From Formalin-fixed Paraffin-embedded Research Articles

Routine application of the Lymph2Cx assay for the subclassification of aggressive B-cell lymphoma: report of a prospective real-world series.

The subclassification of diffuse large B-cell lymphoma (DLBCL) into germinal center B-cell-like (GCB) and activated B-cell-like (ABC) subtypes has become mandatory in the 2017 update of the WHO classification of lymphoid neoplasms and will continue to be used in the WHO 5th edition. The RNA-based Lymph2Cx assay has been validated as a reliable surrogate of high-throughput gene expression profiling assays for distinguishing between GCB and ABC DLBCL and provides reliable results from formalin-fixed, paraffin-embedded (FFPE) material. This test has been previously used in clinical trials, but experience from real-world routine application is rare. We routinely applied the Lymph2Cx assay to day-to-day diagnostics on a series of 147 aggressive B-cell lymphoma cases and correlated our results with the immunohistochemical subclassification using the Hans algorithm and fluorescence in situ hybridization findings using break-apart probes for MYC, BCL2, and BCL6. The routine use of the Lymph2Cx assay had a high technical success rate (94.6%) with a low rate of failure due to poor material and/or RNA quality. The Lymph2Cx assay was discordant with the Hans algorithm in 18% (23 of 128 cases). Discordant cases were mainly classified as GCB by the Hans algorithm and as ABC by Lymph2Cx (n = 11, 8.6%). Only 5 cases (3.9%) were classified as non-GCB by the Hans algorithm and as GCB by Lymph2Cx. Additionally, 5.5% of cases (n = 7) were left unclassified by Lymph2Cx, whereas they were defined as GCB (n = 4) or non-GCB (n = 3) by the Hans algorithm. Our data support the routine applicability of the Lymph2Cx assay.

Comparative Assessment of DNA Extraction Techniques From Formalin-Fixed, Paraffin-Embedded Tumor Specimens and Their Impact on Downstream Analysis.

Good-quality nucleic acid extraction from formalin-fixed, paraffin-embedded (FFPE) specimens remains a challenge in molecular-oncopathology practice. This study evaluates the efficacy of an in-house developed FFPE extraction buffer compared with other commercially available kits. Eighty FFPE specimens processed in different surgical pathology laboratories formed the study sample. DNA extraction was performed using three commercial kits and the in-house developed FFPE extraction buffer. DNA yield was quantified by a NanoDrop spectrophotometer and Qubit Fluorometer, and its purity was measured by the 260/280-nm ratio. A fragment analyzer system was used for accurate sizing of DNA fragments of FFPE DNA. The downstream effects of all extraction methods were evaluated by polymerase chain reaction (PCR) and Sanger sequencing. In comparison with the commercial kits, the in-house buffer yielded higher DNA quantity and quality number (P < .0001). In addition, DNA integrity and fragment size were preserved in a significantly greater number of samples isolated with the in-house buffer (P < .05). The target PCR amplification rate with the in-house buffer extracted samples was also significantly higher, with 98% of the samples showing interpretable sequencing results. The in-house developed FFPE extraction buffer performed superior to other methods in terms of suitability for downstream applications, time, cost-efficiency, and ease of performance.

21P Analytical performance of FusionPlex Dx

FusionPlex Dx (FP Dx) is a 2-part multimodal next-generation sequencing (NGS)-based in vitro diagnostic kitted device that detects structural alterations and oncogenic isoforms in RNA derived from formalin-fixed, paraffin-embedded (FFPE) samples from solid tumors using proprietary Anchored Multiplex PCR (AMP™) chemistry. FP Dx is approved as a companion diagnostic for five indications: METex14 skipping events, ALK, RET, ROS1 gene fusions to guide therapy in non-small cell lung cancer and NTRK1/2/3 gene fusions for all solid tumors.

Formalin Fixation, Delay to Fixation, and Time in Fixative Adversely Impact Copy Number Variation Analysis by aCGH.

Although molecular profiling of DNA isolated from formalin-fixed, paraffin-embedded (FFPE) tumor specimens has become more common in recent years, it remains unclear how discrete FFPE processing variables may affect detection of copy number variation (CNV). To better understand such effects, array comparative genomic hybridization (aCGH) profiles of FFPE renal cell carcinoma specimens that experienced different delays to fixation (DTFs; 1, 2, 3, and 12 hours) and times in fixative (TIFs; 6, 12, 23, and 72 hours) were compared to snap-frozen tumor and blood specimens from the same patients. A greater number of regions containing CNVs relative to commercial reference DNA were detected in DNA from FFPE tumor specimens than snap-frozen tumor specimens even though they originated from the same tumor blocks. Extended DTF and TIF affected the number of DNA segments with a copy number status that differed between FFPE and frozen tumor specimens; a DTF ≥3 hours led to more segments, while a TIF of 72 hours led to fewer segments. Importantly, effects were not random as a higher guanine-cytosine (GC) content and/or a higher percentage of repeats were observed among stable regions. While limiting aCGH analysis to FFPE specimens with a DTF <3 hours and a TIF <72 hours may circumvent some effects, results from FFPE specimens should be validated against fresh or frozen specimens whenever possible.

