (cfDNA) next generation sequencing (NGS) precision oncology workflows are typically limited to targeted and/or disease-specific applications. In advanced cancer, disease burden and cfDNA tumor content are often elevated, yielding unique precision oncology opportunities. We sought to demonstrate the utility of a pan-cancer, rapid, inexpensive, whole genome NGS of cfDNA approach (PRINCe) as a precision oncology screening strategy via ultra-low coverage (~0.01x) tumor content determination through genome-wide copy number alteration (CNA) profiling. We applied PRINCe to a retrospective cohort of 124 cfDNA samples from 100 patients with advanced cancers, including 76 men with metastatic castration-resistant prostate cancer (mCRPC), enabling cfDNA tumor content approximation and actionable focal CNA detection, while facilitating concordance analyses between cfDNA and tissue-based NGS profiles and assessment of cfDNA alteration associations with mCRPC treatment outcomes. Therapeutically relevant focal CNAs were present in 42 (34%) cfDNA samples, including 36 of 93 (39%) mCRPC patient samples harboring AR amplification. PRINCe identified pre-treatment cfDNA CNA profiles facilitating disease monitoring. Combining PRINCe with routine targeted NGS of cfDNA enabled mutation and CNA assessment with coverages tuned to cfDNA tumor content. In mCRPC, genome-wide PRINCe cfDNA and matched tissue CNA profiles showed high concordance (median Pearson correlation = 0.87), and PRINCe detectable AR amplifications predicted reduced time on therapy, independent of therapy type (Kaplan-Meier log-rank test, chi-square = 24.9, p < 0.0001). Our screening approach enables robust, broadly applicable cfDNA-based precision oncology for patients with advanced cancer through scalable identification of therapeutically relevant CNAs and pre-/post-treatment genomic profiles, enabling cfDNA- or tissue-based precision oncology workflow optimization.