Clinical utility gene card for: alveolar rhabdomyosarcoma

1. DISEASE CHARACTERISTICS 1.1 NAME OF THE DISEASE (SYNONYMS) Alveolar rhabdomyosarcoma (ARMS). 1.2 OMIM# OF THE DISEASE 268220. 1.3 NAME OF THE ANALYSED GENES OR DNA/CHROMOSOME SEGMENTS

_FORKHEAD BOX O1A, FOXO1A_ (formerly FORKHEAD IN _RHABDOMYOSARCOMA, FKHR_), gene locus: chromosome 13q14.1 _PAIRED BOX GENE 3_, _PAX3_, gene locus: chromosome 2q35 _PAIRED BOX GENE 7_,

_PAX7_, gene locus: chromosome 1p36 _ALL1-FUSED GENE FROM X CHROMOSOME, AFX1 (MYELOID/LYMPHOID OR MIXED LINEAGE LEUKAEMIA, TRANSLOCATED TO, 7, MLLT7; FORKHEAD BOX O4, FOXO4_), gene locus:

chromosome Xq13.1 _NUCLEAR RECEPTOR COACTIVATOR 1, NCOA1_, gene locus: chromosome 2p23. 1.4 OMIM# OF THE GENE(S) 136533, 606597, 167410, 300033, 602691. 1.5 MUTATIONAL SPECTRUM Recurrent

reciprocal translocations and insertions that form chimeric fusion genes. * t(2;13)(q35;q14) that forms fusion gene _PAX3_–_FOXO1A_; identified in 75% of fusion-positive ARMS cases.1, 2 *

t(1;13)(p36;q14) that forms fusion gene _PAX7_–_FOXO1A_; identified in 25% of fusion-positive ARMS cases.3, 4 * t(2;X)(q35;q13) that forms fusion gene _PAX3_–_AFX1_; identified in <1% of

ARMS cases.5 * t(2;2)(q35;p23) that forms fusion gene _PAX3_–_NCOA1_; identified in <1% of ARMS cases.6, 7 Tetraploidy is common in ARMS and occurs in 77% of ARMS demonstrated in one of

the studies.8 _PAX7_–_FOXO1A_ fusions are commonly amplified.9 Amplification of chromosome 2p24 region including _MYCN_ gene occurs in 13% of cases of fusion-positive ARMS but has no

significant association with clinical outcome.10 Amplification of chromosome 12q13-14 region including _CDK4_ gene occurs in 12% of cases of fusion-positive ARMS (majority are

_PAX3_–_FOXO1A_ fusion) and is associated with worse outcome.10 _TFAP2 β_ (6p24), _CDH3_ (16q22.1) and _CNR1_ (6q14-q15) are highly expressed in fusion-positive ARMS, irrespective of tumour

histology.11 Approximately 20–30% of ARMS have no _PAX_–_FOXO1A_ fusion (ie, fusion negative ARMS). Oligonucleotide microarrays have demonstrated that fusion-negative ARMS has a distinctive

gene expression profile different from fusion-positive ARMS. Some gene expression studies show that fusion-negative ARMS constitutes a heterogeneous group that overlaps with embryonal

rhabdomyosarcoma (ERMS), with frequent whole-chromosome copy number changes, notably gain of chromosome 8 with associated high levels of expression of genes from this chromosome.11, 12, 13

1.6 ANALYTICAL METHODS Routine cytogenetic karyotyping on fresh, unfixed tissue. Reverse transcriptase (RT)-PCR on fresh, frozen or formalin-fixed, paraffin-embedded tissues. Fluorescence

_in situ_ hybridisation (FISH) on either cytologic touch preparations or formalin-fixed, paraffin-embedded tissue. Microarray for gene or protein expression analysis is currently only used

in research field, but may be used as a clinical test in the future. 1.7 ANALYTICAL VALIDATION Although the subclassification of rhabdomyosarcoma has traditionally relied on histological

analysis, cytogenetic and molecular genetic analytic methods are increasingly being used as standard confirmatory tests. All testing should be validated based on histological criteria, but

future treatment protocols may rely on fusion status rather than histology. 1.8 ESTIMATED FREQUENCY OF THE DISEASE (INCIDENCE AT BIRTH (‘BIRTH PREVALENCE’) OR POPULATION PREVALENCE) The

incidence for overall rhabdomyosarcomas is 4.5 cases per million children/adolescents (age 0–19) per year in the United States between 1975 and 2005, of which ARMS account for 23%.14 1.9 IF