Mass spectrometry imaging spatially identifies complex-type N-glycans as putative cartilage degradation markers in human knee osteoarthritis tissue

N-Glycan alterations contribute to the pathophysiology and progression of various diseases. However, the involvement of N-glycans in knee osteoarthritis (KOA) progression at the tissue level, especially within articular cartilage, is still poorly understood. Thus, the aim of this study was to spatially map and identify KOA-specific N-glycans from formalin-fixed paraffin-embedded (FFPE) osteochondral tissue of the tibial plateau relative to cadaveric control (CTL) tissues. Human FFPE osteochondral tissues from end-stage KOA patients (n=3) and CTL individuals (n=3), aged >55 years old, were analyzed by matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI‐MSI) and liquid chromatography–tandem mass spectrometry (LC-MS/MS). Overall, it was revealed that 22 N-glycans were found in the cartilage region of KOA and CTL tissue. Of those, 15 N-glycans were more prominent in KOA cartilage than CTL cartilage. We then compared sub-regions of KOA and CTL tissues based on the Osteoarthritis Research Society International (OARSI) histopathological grade (1 to 6), where 1 is an intact cartilage surface and 6 is cartilage surface deformation. Interestingly, three specific complex-type N-glycans, (Hex)4(HexNAc)3, (Hex)4(HexNAc)4, and (Hex)5(HexNAc)4, were found to be localized to the superficial fibrillated zone of degraded cartilage (KOA OARSI 2.5-4), compared to adjacent cartilage with less degradation (KOA OARSI 1-2) or relatively healthy cartilage (CTL OARSI 1-2). Our results demonstrate that N-glycans specific to degraded cartilage in KOA patients have been identified at the tissue level for the first time. The presence of these N-glycans could further be evaluated as potential diagnostic and prognostic biomarkers.

Targeted Next-Generation Sequencing-Based Multiple Gene Mutation Profiling of Patients with Rectal Adenocarcinoma Receiving or Not Receiving Neoadjuvant Chemoradiotherapy.

This study investigated whether oncogenic and tumor-suppressive gene mutations are involved in the differential outcomes of patients with rectal carcinoma receiving neoadjuvant chemoradiotherapy (nCRT). Genomic DNA was obtained from formalin-fixed paraffin-embedded (FFPE) specimens of patients with rectal carcinoma who received a complete nCRT course. Gene mutation status was examined in specimens from patients before and after nCRT by using the AmpliSeq platform. Our data revealed that the nonsynonymous p53, APC, KRAS, CDKN2A, and EGFR mutations were observed in 93.1%, 65.5%, 48.6%, and 31% of the patients with rectal adenocarcinoma, respectively. BRAF, FBXW7, PTEN, and SMAD4 mutations were observed in 20.7% of patients with rectal carcinoma. The following 12 gene mutations were observed more frequently in the patients exhibiting a complete response than in those demonstrating a poor response before nCRT: ATM, BRAF, CDKN2A, EGFR, FLT3, GNA11, KDR, KIT, PIK3CA, PTEN, PTPN11, SMAD4, and TP53. In addition, APC, BRAF, FBXW7, KRAS, SMAD4, and TP53 mutations were retained after nCRT. Our results indicate a complex mutational profile in rectal carcinoma, suggesting the involvement of BRAF, SMAD4, and TP53 genetic variants in the outcomes of patients with nCRT.

Abstract A40: Evaluation of the tumor microenvironment in mycosis fungoides using the PanCancer IO 360™ assay and nCounter® platform

