Geiersbach, Katherine B.

Context.— Guidelines for HER2 testing in breast cancer have changed over time, from the US Food and Drug Administration guideline to the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines published in 2007, 2013, and 2018. Objective.— To investigate the change in assignment of HER2 status in breast cancers with equivocal (2+) immunohistochemistry (IHC) results by fluorescence in situ hybridization (FISH) following implementation of the ASCO/CAP 2018 guideline. Design.— The study included 3556 invasive breast cancers that were HER2 equivocal (2+) by IHC and were submitted to our FISH laboratory after July 2018. Reflex testing (with repeat IHC staining) was performed on certain categories of FISH results known as groups 2, 3, and 4. Concomitant review of IHC and FISH was performed on these reflex cases per 2018 guideline recommendations. The FISH data were analyzed to compare US Food and Drug Administration and ASCO/CAP 2007, 2013, and 2018 interpretations. Results.— Of 3548 invasive breast cancers with complete data available, the percentage agreement for FISH according to different guidelines was highest for ASCO/CAP 2018 versus US Food and Drug Administration (96.5%), followed by ASCO/CAP 2018 versus 2007 (93.8%), and lowest with ASCO/CAP 2018 versus 2013 (83.7%). Per the 2018 guideline, reflex IHC testing was performed on 633 breast cancers (17.8%); the majority of reflex testing results were negative (541 of 633; 85.5%). The overall distribution of HER2 FISH results (per the 2018 guideline) was 88.5% negative and 11.5% positive. Conclusions.— By eliminating the equivocal FISH category, the 2018 ASCO/CAP guideline significantly reduced the HER2 FISH–positive rate in tumors with equivocal (2+) IHC results.

Abstract Background: Biomarker testing in lung cancer is often limited by a lack of sufficient formalin fixed, paraffin embedded (FFPE) tissue for comprehensive genomic profiling. To promote personalized therapy for lung cancer, a multiplex FISH assay was developed to simultaneously assess aberrations in ROS1, RET, and MET on a single FFPE specimen slide. Methods: Specimens included primary tumor (N = 39) as well as biopsies from a variety of metastatic sites (N = 16). These included 12 samples with ROS1 rearrangements, 3 samples with RET rearrangements, and 11 samples with MET amplification reported by a previously validated laboratory test method. A probe mix contained 6 differentially labeled fluorescent probes: 3' ROS1, 5' ROS1, 3' RET, 5' RET, MET and CEP7. The probes were formulated in Vysis IntelliFISH Hybridization Buffer to allow for a 2 h hybridization time. BioView imaging platform and Duet software algorithm were used to perform automated slide scanning and digital analysis. Specimens were considered positive for ROS1 or RET rearrangement if >15% evaluated cells contained a break apart (rearranged) signal. Specimens were considered positive for MET amplification if >15% of cells had MET/CEP7 ratio >2 and positive for polysomy if >15% of cells had 5 or more MET signals copies. Results: The 6 color FISH assay was 97% concordant for ROS1 rearrangement and 100% concordant for RET rearrangement. The average background percentage of positive tumor cells in cases without known gene rearrangements was approximately 5%, yielding a negative cutoff threshold of approximately 15%, in accordance with cutoff thresholds reported in literature. The 6 color FISH assay was 91% concordant for MET amplification or polysomy. Results were interpretable for 98% of targets analyzed by the 6 color FISH method. Four samples failed on analysis for one of the targets due to lack of sufficient cells or lack of adequate hybridization signal. Conclusion: A newly developed 6-color FISH assay allows simultaneous detection of three genomic abnormalities using only 1 specimen slide. This feature combined with rapid hybridization in IntelliFISH buffer and automated BioView slide imaging and analysis can significantly increase the yield of molecular testing on limited lung cancer tissue samples. Careful pathologic correlation for tumor cell identification and careful assessment of hybridization quality are necessary to optimize the accuracy of this test method. Citation Format: Irina A. Sokolova, Patrick Bedroske, Tatyana A. Grushko, Amber R. Schneider, Kristine Jacobson, Jesse S. Voss, Yishay Tauber, Yossi Avisror, Vitaliy Shkolnik, Katerina Pestova, Katherine B. Geiersbach. Multiplex fast FISH assay for detecting ROS1, RET and MET aberrations in FFPE specimens using BioView image analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4256.

