A case of diagnostic uncertainty: High-grade melanocytoma versus balloon cell melanoma in a pediatric patient.

An 11-year-old female was referred from an outside institution after a diagnostic biopsy and subsequent excision of a progressively enlarging reddish-brown nodule demonstrated features concerning for a balloon cell nevus with severe atypia versus a high-grade melanocytoma. Upon review of the initial biopsy specimen and molecular data, we favored the diagnosis to be consistent with a high-grade melanocytoma with balloon cell changes while considering the possibility of balloon cell melanoma due to concerning histopathologic and genetic abnormalities. In this case study, we discuss critical diagnostic considerations in this rare pediatric case and highlight important pathologic and clinical features of melanocytomas and balloon cell melanoma.

Frequency of HER2 protein overexpression and HER2 gene amplification in endometrial clear cell carcinoma.

HER2 (ERBB2) overexpression and/or HER2 gene amplification has been well established in several tumors types and when present HER2 directed therapy may be to be efficacious. While recent findings suggests that HER2 overexpression and HER2 amplification are a relatively common in serous endometrial carcinoma, similar data regarding clear cell endometrial carcinoma (CCC) is difficult to interpret due to issues such as diagnostic criteria, sample type and HER2 interpretation criteria. Our goals were to study HER2 expression and HER2 copy number status in hysterectomy specimens from a large series of patients with pure CCC to determine the frequency of HER2 overexpression and HER2 amplification and evaluate applicability of current HER2 interpretation criteria. Pure CCC specimens derived from hysterectomy specimens from 26 patients were identified. All diagnoses were confirmed by two gynecologic pathologists. Immunohistochemistry for HER2 protein and fluorescence in situ hybridization (FISH) studies for HER2 were performed on whole-slide sections from all cases. Results were interpreted according to the 2018 ASO/CAP HER2 guidelines for breast cancer and International Society of Gynecologic Pathologists (ISGyP) HER2 guidelines for serous endometrial carcinoma. Additional testing was performed when indicated by the guidelines. HER2 expression by immunohistochemistry was 3+ in 4% and 0% of cases, and 2+ in 46% and 52% of cases, by 2018 ASCO/CAP and ISGyP criteria, respectively, while the remaining cases were negative. HER2 testing by FISH showed a positive result in 27% of tumors with 2018 ASCO/CAP guidelines, while 23% were positive with the ISGyP criteria. Our findings indicate that HER2 overexpression and HER2 amplification occur in a subset of CCC. Therefore, additional study into the potential benefit of HER2 targeted therapy in patients with CCC is warranted.

Clear-cell Variant of Mucoepidermoid Carcinoma Presenting as a Palatal Mass in a 10-Year-old Boy.

BACKGROUND: The clear-cell variant of mucoepidermoid carcinoma (MEC) involving minor salivary glands is extremely rare in children. CASE REPORT: We report a case of clear-cell variant MEC in the minor salivary gland in a 10-year-old boy who presented with a mass of the right hard palate. Fine-needle aspiration showed features suggestive of clear-cell variant of MEC. Microscopically, the tumor cells showed predominant clear cells and scattered mucous cells. There was increased mitotic activity (6/mm2). No tumor necrosis or nuclear pleomorphism was identified. The tumor cells were positive for cytokeratin 7 (CK7), tumor protein p63, P40 (ΔNp63), CK5/6 and mucicarmine. Rearrangement of mastermind-like transcriptional coactivator 2 (MAML2) (11q21) gene was present in the tumor cells by fluorescence in situ hybridization, supporting the diagnosis of an intermediate-grade clear-cell variant of MEC. A right infrastructure maxillectomy for palate carcinoma with negative margins was performed. Grossly, the tumor was a 2.1 cm well-circumscribed, friable, pale tan mass with focal areas of cystic change. The final pathological diagnosis was clear-cell variant of MEC, intermediate grade, pT2. Post surgery, the patient recovered and was doing well, with no tumor recurrence or metastasis at the 6-month follow-up. CONCLUSION: To the best of our knowledge, this is the first documented case of clear-cell variant MEC in a child. Due to low to intermediate tumor grade, an overtly aggressive treatment should be avoided in a child.

Single-Nucleotide Polymorphism Array for Histologically Ambiguous Melanocytic Tumors.