Abstract BACKGROUND: Alterations in the tumor microenvironment (TME) can result in a “T-cell inflamed” TME, which is characterized by activation of the PD1/PD-L1 pathway, immunosuppression, and production of anergic, “exhausted” CD8(+)-T-cells. Similar alterations in the TME of Mycosis Fungoides (MF) have been linked to disease progression. However, the relationship between progression and the TME in MF remains incompletely understood. We aimed to further study the TME in MF using the PanCancer IO 360™ 750-gene expression panel and nCounter® platform (Nanostring; Seattle, WA). DESIGN: Clinical and laboratory data from the electronic medical record were reviewed in combination with microscopic review of biopsies from 27 patients to confirm diagnosis of MF and assign samples as “low-grade” (patch/plaque; n=8) or “high-grade” (tumor/large cell transformation, n=19). Patients were also classified as having “limited stage” (TNMB1 &amp;lt;1B, n=8) or “advanced stage” (TNMB1 &amp;gt;1B, n=19) disease. All patients received prior, heterogenous treatment, but no patients received prior anti-PD1 therapy. RNA was extracted from formalin-fixed, paraffin-embedded (FFPE), punch biopsy sections and analyzed per manufacturer’s instructions. Gene expression scores (log2) and a “tissue inflammation score” (TIS) were calculated for each sample. A TIS of &amp;gt;5 has been associated with the presence of a “T-cell inflamed” microenvironment. Samples scores were compared by histologic group and disease stage as described above. RESULTS: Twenty-nine of 30 samples had sufficient RNA integrity for analysis. TIS was &amp;gt;5 in 26/30 samples (median, 7.6; range, 4.3-9.3). No significant differences in TIS were identified between low-grade/limited-stage and high-grade/advanced-stage samples. High-grade samples had significantly increased expression of PD1 and higher amounts of exhausted CD8(+)-cells than low-grade samples (p= 0.015, 0.027). Low-grade samples showed significantly higher expression of ARG1, B7-H3, and genes associated with tissue hypoxia. Expression of genes associated with tumor proliferation was higher in high-grade lesions (p= 0.0019). Limited stage samples showed higher levels of endothelial cells, mast cells, and T-regulatory cells (p=0.033, 0.024, 0.004,) than advanced stage samples. Proliferation, PD1, and exhausted CD8 scores trended higher in advanced stage disease but did not reach statistical significance. CONCLUSION: Our findings indicate that the IO 360™ panel provides comprehensive analysis of the TME in MF. Most samples showed a TIS of &amp;gt;5, suggesting a T-cell inflamed TME in MF. Increased PD1 and exhausted CD8(+) cells were found in high-grade lesions, supporting a role for this pathway in progression. Increased expression of factors associated with angiogenesis (endothelial cells, ARG1) and immunosuppression (B7-H3, tissue hypoxia, mast cells, T-regulatory cells) suggest these mechanisms may play a role in early stages of disease. These findings highlight the complex and dynamic nature of the TME during MF progression, and the need for further study. Citation Format: Stanton Miller, Akshat Patel, Kiran A Kumar, Farrukh Awan, Heather W Goff, Travis Vandergriff, Marisa Juntilla, Franklin Fuda, Weina Chen, Jesse M Jaso. Evaluation of the tumor microenvironment in mycosis fungoides using the PanCancer IO 360™ assay and nCounter® platform [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A40.

925P Analytical performance of a next-generation sequencing (NGS) assay kit for assessing homologous recombination deficiency (HRD) from solid tumor samples

HRD status is predictive of response to PARP inhibitors (PARPi) in ovarian cancer. Developing reliable methods to assess HRD status is important to enable genomic profiling of tumor types with PARPi indications. Here we describe the analytical performance of the TruSight™ Oncology 500 HRD (TSO 500 HRD) research assay, an in-development NGS kit for assessing HRD using DNA from solid tumor samples. The TSO 500 HRD assay was developed by adding an HRD probe pool targeting genome-wide single nucleotide polymorphisms (SNPs) to the TruSight Oncology 500 (TSO 500) assay to enable Genomic Instability Score (GIS) assessment. For each sample, the DNA library was split into two hybridization reactions, one with TSO 500 probes and the other with HRD probes. Both libraries were pooled for sequencing with 8 samples per NextSeq™ 550 run. The GIS algorithm was licensed from Myriad, re-implemented as part of the DRAGEN Bio-IT software suite and integrated into the TSO 500 analysis workflow. Forty nanograms of DNA extracted from formalin-fixed, paraffin-embedded (FFPE) samples was used as assay input. Samples were also tested with a reference assay as the orthogonal test. Testing of serial dilutions of 8 ovarian cancer samples showed the limit of detection for small variant calling in BRCA1/2 genes is at 5-10% variant allele frequency (VAF), for large rearrangement variant calling in BRCA1/2 genes is at 30-50% VAF, and for GIS is at 30%-40% tumor content. The false positive rate for GIS status and BRCA variants is 0%. Testing of ovarian cancer samples and control samples with multiple operators, reagent lots, and library preparation start days showed >95% concordance in BRCA variant calling and GIS status. The GIS scores were highly concordant with the reference method assay (R = 0.98). The results were robust on both NextSeq 550 and NovaSeq™ 6000 with SP flowcells. The TSO 500 HRD assay demonstrated high analytical sensitivity and specificity in detecting BRCA variants and GIS status for HRD status assessment in ovarian cancer samples. TSO 500 HRD enables high resolution comprehensive genomic profiling alongside HRD assessment to maximize biomarker detection from limited amount of sample.

Improving gene expression analysis efficacy from formalin-fixed paraffin embedded tissues.

Improving RNA isolation and cDNA synthesis techniques has emerged due to advancements in the knowledge of molecular basis of most diseases. This in turn increased the need of higher quantity and quality of the extracted genetic material to be used for a variety of diagnostic tests and experiments. The aim of the study was to compare three modified methods for RNA extraction from formalin-fixed paraffin embedded (FFPE) biopsied tissue and different cDNA synthesis strategies to facilitate study of gene expression. Compared RNA extraction methods were: lysis buffer, phenol-based extraction, and combination of both with concomitant use of silica-based spin columns. RNA quantity and purity were estimated spectrophotometrically. Different priming strategies for cDNA synthesis were applied: oligodT, combination of oligodT and random hexamer, and gene specific primer. Two-step RT-qPCR of ribosomal protein L37A on preamplified and non-preamplified cDNA templates was performed. The combination of lysis buffer with phenol based extraction gave higher RNA yield. By doing cDNA preamplification, the confidence of detection by qPCR was raised, and efficiency was improved. The preamplified template increased the sensitivity of analysis. Together, the combination of approaches improved substantially the reproducibility and validity of quantitative gene expression analyses from FFPE tissues.