3144 Background: Updated ASCO/CAP Guidelines for HER2 testing in breast cancer have been most impactful on the resolution of certain challenging groups of FISH results. We review the change in assignment of HER2 status in a large series of breast cancers referred to a large national reference laboratory for FISH testing since the introduction of the 2018 updated guidelines. Methods: Patient samples submitted to the Mayo Clinic Cytogenetics Laboratory (N = 2208) were analyzed by FISH. Samples with Group 2, Group 3, or Group 4 FISH results were reflexed to immunohistochemistry (IHC) in our central laboratory; FISH slides for those cases with equivocal 2+ IHC results were re-scored in the regions of invasive cancer showing more intense membranous staining. A subset of 202 samples with Group 4 FISH results were also reflexed to the previously employed reflex FISH assay (HER2/D17S122), and these were also re-analyzed according to the new reflex IHC/FISH process. Results: 382 of 2208 breast cancer samples tested (17.3%) had FISH results categorized as Group 2 (N = 17, 0.8%), Group 3 (N = 34, 1.5%), or Group 4 (N = 331, 15%) and required reflex IHC testing, and of those, 75% were 2+ equivocal and required targeted re-analysis of the FISH slide according to the 2018 updated guidelines. Re-analysis of the FISH slide resulted in switching between Groups 1-5 in 19.4% of cases, but HER2 status was changed by FISH re-scoring in only 7.7% of cases re-scored (1.0% of all samples), generally due to only minor shifts in HER2 copy number and HER2/control ratios between the initial and IHC-guided reflex FISH scores. In the subset of 202 cases tested by both reflex methods, the previously employed HER2/D17S122 reflex probe set was positive in 123 cases (60.9%), whereas reflex IHC/FISH was positive in only 10 cases (7.9%). Including positive reflex IHC (0.4%) and positive reflex FISH results (2.1%), the overall assignment of positive HER2 status on our series of 2208 cases was 11.5%. Conclusions: Overall rates of HER2 positive FISH results have declined under the most recent ASCO/CAP guideline update as a consequence of new recommendations for reflex testing for Groups 2-4. This change is largely due to reassignment of Group 2 and Group 4 results as negative in the absence of positive IHC.

AbstractPrimary aneurysmal bone cyst (ABC) is a benign multiloculated cystic lesion of bone that is defined cytogenetically by USP6 gene rearrangements. Rearrangements involving USP6 are promoter swaps, usually generated by fusion of the noncoding upstream exons of different partner genes with exon 1 or 2 of USP6, thus leading to transcriptional upregulation of full‐length USP6 coding sequence. Testing for USP6 rearrangements is used diagnostically to distinguish it from secondary ABC and other giant cell‐rich primary bone tumors. In this report, we present a case of a 16‐year‐old male with a primary ABC of the left distal femur. USP6 break apart fluorescence in situ hybridization was positive for a rearrangement and conventional chromosome analysis identified a reciprocal X;17 translocation. In order to identify the putative USP6 fusion partner, we performed RNA sequencing and uncovered a novel USP9X‐USP6 promoter swap fusion. This result was confirmed by reverse transcription‐polymerase chain reaction (RT‐PCR) and by mate pair sequencing thus showing the utility of these alternative methodologies in identifying novel fusion candidates. Ubiquitin‐specific protease 9X (USP9X), like USP6, encodes a highly conserved substrate‐specific deubiquitylating enzyme. USP9X is highly expressed in a number of tissue types and acts as both an oncogene and tumor suppressor in several human cancers. We conclude that oncogenic activation of USP6 via USP9X promoter exchange represents a novel driver of primary ABC formation.

