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- et al.
- Working Group of the American College of Medical Genetics and Genomics (ACMG) Laboratory Quality Assurance Committee.
- 1.Standardized definitions of terms used to describe single variants or patterns of variants detected by CMA
- 2.A four-tier evidence-based categorization system for acquired CNAs and CN-LOH, which is based on the level of available evidence regarding their diagnostic, prognostic, and therapeutic relevance
- 3.CNA and CN-LOH examples in tiers 1 and 2 in various hematologic malignancies and solid tumors
- 4.Considerations regarding the interpretation and reporting of unanticipated clinically significant germline variants
- 5.A framework to standardize the clinical reporting of acquired CNAs and CN-LOH
Definitions of specific terms used to describe single variants or patterns of variants detected by CMA
Proposed four-tier evidence-based categorization system for acquired CNAs and CN-LOH
- Li M.M.
- Datto M.
- Duncavage E.J.
- et al.
- I.Tier 1 (variants with strong clinical significance): Variants with strong diagnostic, prognostic, and/or therapeutic clinical significance. They have been demonstrated to play a critical role in the oncogenic process under investigation. Based on the level of evidence available, tier 1 variants are further subdivided into:
- a.Tier 1A: Acquired variants or a specific pattern of acquired variants that fulfill one or more of the following criteria:
- –Define a specific entity in the WHO classification.
- –Are included in professional clinical practice guidelines as clinically significant variants (e.g., NCCN, Children’s Oncology Group (COG), Myelodysplastic Syndromes (MDS) International Prognostic Scoring System, International Myeloma Working Group Criteria).
- –Can be treated by a targeted FDA approved drug.Tier 1A also includes germline pathogenic variants associated with cancer predisposition.
- b.Tier 1B: Acquired variants or a specific pattern of acquired variants with either:
- –High quality evidence (levels 1 and 2 CEBM evidence) in the literature that shows association with a specific neoplasm, prognosis, or treatment response. This includes well-powered studies in the form of randomized controlled clinical trials, systematic review and meta-analysis of these studies, and cohort studies with consensus from experts in the field.
- –Good quality evidence (level 3 CEBM evidence) in the literature that shows association with a specific neoplasm, prognosis, or treatment response. This includes multiple (at least two) smaller clinical studies in the form of cohort or case–control studies that have been confirmed and reproduced by different independent groups.
- II.Tier 2 (variants with some clinical significance): Acquired variants or a specific pattern of acquired variants with some diagnostic, prognostic, and/or therapeutic clinical significance. They include:
- –Recurrent variants observed in different neoplasms but not specific to a particular tumor type, and usually encompassing Catalogue of Somatic Mutations in Cancer (COSMIC) census cancer genes(s).
- –Acquired variants or a specific pattern of acquired variants with average quality evidence (levels 4 and 5 CEBM evidence) in the literature that shows association with a specific neoplasm, prognosis, or treatment response. This includes a small case series or multiple (at least two) case reports that describe the association.
- III.Tier 3 (clonal variants with no documented neoplastic disorder association): Acquired clonal variants with no documented neoplastic disorder association. All variants that do not meet the criteria for tiers 1 and 2 and cannot be classified as constitutional benign or likely benign, can be classified as tier 3 variants. Tier 3 variants are defined as “acquired clonal variants with no documented neoplastic disorder association” rather than “acquired clonal variants with uncertain clinical significance.” This is because an “acquired clonal variant” is by default significant for this particular patient because it can be used as a marker for the neoplastic clone to monitor residual disease and/or relapse.
- IV.Tier 4 (benign or likely benign variants): Constitutional benign or likely benign variants that are listed in the ClinGen curated benign variants and/or in the Database of Genomic Variants (DGV) with ≥1% population frequency, and usually do not encompass COSMIC cancer gene(s). It is not recommended to report tier 4 variants.
General and special considerations
- 1.The interpretation of clinical significance of CNAs and CN-LOH using this tier system should be performed in the context of the clinical/pathologic diagnosis, as well as other laboratory tests including G-banded karyotype, FISH, and other relevant tests. This is crucial because some acquired variants will have different clinical significance in different neoplastic disorders. For example, 1q gain is associated with adverse prognosis in multiple myeloma (MM) (tier 1A),14.,
Updated diagnostic criteria and staging system for multiple myeloma.Am Soc Clin Oncol Educ Book. 2016; 35: e418-e42315.while it does not have major prognostic significance in MDS (tier 2).16.CNAs may also have different clinical significance depending on other cytogenetic or molecular diagnostic abnormalities present in the tumor. For example, loss of chromosome 7 or 7q deletion are typically associated with an inferior outcome in myeloid malignancies (tier 1A), but in acute myeloid leukemia (AML) with aCBFB gene rearrangement, they do not appear to significantly change the prognosis (tier 2).17.