Genome-wide copy number profiling by single-nucleotide polymorphism (SNP) array is increasingly employed in the clinical diagnostic workup of melanocytic tumors. We present our SNP array results on 675 melanocytic tumors, including 615 histologically ambiguous tumors evaluated by our institution's dermatopathology consultation service and a separate validation cohort of 26 known benign nevi and 34 known malignant melanomas. The total number of somatic copy number abnormalities, sub-chromosomal copy number abnormalities, regions of homozygosity, and abnormalities at disease-associated regions was significantly associated with a diagnosis of malignancy across disease categories. In our study, the number of copy number abnormalities was the factor that best discriminated between benign versus malignant diagnoses, confirming recent published research. Histologically ambiguous tumors had a range and spectrum of abnormalities, including recurrent 11p gains, copy state transitions over kinase genes, and 3p deletions overlapping BAP1 in neoplasms with Spitzoid morphology. Our data suggest that histologically ambiguous melanocytic neoplasms and early primary melanomas have a range of abnormalities that is intermediate between unambiguous benign or malignant melanocytic neoplasms. Careful technical review and an integrated diagnostic approach are essential for the accurate interpretation of SNP array results on histologically ambiguous melanocytic tumors.

Mammary analogue secretory carcinoma: A challenging case arising in a young man with radiation exposure.

Mammary Analogue Secretory Carcinoma (MASC) is a recently described salivary gland tumor frequently sampled via fine-needle aspiration. The cytologic features of MASC are not entirely distinctive and can simulate acinic cell carcinoma, but the tumor harbors an ETV6 gene rearrangement resulting in an ETV6-NTRK3 fusion gene. We present a case of MASC arising in a 31 year old man with a history of multiple radio-embolization procedures.

Detailed Reanalysis of 500 Breast Cancers With Equivocal HER2 Immunohistochemistry and Borderline ERBB2 Fluorescence In Situ Hybridization Results.

OBJECTIVES: We investigated the impact of our laboratory's reflex testing process for resolving ERBB2 (HER2) status on breast cancer samples that require additional workup after fluorescence in situ hybridization (FISH), per guideline recommendations published in 2018 by the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP). METHODS: In total, 500 breast cancer specimens with ERBB2 FISH results in groups 2 through 4 (all reported as immunohistochemistry [IHC] equivocal [2+] at external laboratories) were resubmitted for IHC testing in our laboratory. Per the ASCO/CAP guideline, FISH was rescored when internal IHC was also equivocal (2+), targeted to tumor areas demonstrating more intense IHC staining, if observed. RESULTS: Reflex IHC/FISH testing changed the final reported ERBB2 status in 185 of 500 (37.0%) samples. Result changes included discordant IHC (n = 4 score 0, n = 132 score 1+, and n = 16 score 3+) and discordant FISH (n = 33). Numerical differences in FISH scores were comparable for targeted vs nontargeted FISH rescoring (P = .086 for ERBB2 copy number; P = .49 for ERBB2 ratio). Two cases showed larger differences in FISH scores, suggesting heterogeneity. CONCLUSIONS: Retesting of breast cancer samples with equivocal IHC frequently changes IHC results, but targeted reanalysis of borderline FISH results rarely identifies significant differences in ERBB2 copy number or ratio.

HER2 Testing for Breast Cancer in the Genomics Laboratory: A Sea Change for Fluorescence In Situ Hybridization.

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.

Current concepts in breast cancer genomics: An evidence based review by the CGC breast cancer working group.

BACKGROUND: Genomic abnormalities in breast cancer have been described according to diverse conceptual frameworks, including histologic subtypes, clinical molecular subtypes, intrinsic DNA, RNA, and epigenetic profiles, and activated molecular pathways. METHODS: The Cancer Genomics Consortium (CGC) Breast Cancer Workgroup performed an evidence based literature review to summarize current knowledge of clinically significant genomic alterations in breast cancer using CGC levels of evidence. Targetable or disease-defining alterations were prioritized. RESULTS: We summarized genomic alterations in breast cancer within a framework of existing clinical tools for diagnosis, risk stratification, and therapeutic management. Using CGC levels of evidence, we catalog copy number profiles, gene expression profiles, and mutations in clinically significant genes. We also describe emerging molecular markers such as methylation profiling and immunotherapy biomarkers. CONCLUSION: A summary of currently available information on breast cancer genomics will enhance precision medicine by serving as an interpretive resource for clinical laboratory geneticists, providing a foundation for future practice guidelines, and identifying knowledge gaps to address in future research.