The extraction of Peste Des Petits Ruminants Virus RNA from paraffin-embedded tissues using a modified extraction method

Peste des petits ruminants (PPR) which is caused by small ruminant morbillivirus (PPRV) has an important economic impact on small ruminant farming due to high mortality rates, weight loss and restrictions on the export of small ruminants products. Molecular assays are commonly used in the diagnosis of the disease. Extraction of RNA from formalin-fixed paraffin-embedded (FFPE) tissues is challenging because of the RNA is often degraded by formalin fixation process. Although commercial kits have been developed for extraction of nucleic acids from FFPE tissues, they are expensive than other extraction kits. In this study, a modified extraction method was evaluated for detection of PPRV from FFPE tissues. A total of 20 FFPE tissue samples including 15 PPRV positive and 5 PPRV negative FFPE tissue samples were used. Two years ago, these selected FFPE tissue samples were analysed by nucleoprotein gene based one step real time RT-PCR method before they were fixed with formalin and embedded in paraffin. FFPE tissue samples were extracted using modified extraction method and were tested by fusion (F) gene based one step RT-PCR. PPRV specific RNA was detected in 12 FFPE tissue samples whereas 3 positive samples were found negative by one-step RT-PCR. Furthermore, 5 negative FFPE tissue samples were also found negative. Three false negative results were from samples with high real-time RT-PCR cycle threshold. Therefore, false negative results could be related with lower viral loads which might be lower than detection limit of the one-step RT-PCR. The results of the study show that modified extraction method could be used for RNA extraction from FFPE tissues which had been stored for 2 years.

Identification of fusion transcripts in sarcoma from archival formalin-fixed paraffin-embedded tissues: A next-generation sequencing approach.

Most sarcomas are highly aggressive, and cause necrosis and hemorrhage. The diagnosis of sarcoma is challenging because of the lack of specificity of immunohistochemical staining; however, molecular biological approaches, such as genetic mutation, chromosomal translocation, and gene amplification, are promising. In this study, we extracted RNA from formalin-fixed paraffin-embedded (FFPE) tissue derived from surgically resected specimens of sarcoma stored for various periods and performed next-generation sequencing (NGS) analysis by MiniSeq using the Archer Fusion-Plex Sarcoma Panel. RNA was extracted from 63 FFPE tissue samples, and the degree of RNA degradation was assessed. The number of reads and fragment lengths were evaluated by NGS analysis. RNA extraction and cDNA synthesis were successful in 56 cases and library preparation was possible. Fusion genes were detected in 16 of 63 archived FFPE tissue samples in this study. However, in 18 cases, fragmentation was strong, and high-quality libraries could not be obtained. Nevertheless, comprehensive analysis of fusion genes with high sequence specificity by NGS can be a powerful alternative to reverse transcription-polymerase chain reaction and fluorescence in situ hybridization methods.

Clinicopathological characteristics and prognostic analysis of PIK3CA mutation in breast cancer patients in Northwest China

PurposeMultiple studies on PIK3CA mutations in breast cancer (BC) had been performed, which showed the controversial results among different countries and races even those from the same country. The present study aimed to explore the PIK3CA gene mutation status in BC patients in Northwest China and reveal the relationship between PIK3CA mutations and clinicopathological features along with prognosis. Materials and methods1002 BC patients from Northwest China were recruited in this study, genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissues, and hotspot mutations in the exon 9 and 20 of PIK3CA gene were detected by ARMS-PCR. ResultsPIK3CA mutations were found in 31.2% (313/1002) of BC patients, among them 66.1% were mutations in exon 20% and 32.6% were mutations in exon 9. H1047R was the most common mutation type, accounting for 56.5% of the total mutated samples. Significant correlations were observed between PIK3CA mutation status and age (P = 0.035), histopathologic types (P = 0.004), pathological grade (P = 0.013), ER positivity (P < 0.001), PR positivity (P < 0.001), molecular subtypes (P = 0.004) and family history (P = 0.007). Cox multivariate analysis showed that patients with mutations in exon 9 or 20 had shorter DFS and OS than wild-type patients. Those with exon 9 mutations subgroup had the worst prognosis. Interestingly, patients with H1047L mutation had the best prognosis than others. ConclusionPIK3CA mutations could be used as an indicator of clinical outcome or targeted therapy for multiple breast cancer subgroups in Northwest China.

Prognostic Role of Targeted Methylation Analysis in Paraffin-embedded Samples of Adrenocortical Carcinoma.