- Rajkumar S.V.
Refinement of cytogenetic classification in acute myeloid leukemia: determination of prognostic significance of rare recurring chromosomal abnormalities amongst 5,876 younger adult patients treated in the UK Medical Research Council trials.
- Grimwade D.
- Hills R.K.
- Moorman A.V.
- et al.
- 2.This tier system can be used to classify a specific pattern of CNAs and/or CN-LOH that is diagnostic of a specific neoplastic disease entity. This includes a characteristic pattern of whole chromosome gains/losses (e.g., hyperdiploid and hypodiploid B-ALL) and whole chromosome CN-LOH (e.g., doubled hypodiploid/near-haploid B-ALL).18.It also includes a characteristic signature of gains and losses along one chromosome (e.g., intrachromosomal amplification of chromosome 21 [iAMP21] in B-ALL).
- 3.Diagnostic balanced chromosomal abnormalities (e.g., translocations, inversions, and insertions) detected by G-banded karyotype and/or FISH testing but not by CMA should be discussed in the CMA report but should not be included in the classification using the tier system or listed in the results table/nomenclature string. When present in the unbalanced form and detected by CMA with breakpoints mapping within genes known to be associated with a specific gene fusion, these abnormalities can be classified using the tier system and listed in the results table/nomenclature string (e.g., the presence of an extra copy of the Philadelphia chromosome der(22)t(9;22)(q34;q11.2) in CML or ALL,18.or an extra copy of the der(21)t(12;21)(p13;q22) in B-ALL, and the unbalanced der(19)t(1;19)(q23;p13) in B-ALL).20.
- Baughn L.B.
- Biegel J.A.
- South S.T.
- et al.
- 4.An interstitial loss or gain involving one chromosome arm with recurring breakpoints in genes known to be involved in a specific gene fusion can be classified using this tier system (e.g., 4q12 deletion that results in FIP1L1-PDGFRA fusion, PAR1 deletion at Xp22.33/Yp11.32 that results in P2RY8-CRLF2 fusion, and 9q34.1 gain that results inNUP214-ABL1 fusion).18.,
- 5.Interstitial or terminal losses or gains involving two chromosome arms with breakpoints within genes known to be associated with a specific gene fusion as a result of an interchromosomal rearrangement (e.g., translocation or insertion) or intrachromosomal rearrangement (e.g., inversion) should be interpreted according to the level of supporting evidence. They can be classified using this tier system with later confirmation of the gene fusion by other molecular techniques if there is enough supporting evidence, including the clinical/pathologic diagnosis, visible recurrent rearrangement by G-banded karyotype, and/or other acquired variant known to be associated with the gene fusion in question. In the absence of such supporting evidence, the report should describe the possibility of a gene fusion but without classifying the variants using the tier system until the fusion is confirmed by other molecular techniques.
- 6.Correlation of the CMA results with the G-banded karyotype and FISH results is strongly recommended because some professional clinical practice guidelines used to classify tier 1A variants are technique specific. For example, some chromosomal abnormalities can only be considered diagnostic/prognostic if detected by G-banded karyotype (e.g., MDS and MM prognostic criteria).
- 7.CMA has the potential to identify acquired variants associated with comorbid neoplastic disorders. For example, comorbid MDS-related variants may be identified in patients treated for chronic lymphocytic leukemia (CLL) or MM either because of prior therapy or age-related disease. These variants should be interpreted in the context of the clinical/pathologic diagnosis and correlated with G-banded karyotypes from both stimulated and unstimulated CLL or MM cultures. CMA performed in MM on CD138+ enriched cells is helpful in identifying MM-specific acquired variants.7.
Assessing genome-wide copy number aberrations and copy-neutral loss-of-heterozygosity as best practice: an evidence-based review from the Cancer Genomics Consortium working group for plasma cell disorders.
- Pugh T.J.
- Fink J.M.
- Lu X.
- et al.
- 8.The term “CN-LOH” is used in this document to refer to a region with acquired allelic imbalance (i.e., homozygosity) without an associated copy-number change (i.e., copy-neutral), which is a common finding in cancer. The term “copy-neutral” is used to allow distinction from loss of heterozygous single-nucleotide polymorphism (SNP) calls due to a one copy-number loss (i.e., heterozygous deletion). However, in some cases LOH can also be observed with a copy-number gain. Examples include high-level amplification involving only one allele, and the copresence of a clone with trisomy of a particular chromosome and a subclone that lost one copy of that chromosome resulting in whole chromosome LOH.
