Zou, Ying S

Context.— One objective of the College of American Pathologists/American College of Medical Genetics Cytogenetics Committee is to ensure the accurate detection and clinical interpretation of abnormalities observed by fluorescence in situ hybridization (FISH) analysis when evaluating hematologic neoplasms. Objective.— To report a 20-year performance summary (2005–2024) of FISH challenges focusing on hematologic neoplasms. Design.— A retrospective review was performed from 2005 through 2024 to identify FISH challenges specifically addressing hematologic neoplasms. The overall performance of participants was evaluated to identify potential recurring errors of clinical relevance. Results.— A total of 124 hematologic neoplasm FISH challenges from 2005 to 2024 were provided to participating laboratories, including 45 paper challenges (results and/or images were provided for interpretation purposes), 43 liquid challenges (bone marrow aspirate or peripheral blood), and 36 formalin-fixed paraffin-embedded (FFPE) tissue challenges. Of the 124 challenges, 120 (97%) exceeded the required 80% consensus for grading purposes. Of the 4 failed challenges (3%), 2 (1 liquid, 1 paper) involved the interpretation of atypical FISH signal patterns; 1 (paper) involved the interpretation of FISH signal patterns correlating involvement by myeloid versus lymphoid interphase nuclei; and 1 (liquid) likely involved a specimen processing or handling error that resulted in discordant participant responses. All 36 FFPE tissue challenges exceeded the 80% consensus for grading purposes. Conclusions.— This 20-year retrospective review demonstrates that clinical cytogenomic laboratories have been and continue to be highly proficient in the detection and interpretation of FISH abnormalities associated with hematologic neoplasms.

Background/Objective: Although most melanocytic lesions are diagnosed as benign or malignant by histopathologic evaluation, with or without the aid of immunohistochemistry, diagnosis may remain uncertain in a minority of cases. Assessment of copy number abnormalities (CNAs) may provide sufficient additional evidence to favor either a benign or malignant diagnosis in both pediatric and adult cases and in melanocytic lesions of various subtypes, including Spitzoid, mucosal, and acral. CNAs are common in melanomas, while they are rare, with few exceptions, in benign lesions. Detection of CNAs by fluorescence in situ hybridization (FISH) and chromosomal microarray (CMA) has been well established for melanocytic lesions, with advantages and disadvantages for each. The objective of this meta-analysis was to evaluate the utility of CNA testing for the diagnosis of melanoma, across subtypes, when a lesion remains ambiguous after histopathologic and immunohistochemical assessment. In addition, the utility of CNAs to determine prognosis in established diagnoses of melanoma was also evaluated. Methods: The Cancer Genomics Consortium Working Group for Melanocytic Lesions reviewed published data from January 1998 through September 2022 of CNAs in melanocytic lesions detected by either FISH or CMA and conducted a meta-analysis of the findings. Results: Specific abnormalities common in primary cutaneous melanomas of various subtypes and uveal melanomas were enumerated. Differences in CNAs found in primary versus metastatic lesions were also determined, and published evidence for prognosis was summarized. Conclusions: The working group established evidence-based recommendations for the use of CNA testing for evaluation of ambiguous melanocytic lesions.

Background: The joint College of American Pathologists/American College of Medical Genetics and Genomics Cytogenetics Committee works to ensure the competency and proficiency of clinical cytogenetic testing laboratories through proficiency testing (PT) programs for various clinical tests offered by such laboratories, including the evaluation of cytogenetic abnormalities in solid tumors. Methods: Review and analyze 25 years (1999–2023) of solid tumor chromosome analysis PT results, utilizing G-banded karyograms. A retrospective review of results from 1999 to 2023 was performed, identifying the challenges addressing solid tumors. The chromosomal abnormalities and overall performance were evaluated. Results: A total of 21 solid tumor challenges were administered during the period 1999–2018. No solid tumor challenges were administered during the period 2019–2023. Challenges consisted of metaphase images and accompanying clinical history for the evaluation of numerical and/or structural abnormalities. All 21 cases reached 80% grading consensus for abnormality recognition. However, five cases (24%) failed to reach consensus for nomenclature reporting by participating laboratories. These cases illustrate errors in reporting chromosomal abnormalities, including whole-arm translocations and those involving sex chromosomes. In addition, they highlight the challenges with differentiation of terminal and interstitial deletions, difficulties in identifying correct breakpoints, and omission of brackets in neoplastic cases. Conclusions: This comprehensive 25-year review demonstrates the exceptional proficiency of cytogenetic laboratories in accurately identifying chromosome abnormalities in solid tumors, while also highlighting the challenges of reporting specific types of chromosomal abnormalities.