ContextAdrenocortical carcinoma (ACC) is a rare aggressive disease with heterogeneous prognoses. Previous studies identified hypermethylation in the promoter region of specific genes to be associated with poor clinical outcome.ObjectiveComparative analysis of promising hypermethylated genes as prognostic markers and evaluation of their added value to established clinical prognostic tools.DesignWe included 237 patients with ACCs. Tumor DNA was isolated from formalin-fixed paraffin-embedded (FFPE) samples. Targeted pyrosequencing was used to detect promoter region methylation in 5 preselected genes (PAX5, GSTP1, PYCARD, PAX6, G0S2). The prognostic role of hypermethylation pattern was compared with the Stage, Grade, Resection status, Age, Symptoms (S-GRAS) score. Primary endpoints were progression-free (PFS) and overall survival (OS), with disease-free (DFS) as secondary endpoint.ResultsA total of 27.9%, 13.9%, 49%, 49%, and 25.3% of cases showed hypermethylation in PAX5, GSTP1, PYCARD, PAX6, and G0S2, respectively. Hypermethylation in all individual genes—except GSTP1—was significantly associated with both PFS and OS—with hazard ratios (HR) between 1.4 and 2.3. However, only hypermethylation of PAX5 remained significantly associated with OS (P = 0.013; HR = 1.95, 95% CI, 1.2-3.3) in multivariable analysis. A model for risk stratification was developed, combining PAX5 methylation status and S-GRAS groups, showing improved prognostic performance compared to S-GRAS alone (Harrell’s C index: OS = 0.751, PFS = 0.711, DFS = 0.688).ConclusionsThis study demonstrated that hypermethylation in PAX5 is associated with worst clinical outcome in ACC, even after accounting for S-GRAS score. Assessing methylation in FFPE material is straightforward in the clinical setting and could be used to improve accuracy of prognostic classification, enabling the direction of personalized management.

Clinical performance of quantitative PCR for the molecular identification of skeletal tuberculosis from formalin-fixed paraffin-embedded tissues

BackgroundAt present, skeletal tuberculosis (TB) diagnosis is mostly by histopathology, but the positivity rate is low. There is a need to develop new methods for the molecular identification of this disorder. Therefore, we aimed to investigate the clinical utility of quantitative PCR (qPCR)-based diagnosis of skeletal TB from formalin-fixed paraffin-embedded (FFPE) tissues and its comparative evaluation with acid-fast bacillus staining (AFS).MethodsWe detected Mycobacterium tuberculosis (M. tuberculosis/MTB) DNA using qPCR and AFS in FFPE tissue samples from 129 patients suspected of having skeletal TB. The sensitivity, specificity as well as area under the curve (AUC) of qPCR and AFS were calculated. Meanwhile, some factors potentially affecting qPCR and AFS results were investigated.ResultsOverall, qPCR outperformed AFS in detecting M. tuberculosis. The AUC of qPCR was higher than that of AFS (0.744 vs.0.561, p < 0.001). Furthermore, decalcification of bone tissues did not affect the sensitivity and specificity of qPCR tests. Whereas it impacted the performance of AFS, decalcification increased AFS's specificity and decreased its sensitivity (p < 0.05). Moreover, qPCR had a significantly larger AUC than AFS in decalcified and non-decalcified groups (0.735/0.756 vs. 0.582/0.534, p < 0.001) respectively. Similarly, the AUC of PCR was more extensive than that of AFS regardless of skeletal TB patients with concomitant pulmonary TB or not (0.929 vs. 0.762; 0.688 vs. 0.524, p < 0.01).ConclusionsOur data demonstrate that qPCR offers superior accuracy for the detection of mycobacteria in FFPE tissues compared to traditional AFS, indicating its clinical value in osteoarticular TB diagnosis.

Expression of HLA and Autoimmune Pathway Genes in Liver Biopsies of Young Subjects With Autoimmune Hepatitis Type 1.

Objectives:To test the hypothesis that autoimmune hepatitis (AIH type I) in young subjects is due to genetic differences in proinflammatory genes responding to viral triggers in patients and controls.Methods:Intrahepatic gene expression was compared between AIH type I (n = 24, age 9–30 years) patients (hereafter referred to as the AIH group) and controls (n = 21, age 4–25 years). RNA sequencing was performed on complementary DNA (cDNA) libraries made from total RNA extracted from formalin-fixed paraffin-embedded (FFPE) liver biopsy samples. Gene expression levels were quantified, and differentially expressed genes were functionally analyzed. Pathway analysis was performed using the databases Kyoto Encyclopedia of Genes and Genomes (KEGG) and PANTHER. The remaining sequences were mapped to the RefSeq complete set of viral genomes.Results:Differential gene analysis identified 181 genes that were significantly differentially expressed (136 upregulated in the AIH group). Autoimmune pathway genes such as CD19 and CD20 which are important in B cell regulation and maturation as well as, CD8 and LY9, which are T-cell related, were upregulated in our AIH group. Genes implicated in AIH pathogenesis including CXCL10, which is thought to be associated with AIH severity and progression, complement genes (C1QA, C1QB, and C1QC), and human leucocyte antigen (HLA) genes (HLA-DRB1, HLA-DRA, HLA-B, and HLA-C) were upregulated in samples from the AIH group. Specific viral etiologies were not found.Conclusions:Unbiased next-generation sequencing and differential gene expression analysis of the AIH group has not only added support for the role of B cells in the pathogenesis and treatment of AIH but also has introduced potential new therapeutic targets: CXCL10 (anti-CXCL10) and several complement system–related genes.