- 9.Regions of CN-LOH may have a higher level of clinical significance if they span a gain-of-function variant in an oncogene and/or loss-of-function variant in a tumor suppressor gene documented in this patient. This is especially relevant in laboratories that do integrated reporting of CNAs, regions of CN-LOH, and sequence variants results.
Databases and resources for interpretation of CNAs and CN-LOH in neoplastic disorders
- 1.Databases and data portals focusing directly on acquired CNAs and CN-LOH
- 2.Databases and data portals focusing on acquired sequence variants, which allow the evaluation of whether specific genes within the region affected by a CNA have been associated with the tumor type of interest
- 3.Knowledge bases that contain curated information on the significance of individual genes and acquired sequence variants in different tumor types
- 4.Chromosome-level databases and knowledge bases that compile data from conventional cytogenetic analysis and curations regarding the significance of chromosome aberrations detected by karyotyping
- 5.Databases of benign and pathogenic germline variants that allow exclusion of benign germline variants and interpretation of germline secondary findings
Considerations regarding interpretation and reporting of unanticipated clinically significant germline variants
Indications that a detected CNV or AOH may be germline
- 1.Involvement of 100% of the cells in a sample. Often, acquired variants involve only a subset of cells corresponding to the tumor clone. Review of the log2 ratio and SNP data allows determining if a variant is present in all or only a subset of cells in a sample. However, it is important to be aware of the following caveats:
- a.Some specimens may consist of pure tumor tissue and have acquired variants that involve close to 100% of the cells (e.g., a bone marrow specimen packed with leukemic blasts or a dissected tumor section).
- b.Copy-number losses encompassing cancer predisposition genes are particularly difficult to interpret by CMA alone. For small abnormalities with insufficient SNP data, CMA may not reliably differentiate a heterozygous loss in 100% of the cells from a homozygous loss in 50% of the cells. If CMA shows copy-number losses encompassing cancer predisposition genes, it might not be possible to distinguish between a germline heterozygous deletion of the gene in question in 100% of the cells versus acquired biallelic loss of the gene in 50% of the cells. Frequently encountered examples include the Fanconi anemia/DNA repair pathway genes (includingBRCA1 andBRCA2),NF1, RB1, and PAX5. Follow-up interphase FISH analysis using gene-specific probes can be helpful in distinguishing between these two possibilities.
- 2.Higher proportion of cells involved by a variant than expected by pathologic findings. For hematologic malignancies, a finding may be germline if it appears to involve a significantly greater proportion of cells than that expected based on the blast cell count or degree of involvement determined by morphology or flow cytometry. Correlation with hematopathology and flow cytometry/immunophenotyping data is valuable, and efforts to obtain this information are recommended. For solid tumors, a finding may be germline if the estimate of involvement by CMA is significantly greater than the estimation of tumor cell fraction provided by the submitting pathologist. However, estimating tumor fraction in solid tumors is often challenging and involves subjective judgment; this estimate may not always be perfectly correlated with CMA results.
- 3.Supporting clinical information may suggest that a CMA variant is germline:
- a.Some tumor types are frequently associated with the presence of predisposing germline variants. Examples include Wilms tumor, tuberous sclerosis complex (TSC1/TSC2) tumors, neurofibromas, adrenocortical carcinoma, and rhabdoid tumor (Supplementary Table 1). Laboratories should have an increased level of suspicion for germline variants when performing CMA for these tumor types.
- b.CNVs/AOH including known cancer predisposition genes (Supplementary Table 1) may be suspected as germline in patients with features of hereditary cancer syndromes, including diagnosis at unusually young age, development of bilateral or multifocal tumors, or family/personal history of cancer.
- c.CNVs involving genes and regions associated with known pathogenic microdeletion/microduplication syndromes may be suspected as germline in patients who have reported features consistent with the disorder in question. If the provided clinical information is limited, the laboratory may request additional details to allow accurate interpretation of the findings.
Interpretation and reporting of suspected germline variants
- a.Laboratories should have an established policy for reporting CNVs that are likely germline and have been curated as pathogenic by ClinGen (including pathogenic CNVs associated with disorders that show incomplete penetrance) and/or span known haploinsufficient or triplosensitive genes.
- b.Possibly constitutional CNVs unrelated to the patient’s cancer diagnosis should not be classified into the tier system. For unambiguous reporting, the laboratory may have a separate section of the report for describing these variants.