RNA sequencing identifies a novel USP9X-USP6 promoter swap gene fusion in a primary aneurysmal bone cyst.

Primary 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.

Mate pair sequencing improves detection of genomic abnormalities in acute myeloid leukemia.

OBJECTIVE: Acute 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. METHOD: We 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. RESULTS: Using 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. CONCLUSION: Utilization 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.

Chromoanasynthesis is a common mechanism that leads to ERBB2 amplifications in a cohort of early stage HER2+ breast cancer samples.

BACKGROUND: HER2 positive (HER2+) breast cancers involve chromosomal structural alterations that act as oncogenic driver events. METHODS: We interrogated the genomic structure of 18 clinically-defined HER2+ breast tumors through integrated analysis of whole genome and transcriptome sequencing, coupled with clinical information. RESULTS: ERBB2 overexpression in 15 of these tumors was associated with ERBB2 amplification due to chromoanasynthesis with six of them containing single events and the other nine exhibiting multiple events. Two of the more complex cases had adverse clinical outcomes. Chromosomes 8 was commonly involved in the same chromoanasynthesis with 17. In ten cases where chromosome 8 was involved we observed NRG1 fusions (two cases), NRG1 amplification (one case), FGFR1 amplification and ADAM32 or ADAM5 fusions. ERBB3 over-expression was associated with NRG1 fusions and EGFR and ERBB3 expressions were anti-correlated. Of the remaining three cases, one had a small duplication fully encompassing ERBB2 and was accompanied with a pathogenic mutation. CONCLUSION: Chromoanasynthesis involving chromosome 17 can lead to ERBB2 amplifications in HER2+ breast cancer. However, additional large genomic alterations contribute to a high level of genomic complexity, generating the hypothesis that worse outcome could be associated with multiple chromoanasynthetic events.

Comparative Performance of Breast Cancer Human Epidermal Growth Factor Receptor 2 Fluorescence In Situ Hybridization and Brightfield In Situ Hybridization on College of American Pathologists Proficiency Tests.

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.

Integrated Analysis of HER2 Copy Number by Cytogenomic Microarray in Breast Cancers With Nonclassical In Situ Hybridization Results.

OBJECTIVES: To develop and test an integrated approach to human epidermal growth factor receptor 2 (HER2) copy number analysis in breast cancer using in situ hybridization (ISH) and cytogenomic microarray (CMA). METHODS: CMA was performed on four clinical breast cancer samples with nonclassical patterns of HER2 ISH results. Integrated analysis was performed by correlating the data from pathology review, ISH, and CMA. RESULTS: Integrated analysis provided a more comprehensive view of the genomic copy number landscape that informed HER2 copy number analysis, but ISH provided essential data in all cases. CONCLUSIONS: CMA can be helpful for clarifying HER2 amplification status in breast cancer. However, uncertainties over tumor percentage, clonal heterogeneity, and varying ploidy levels present challenges for genomic methods such as CMA. Accurate interpretation of HER2 copy number by CMA requires correlation with the pathology and ISH data.

PD-L1 protein expression in tumour cells and immune cells in mismatch repair protein-deficient and -proficient colorectal cancer: the foundation study using the SP142 antibody and whole section immunohistochemistry.

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.

HER2 immunohistochemical and fluorescence in situ hybridization discordances in invasive breast carcinoma with micropapillary features.