P1284: LIQUID BIOPSY BY ULTRA-DEEP SEQUENCING PLUS PET-TC MONITORING IN REAL-LIFE FOLLICULAR LYMPHOMA PATIENTS

Background: Follicular lymphoma (FL) is considered an indolent disorder with a relatively favorable course. With modern day treatments, long remissions are often achieved both in front-line and relapsed setting. However, a subset of patients has a more aggressive course and a poorer outcome. Although understanding prognosis is important, ultimately the goal in FL should be to guide therapy. Studies of risk-adapted therapy based on MRD remains investigational. Aims: To prospectively evaluate the predictive value of a liquid Biopsy MRD (LiqBio-MRD) SNV-NGS method in combination with the Deauville criteria using the 5-point scale (D5PS) in the evaluation of response in a cohort of 72 FL patients that require treatment from 2018-2021. Methods: 57 patients received first-line therapy (39 R-CHOP, 10 R-B, 5 Rituximab in monotherapy and 3 radiotherapy) and 15 patients salvage treatment. Genomic DNA from formalin-fixed paraffin embedded (FFPE) lymph node biopsies and/or cell-free DNA (cfDNA) was obtained before start treatment. These samples were sequenced with short length Ampliseq Custom Panel targeting 56 important genes in B-cell dyscrasias. Somatic mutations were selected as disease biomarkers, and cfDNA samples were analysed at mid-induction (n = 26) and at the end of treatment (n= 41). PET/CT examinations were usually performed after 4 cycles (n = 44) and at the end of treatment (n = 63). PET positive was considered when D5PS was 4 or 5. Statistical analysis was performed with SPSS® using Cox regression, Kaplan-Meier survival curves and log-rank test. Results: Median age at diagnosis was 66 years (35 – 90) and 58% were female. At diagnosis only the lymphocyte to monocyte ratio < 2.5 had prognostic value (p=0.04). Median PFS was 13 months for PETEOT (+) cases and 10 months for MRDEOT (+) ones. 71% of patients with PETEOT (+) presented POD24, in contrast to only 9% of patients with PETEOT (-), p < 0.001. Regarding MRD, 73% of patients with MRDEOT (+) presented POD24 vs 12% of patients with MRDEOT (-), (p < 0,001; HR 8.30, 95%CI 1.70–40.38). The sensitivity (SE) and specificity (SP) of both tests to predict POD24 were: PETEOT (SE77%, SP68%) and MRD (SE77%, SP86%). 40 patients had MRDEOT and PETEOT available and results were concordant in 80% of cases. In a stratified analysis combining the information on MRD and PET, the 2-yr PFS were; 96% for MRD/PET -/-, 71% for MRD/PET -/+, and 58% MRD/PET +/-. Strikingly, patients that were positive by both tests presented a 2-yr PFS of only 10%. Using only concordant results, SE91% and SP96% was achieved. Similar results in PFS and in predicting POD24 were obtained when including only patients with first line therapy. Figure 1. When interim results were analysed, also PETINT(+) and MRDINT(+) were associated with higher risk of relapse (p<0,001 and p=0.009, respectively). The negative predictive value of PETINT results was 96%. Concordance between both tests was clearly inferior (47%), although no patient with PETINT(-) and MRDINT(-) results relapsed. Image:Summary/Conclusion: Our results demonstrate for the first time that NGS based MRD quantification is feasible in Liquid Biopsies from FL. The achievement of negative MRD after treatment, but also in an interim analysis, enhances prognostic information on patient outcome. PET/CT and cfDNA are complementary increasing SE and SP to predict progression and POD 24 with a high accuracy. Although, more patients are necessary to draw meaningful conclusion, the combination of these techniques could aid to the develop clinical trials with a response-adapted precision therapy.

Abstract 84: Sensitive and unbiased detection of clinically actionable gene fusions from FFPE tumor biopsies using the Arima-HiC platform