Complex structural chromosome abnormalities such as chromoanagenesis have been reported in acute myeloid leukemia (AML). They are usually not well characterized by conventional genetic methods, and the characterization of chromoanagenesis structural abnormalities from short-read sequencing still presents challenges. Here, we characterized complex structural abnormalities involving chromosomes 2, 3, and 7 in an AML patient using an integrated approach including CRISPR/Cas9-mediated nanopore sequencing, mate pair sequencing (MPseq), and SNP microarray analysis along with cytogenetic methods. SNP microarray analysis revealed chromoanagenesis involving chromosomes 3 and 7, and a pseudotricentric chromosome 7 was revealed by cytogenetic methods. MPseq revealed 138 structural variants (SVs) as putative junctions of complex rearrangements involving chromosomes 2, 3, and 7, which led to 16 novel gene fusions and 33 truncated genes. Thirty CRISPR RNA (crRNA) sequences were designed to map 29 SVs, of which 27 (93.1%) were on-target based on CRISPR/Cas9 crRNA nanopore sequencing. In addition to simple SVs, complex SVs involving over two breakpoints were also revealed. Twenty-one SVs (77.8% of the on-target SVs) were also revealed by MPseq with shared SV breakpoints. Approximately three-quarters of breakpoints were located within genes, especially intronic regions, and one-quarter of breakpoints were intergenic. Alu and LINE repeat elements were frequent among breakpoints. Amplification of the chromosome 7 centromere was also detected by nanopore sequencing. Given the high amplification of the chromosome 7 centromere, extra chromosome 7 centromere sequences (tricentric), and more gains than losses of genomic material, chromoanasynthesis and chromothripsis may be responsible for forming this highly complex structural abnormality. We showed this combination approach’s value in characterizing complex structural abnormalities for clinical and research applications. Characterization of these complex structural chromosome abnormalities not only will help understand the molecular mechanisms responsible for the process of chromoanagenesis, but also may identify specific molecular targets and their impact on therapy and overall survival.

Abstract Objectives To demonstrate clinicopathologic features and evaluate the clonality of double PIK3CA alterations in colorectal cancers (CRCs). Methods Clonality was examined in 13 CRCs with double PIK3CA alterations (1.7% of CRCs or 9.6% of PIK3CA-mutated CRCs). Multiregional analyses were performed to confirm subclonal PIK3CA alterations. Results PIK3CA alterations were detected within exon 9 (51%), exon 20 (23%), exon 1 (15%), and exon 7 (6.0%). CRCs with exon 7 alterations showed a significantly higher incidence of double PIK3CA alterations. Most double PIK3CA alterations consisted of a hotpsot alteration and an uncommon alteration; they were often clonal and present within a single tumor population. Multiregional analyses of CRCs with predicted subclonal double-alterations revealed multiclonal CRCs with divergent PIK3CA variant status originating from a common APC- and KRAS-mutated founder lineage of adenoma. Conclusions The findings supported multiclonal CRCs resulting from parallel evolution during the progression from adenoma to adenocarcinoma within the mitogen-activated protein kinase pathway, as previously demonstrated, or the mammalian target of rapamycin pathway. Further studies are warranted to elucidate clinical significance and potential targeted therapy for CRC patients with double PIK3CA alterations and impacts on clinical decision-making in patients with multiclonal CRCs harboring divergent PIK3CA mutational status.