The 2013 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) recommendations for HER2 testing contain a recommendation for pathologists with respect to invasive micropapillary carcinoma. The guidelines suggest that HER2 immunohistochemical staining that is intense but incomplete and would be considered 1+ may actually be HER2-amplified by fluorescence in situ hybridization. Thus, pathologists should consider reporting the immunohistochemistry as equivocal (2+) and employ an alternative testing methodology. This recommendation is based largely on one paper wherein the authors tested a series of 22 micropapillary carcinomas that were considered 1+ by immunohistochemistry and identified HER2 amplification in one case (5%). In order to assess for a possible discordance between HER2 immunohistochemistry and fluorescence in situ hybridization, we evaluated a series of invasive carcinomas with micropapillary features using both methodologies. As described by the WHO, invasive carcinomas with micropapillary features have small, hollow, or morula-like clusters of cells surrounded by clear stromal spaces. All cases had HER2 immunohistochemistry and fluorescence in situ hybridization performed, and for cases with equivocal fluorescence in situ hybridization results, an alternative Chromosome 17 probe (RAI1) was employed. All assays were scored according to the 2013 ASCO/CAP guidelines. Specifically for this study, immunohistochemistry was scored irrespective of the presence of micropapillary features. Overall, we identified HER2 amplification in 21 (47%) of the cases assayed, with the corresponding immunohistochemistry being 1+ (n=9), 2+ (n=11), and 3+ (n=1). The ASCO/CAP recommendation that this morphology may deviate from the typical staining pattern is highlighted, as we found that 43% of cases with micropapillary features and HER2 staining that would otherwise be scored as 1+ were HER2-amplified by fluorescence in situ hybridization. This study supports the ASCO/CAP recommendation that pathologists should consider reporting immunohistochemistry in this morphology as equivocal and perform reflex testing using in situ hybridization.

RAI1 Alternate Probe Identifies Additional Breast Cancer Cases as Amplified Following Equivocal HER2 Fluorescence In Situ Hybridization Testing: Experience From a National Reference Laboratory.

CONTEXT: -In 2013 the American Society of Clinical Oncology and College of American Pathologists updated the HER2 guidelines and changed the equivocal category for HER2 in situ hybridization testing to an average HER2 copy number of 4.0 to 5.9 with a HER2:CEP17 ratio of less than 2.0 and proposed retesting, with an option of using another control probe to avoid false-negative results. RAI1, located at band position 17p11.2, is a popular alternate probe locus for retesting equivocal changes. OBJECTIVE: -To review experience with the RAI1 alternate probe in HER2 fluorescence in situ hybridization equivocal breast cancers. DESIGN: -Primary and metastatic breast cancers with equivocal HER2 fluorescence in situ hybridization, retested with an alternate (RAI1) probe, were identified. HER2, RAI1, and CEP17 copy numbers, HER2 to control probe ratios, and genetic heterogeneity were recorded. Hematoxylin-eosin-stained slides were reviewed for type and grade of cancer. RESULTS: -Of 876 cases tested with CEP17 as the reference probe, 97 (11.1%) had equivocal HER2 fluorescence in situ hybridization results. Additional testing with the RAI1 probe classified 39.2% cases (38 of 97) as amplified with a HER2:RAI1 ratio ranging from 2.0 to 3.2 (mean, 2.37); 3.1% (3 of 97) were still unclassifiable because of a deletion of RAI1. CONCLUSIONS: -RAI1 identified close to 40% of original HER2 fluorescence in situ hybridization equivocal cases as amplified, making these patients eligible for targeted therapies. It is not known whether guidelines for US Food and Drug Administration-approved probes can be extrapolated to alternate probes when an alternate control probe shows losses or gains. Because of the lack of guidelines for reporting HER2 status with alternate probes, laboratories face challenges in interpreting results.

Inconsistent Results With Different Secondary Reflex Assays for Resolving HER2 Status.

OBJECTIVES: Guidelines suggest that secondary reflex testing may be useful for resolving HER2 status in breast cancers with equivocal results by both immunohistochemistry (IHC) and in situ hybridization (ISH). We compared two reflex ISH assays and a polymerase chain reaction (PCR) assay for this application. METHODS: Twenty-nine breast cancers with equivocal IHC and ISH results were retested two ways: (1) ISH using differentially labeled probes targeting ERBB2 ( HER2 , 17q12) and either RAI1 (17p11.2) or ORC4 (2q22.3-2q23.1) in two separate assays and (2) real-time quantitative PCR amplification of ERBB2 and a control locus ( EIF5B , 2q11.2). RESULTS: Results of the HER2 / RAI1 and HER2 / ORC4 ISH assays were concordant for 21 (72%) cases, and results of all three secondary reflex assays were concordant for only 18 (62%) cases. Result discrepancies between the two ISH readers were observed for cases close to the cutoff threshold. CONCLUSIONS: Use of different control loci for ISH is associated with discordant results, and PCR is more likely to classify cases as nonamplified, possibly due to contamination with nontumor cells. While resolution of HER2 -equivocal results is desirable from a clinical perspective, different secondary reflex assays yield different results, and the correlation of these results with clinical outcomes is unknown.