Abstract Accurate and sensitive identification of gene fusions is critical to understanding disease mechanisms and the development and selection of optimal treatment regimens for cancer patients. Typically, these fusions are detected using low-resolution karyotyping, low throughput and biased FISH assays, or RNA sequencing approaches; however, the accuracy and sensitivity of gene fusion detection can be limited by factors such as low transcript abundance, transcript length, RNA degradation from formalin fixed paraffin embedded (FFPE) tissues, or the limited availability of fresh biopsy samples for RNA extraction. To address these limitations, we developed a novel approach to identifying gene fusions from FFPE samples using the Arima-HiC platform and short-read sequencing. We performed pan-cancer analysis on 12 FFPE adult tumor biopsies, each with gene fusions known to be clinically actionable. With this newly developed Hi-C workflow we identified all the known gene fusions with 100% sensitivity in the samples including those involving ALK, NTRK3, ROS1, FGFR2, and SS18 genes. This approach also revealed that the NTRK3 gene fusion was the result of a more complex rearrangement within chr12, within chr15, and between chr12 and chr15. Additionally, we detected the presence of numerous structural variants per sample in addition to the known gene fusion in each sample. For example in a bile duct tumor, Arima-HiC detected an FGFR2-EEA1 gene fusion, as well as 25 other structural variants genome-wide including in a CPD-LASP1 gene fusion This gene fusion has not been reported to our knowledge, however, LASP1 is a reported 3’ fusion partner with MLL in leukemia. While these findings are not clinically actionable today, the unbiased, accurate, and sensitive detection of structural variants from frozen and FFPE solid tumor biopsies may facilitate further research into their relationship between disease mechanisms and clinical outcomes. Taken together, these findings demonstrate the analytical utility of Arima Hi-C sequencing technology to provide both chromosome-scale and gene-level resolution for the detection of structural variants in tumor biopsies samples. This workflow can provide improved access to critical genomic information from FFPE blocks for the identification of pathognomonic and druggable gene fusion events and other structural variants across tumor types. Citation Format: David Jacob Hermel, Kristin Sikkink, Derek Reid, Anthony Schmitt, Darren S. Sigal. Sensitive and unbiased detection of clinically actionable gene fusions from FFPE tumor biopsies using the Arima-HiC platform [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 84.

Abstract 3739: EM-seq permits accurate and precise methylome analysis of cfDNA and FFPE DNA

Abstract The study of DNA methylation from cell-free DNA (cfDNA) and DNA extracted from formalin fixed paraffin embedded (FFPE) tissues are notoriously difficult. The use of these DNA inputs is becoming increasingly important in the field of cancer diagnostics. The cytosine modifications, 5-methylcytosines (5mC) and 5-hydroxymethylcytosines (5hmC), are key epigenetic factors that play an important role in cellular processes and their mis-regulation results in diseased states like cancer. Advances in the study of these epigenetic factors in combination with improvements in sample preparation have enabled cancer biomarker identification based on methylation profiling. The historical gold standard for detecting cytosine methylation has been bisulfite sequencing. This method uses chemical conversion of cytosines into uracils and has been used for both targeted and whole genome methylation analysis. However, bisulfite conversion leads to DNA damage which results in shorter DNA insert sizes as well as biases in the data. It is challenging to make bisulfite libraries from cfDNA and FFPE DNA as typically the DNA is of low quantity and quality. We developed an enzyme-based methylation detection technology (EM-seq) to address the flaws inherent to bisulfite sequencing. This method, primarily due to the intact nature of DNA after conversion, results in better quality libraries with longer insert sizes, lower duplication rates and minimal GC bias. EM-seq libraries prepared from cfDNA and lung FFPE DNA had longer inserts, lower duplication rates, higher percentages of mapped reads and less GC bias compared to bisulfite libraries. EM-seq libraries also identified a higher number of CpGs with even coverage facilitating accurate methylation calling. EM-seq’s superior sequencing metrics establish it as a standard method for robust methylation profiling, particularly for these types of challenging DNA samples. Citation Format: Louise Williams. EM-seq permits accurate and precise methylome analysis of cfDNA and FFPE DNA [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 3739.

Abstract 4104: Low-cost, simple and rapid assay for single-molecule detection of gene fusions from RNA with ASPYRE technology