Follow-up recommendations for suspected germline variants
- a.Referral to a genetic specialist for evaluation and counseling.
- b.Confirmation of germline status by testing noninvolved tissue. For patients with solid tumors, a peripheral blood sample may be tested. For patients with hematologic malignancies, the optimal samples for germline testing are cultured skin fibroblasts, although a buccal swab or a peripheral blood sample at the time of complete remission may be acceptable.
Suspected germline variant
Reporting recommendations for acquired CNAs and CN-LOH
- –Chromosomes and corresponding bands involved in the variant
- –Type of variant (loss, gain, amplification, CN-LOH)
- –Genomic coordinates with designated genome build
- –Copy-number state and percentage of cells involved, estimated based on the log2 ratio and SNP data
- –Tier classification
- –Variant size in kb or Mb
- –COSMIC cancer census genes within the affected region
Full interpretation of clinically significant variants and a text summary integrating results
- •Clinically significant CNAs and/or CN-LOH (tier 1 and 2 variants).
- •Clinically significant pattern of CNAs and/or CN-LOH (tier 1 and 2 variants).
- •CNAs and/or CN-LOH of potential clinical significance (cannot be tiered at the time of reporting). This category addresses point 5 in “General and special considerations” when there is uncertainty about an acquired variant being indicative of a specific gene fusion in the absence of supporting evidence at the time of reporting.
- •Optional: other clonal variants (tier 3 variants).
Methodology and disclaimers
- •Supplementary Table 1 illustrating selected tumor suppressor genes associated with germline predisposition to cancer.
- •CMA report examples in hematologic malignancies and solid tumors.
- •Supplementary figures illustrating examples of amplification, chromothripsis, intrachromosomal complexity, and genomic complexity. The same pattern of acquired CNAs suggestive of a specific disease entity is demonstrated using different CMA platforms. To illustrate the clinical utility of this tier classification system in the interpretation of acquired CNAs derived from whole genome sequencing (WGS), examples of such abnormalities derived from WGS data are also included.
Box 1: Specific terms used to describe single variants or patterns of variants detected by CMA
- •Size/location of variant:
- Focal: Relatively small change (typically less than 5Mb) that usually contains a known or suspected driver cancer gene
- Whole arm: Change that involves the entire chromosome short (p) or long (q) arm
- Whole chromosome: Change that involves the entire chromosome
- Interstitial: Change mediated by at least two breaks within a chromosome p or q arm
- Terminal: Change that includes the end of the p or q arm of the chromosome
- Intragenic: Change that occurs within a single gene
- Proximal/distal: Describes a position relative to the centromere and moving outward on the chromosome p or q arm
- •Type of variant:
- Gain/loss: Type of copy-number change observed. It is recommended that the term “gain” be used rather than “duplication.” Attempts should be made to determine the relative gain/loss in polyploid samples.
- Copy-number abnormalities (CNAs): Neoplastic disease-associated changes that represent acquired gains or losses of chromosome material.
- Copy-neutral loss of heterozygosity (CN-LOH): Allelic imbalance without an associated copy-number change. This is a somatic process occurring in tumors, and terms such as absence of heterozygosity (AOH), identity by descent (IBD), and uniparental disomy (UPD) should be used when the change is germline.
- Amplification: High copy-number gain of sequences, typically containing oncogene(s) that are important for the cancer being studied. Note that the term should not be used to describe a single copy gain of chromosomal material or to describe gain due to polysomy. Standard thresholds used to represent amplification typically range from 3–5 fold increases over baseline ploidy (e.g., intrachromosomal amplification of chromosome 21 [iAMP21] in B-ALL) to >100 copies per genome (e.g., MYCN amplified neuroblastoma) and will vary depending on the type of tumor. The laboratory should establish specific copy-number threshold cutoffs that will be used to identify clonally amplified regions by correlating CMA results to established methodologies for different tumor types.
- Chromothripsis: A copy-number profile that has alternating copy states in a single region—typically a single chromosome or chromosome arm—that contains at least ten distinct alternating copy-number segments.9.,10.,
- Intrachromosomal complexity: Summary of chromosomal regions that include more than two copy-number states, are largely confined to a single chromosome or chromosome arm, and contain at least five distinct copy-number segments. If clinically significant abnormalities (tiers 1 or 2) fall within a complex region, they may be reported individually.
- Genomic complexity: Pattern of chromosome instability predominantly due to structural alterations resulting in widespread gains and losses of chromosomes or chromosomal regions in the majority of chromosomes.
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