AbstractObjectiveAcute myeloid leukemia (AML) can be subtyped based on recurrent cytogenetic and molecular genetic abnormalities with diagnostic and prognostic significance. Although cytogenetic characterization classically involves conventional chromosome and/or fluorescence in situ hybridization (FISH) assays, limitations of these techniques include poor resolution and the inability to precisely identify breakpoints.MethodWe evaluated whether an NGS‐based methodology that detects structural abnormalities and copy number changes using mate pair sequencing (MPseq) can enhance the diagnostic yield for patients with AML.ResultsUsing 68 known abnormal and 20 karyotypically normal AML samples, each recurrent primary AML‐specific abnormality previously identified in the abnormal samples was confirmed using MPseq. Importantly, in eight cases with abnormalities that could not be resolved by conventional cytogenetic studies, MPseq was utilized to molecularly define eight recurrent AML‐fusion events. In addition, MPseq uncovered two cryptic abnormalities that were missed by conventional cytogenetic studies. Thus, MPseq improved the diagnostic yield in the detection of AML‐specific structural rearrangements in 10/88 (11%) of cases analyzed.ConclusionUtilization of MPseq represents a precise, molecular‐based technique that can be used as an alternative to conventional cytogenetic studies for newly diagnosed AML patients with the potential to revolutionize the diagnosis of hematologic malignancies.

Context.— Fluorescence in situ hybridization (FISH) and brightfield in situ hybridization (ISH) are 2 clinically approved laboratory methods for detecting ERBB2 (HER2) amplification in breast cancer. Objective.— To compare the performance of FISH and brightfield ISH on proficiency testing administered by the College of American Pathologists Laboratory Accreditation Program. Design.— Retrospective review was performed on 70 tissue core samples in 7 separate proficiency testing surveys conducted between 2009 and 2013. Results.— The samples included 13 consensus-amplified tissue cores, 53 consensus-nonamplified cores, and 4 cores that did not reach consensus for FISH and/or brightfield ISH. There were 2552 individual responses for FISH and 1871 individual responses for brightfield ISH. Consensus response rates were comparable for FISH (2474 of 2524; 98.0%) and brightfield ISH (2135 of 2189; 97.5%). The FISH analysis yielded an average HER2 copy number per cell that was significantly higher (by 2.86; P = .02) compared with brightfield ISH for amplified cores. For nonamplified cores, FISH yielded slightly, but not significantly, higher (by 0.17; P = .10) HER2 copy numbers per cell. There was no significant difference in the average HER2 to control ratio for either consensus-amplified or consensus-nonamplified cores. Participants reported “unable to analyze” more frequently for brightfield ISH (244 of 2453; 9.9%) than they did for FISH (160 of 2684; 6.0%). Conclusions.— Our study indicates a high concordance rate in proficiency testing surveys, with some significant differences noted in the technical performance of these assays. In borderline cases, updated American Society of Clinical Oncology/College of American Pathologists cutoff thresholds that place greater emphasis on HER2 copy number per cell could accentuate those differences between FISH and brightfield ISH.

Aims Routine application of PD-L1 immunohistochemistry (IHC) in colorectal cancer (CRC) is limited due to lack of standardized scoring criteria, antibody clones, and intratumoral staining heterogeneity. We assessed PD-L1 protein expression on full face CRC tissue sections and applied two algorithms based on the published clinical trials that support the recent FDA approval for immune checkpoint inhibitors (ICPI) therapy in non-small cell lung cancer (NSCLC). Methods PD-L1/CD274 IHC (Roche/Ventana, clone SP142) was performed on representative tumour blocks from 52 mismatch repair-deficient (MMR-D) and 52 MMR-proficient (MMR-P) CRCs. Membranous PD-L1 expression was scored for the tumour cell (TC) and tumour-infiltrating immune cell (IC) components. PD-L1 positivity status was determined based on the published NSCLC clinical trials that utilized the Ventana SP142 assay. Hybrid capture-based comprehensive genomic profiling (CGP) was performed on a separate set of 2268 clinically advanced CRCs and the frequency of PD-L1/PD-L2 amplification was determined. Results PD-L1 expression in the TC and IC correlated with MMR-D (p=0.013, p<0.0001), T stage (p=0.036, p=0.0036) and clinical stage (p=0.022, p=0.0037). PD-L1 positivity status correlated with MMR-D by two algorithms. Five of 2268 (<1%) advansced CRCs demonstrated amplification of either the PD-L1 or PD-L2 genes by CGP. Conclusions PD-L1 expression in TC and IC is associated with advanced stage and MMR-D. PD-L1 positivity status by the published algorithm is associated with MMR-D. PD-L1 amplification is extremely uncommon in CRC. Evaluation of whole tissue section and incorporation of IC staining enhance the sensitivity to screen patients who may benefit from ICPI therapy.