Abstract Introduction: The FDA has approved 11 therapies for non-small cell lung cancer (NSCLC) patients diagnosed with gene fusions. Importantly, these life-saving targeted therapies remain underutilized due to a lack of cost-effective, comprehensive, and simple-to-use technologies for gene fusion detection. Our previous work using ASPYRE technology (allele-specific pyrophosphorolysis reaction; Silva et al, 2020) demonstrated detection of somatic variants at single-molecule level from DNA extracted from tumor tissue. However, DNA-based fusion detection requires tiling across large introns, which limits sensitivity particularly if breakpoints are located in repetitive regions of the genome. Here, we show that ASPYRE technology can also be utilized for highly sensitive detection of RNA fusions, scaling to over 30 fusion targets covering the main 3’ fusion partners (ALK, ROS1, RET, NTRK1, NTRK3) and from a single reverse transcription PCR (RT-PCR) amplification reaction. Methods: The ASPYRE assay consists of four sequential enzymatic stages: RT-PCR; enzymatic clean-up; combined exonuclease digestion, hybridization, pyrophosphorolysis and ligation; rolling circle isothermal amplification and detection. All targets are detected across two reaction wells using four channels on a standard real-time PCR instrument. A panel of synthetic RNA oligonucleotides comprising gene fusion boundaries was spiked into a background pool of lung-RNA, quantified using digital PCR (dPCR) and compared against a commercial reference standard to validate its suitability for assessing assay performance. RNA was extracted from formalin-fixed paraffin-embedded (FFPE) lung tissue from healthy donors to determine assay specificity, and from FFPE lung tissue from NSCLC patients with known fusions to exemplify end-to-end sample analysis. Results: We demonstrate equivalence of our in-house reference samples to commercial reference standards. Using serial dilutions of input RNA standards, we show that detection by ASPYRE is consistent with detection of single molecules as input for common ROS1, ALK, RET and NTRK3 fusions. In addition, ASPYRE was able to detect previously confirmed ROS1 and ALK fusions and MET exon skipping variants in FFPE samples. ASPYRE had high specificity, correctly confirming the absence of RNA fusions and exon skipping variants in healthy control FFPE samples. Conclusions: ASPYRE approached single-molecule sensitivity for over 30 RNA fusion targets in a single reaction. The assay required only 1 ng RNA input, with a turn-around time of &amp;lt;4 hours from extracted RNA to result, opening the door to routine detection of RNA fusions from limited specimens. The simplicity and ease of use of ASPYRE, combined with its rapid turnaround time and analytical performance characteristics, provides a new tool for rapid and comprehensive treatment selection in NSCLC. Citation Format: Eleanor Gray, Justyna Mordaka, Efthimia Christoforou, Kristine von Bargen, Nicola Potts, Rebecca Palmer, Barnaby Balmforth. Low-cost, simple and rapid assay for single-molecule detection of gene fusions from RNA with ASPYRE technology [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 4104.

Abstract 5795: Validation of FDA approved oncoReveal Dx lung and colon cancer assay (oRDx-LCCA)

Abstract Introduction: The oncoReveal™ Dx Lung and Colon Cancer Assay (oRDx-LCCA) is an amplicon-based targeted next generation sequencing (NGS), in vitro diagnostic test that detects DNA variants across 22 genes, including single nucleotide variants (SNVs) and insertions and deletions (INDELs) in KRAS and EGFR, from DNA isolated from formalin-fixed paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) tumor tissue specimens. It is approved by the FDA as a companion diagnostic (CDx) for identifying approved therapeutic treatments for KRAS G12/G13 wild-type variants in CRC, and EGFR L858R variants and exon 19 in-frame deletions for NSCLC. oRDx-LCCA can detect non-CDx variants such as EGFR T790M, G719X and exon 20 insertions in NSCLC and KRAS Q61X and A146T and BRAF V600E in CRC. Variants are detected using Pillar Biosciences’ proprietary PiVAT® (Pillar Variant Analysis Toolkit) software. Methods: DNA input range was evaluated at 6 concentrations between 5 - 160ng in duplicate using DNA extracted from 15 clinical FFPE samples containing SNVs and INDELs from representative targeted mutations. Accuracy was determined by comparing detected variants with an externally validated comparator (CompO) across 208 samples (84 CRC, 124 NSCLC). Limit of detection (LoD) was determined by serial dilution of KRAS, EGFR, or BRAF positive FFPE samples in normal DNA to detect variant allele frequency (VAF) between 1 - 10%. Reproducibility tests were performed across 3 sites, 9 runs, 6 operators, 3 reagent lots and 3 thermocyclers, resulting in a total of 360 libraries. The non-inferiority (NI) statistical test was used to compare the oRDx-LCCA with an FDA approved CDx assay for EGFR exon 19 deletions and L858R mutations (CompQ) and for KRAS G12/G13 mutations (CompC). Synthetic FASTQ files were created at VAFs above and below the variant detection threshold for CDx mutations and used to validate quality control parameters, mean coverage, and on-target rate. Results: Effective DNA input range was determined to be between 10 - 80ng. LoD’s for KRAS G12/G13, EGFR L858 and exon 19 deletion, and BRAF V600E were established at &amp;lt; 4%. Positive percent agreement (PPA) and negative percent agreement (NPA) between oRDx-LCCA and CompO were &amp;gt; 99% each. Across all variables tested for reproducibility, the average percent positive agreement (APA) and average percent negative agreement (ANA) were each &amp;gt; 95%. NI margin was calculated to be &amp;lt; 10% between oRDx-LCCA and CompQ across all NSCLC samples and between oRDx-LCCA and CompC across all CRC samples. A total of 61 synthetic FASTQ datasets were created to validate the functionality and accuracy of PiVAT including VAF thresholds for 126 SNV and INDEL CDx variants. Conclusion: oRDx-LCCA is a highly accurate FDA approved IVD assay and PiVAT is a powerful FDA approved software tool for the detection of clinically relevant KRAS variants in CRC and EGFR variants in NSCLC and determination of approved therapy. Citation Format: Gloria Chan Johnson, Jordan Aldersley, Nicholas Lodato, Jamie Lim, Ian Flockhart, Mark Urbin, Yue Ke, Sean Polvino, Martin Zillmann, Gang Song, Zhaohui Wang. Validation of FDA approved oncoReveal Dx lung and colon cancer assay (oRDx-LCCA) [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 5795.