APPLICABLE, PREVALENCE IN THE ETHNIC GROUP OF INVESTIGATED PERSON In the United States between 1975 and 2005, African-American children had slightly higher rates of ARMS than Caucasian

children (1.3 of 1 000 000 _vs_ 1.0 of 1 000 000, respectively).14 1.10 DIAGNOSTIC SETTING Comment: Patients with fusion-positive ARMS have a significantly worse outcome than those with

fusion-negative lesions having similar histology.15 Some studies suggest that tumours with _PAX7_ fusions have a better prognosis than other ARMS.13, 16 2. TEST CHARACTERISTICS 2.1

ANALYTICAL SENSITIVITY (PROPORTION OF POSITIVE TESTS IF THE GENOTYPE IS PRESENT) Routine cytogenetic karyotyping: 28%17 RT-PCR: 25–86%17, 18 FISH: 38–88%.18, 19 Comment: Depends on the

technique and methods used in each laboratory, the sensitivity may vary. False negative results with routine cytogenetic method may be associated with normal cellular components overgrowing

tumour cells and low-level gene expression may cause false negative results associated with RT-PCR. 2.2 ANALYTICAL SPECIFICITY (PROPORTION OF NEGATIVE TESTS IF THE GENOTYPE IS NOT PRESENT)

Routine cytogenetic karyotyping: 100%17 RT-PCR: 93–100%17, 18 FISH 100%.18 Comment: Depends on the technique and methods used in each laboratory, the specificity may vary. 2.3 CLINICAL

SENSITIVITY (PROPORTION OF POSITIVE TESTS IF THE DISEASE IS PRESENT) The clinical sensitivity can be dependent on variable factors such as age or family history. In such cases a general

statement should be given, even if a quantification can only be made case by case. Approximately 70–80%. Around 70–80% ARMS (so called fusion-positive ARMS) possess either _PAX3_-_FOXO1A_ or

_PAX7_-_FOXO1A_ translocations. Approximately 25% of cases have classic ARMS histology, but do not contain a fusion gene (so called fusion-negative ARMS). Gene expression arrays indicate

that fusion-negative ARMS constitute a heterogeneous group that overlaps with ERMS. Although _PAX3_-_FOXO1A_ tumours comprise a molecularly homogeneous entity with a uniformly poor

prognosis, _PAX7_-_FOXO1A_-positive tumours exhibit gene amplification rather than overexpression. This subset may have a better prognosis than other alveolar genetic subtypes.13, 16 2.4

CLINICAL SPECIFICITY (PROPORTION OF NEGATIVE TESTS IF THE DISEASE IS NOT PRESENT) The clinical specificity can be dependent on variable factors such as age or family history. In such cases a

general statement should be given, even if a quantification can only be made case by case. Approximately 100%. 2.5 POSITIVE CLINICAL PREDICTIVE VALUE (LIFE-TIME RISK TO DEVELOP THE DISEASE

IF THE TEST IS POSITIVE) Routine cytogenetic karyotyping: 100%17 RT-PCR: 90–100%17, 18 FISH: 100%.18 2.6 NEGATIVE CLINICAL PREDICTIVE VALUE (PROBABILITY NOT TO DEVELOP THE DISEASE IF THE

TEST IS NEGATIVE) Assume an increased risk based on family history for a non-affected person. Allelic and locus heterogeneity may need to be considered. Routine cytogenetic karyotyping: no

reference available (or 0% based on limited data from Ref. 18). RT-PCR: 26–90%.17, 18 FISH: 38%.18 3. CLINICAL UTILITY 3.1 (DIFFERENTIAL) DIAGNOSTICS: THE TESTED PERSON IS CLINICALLY

AFFECTED (To be answered if in 1.10 ‘A’ was marked) _3.1.1 CAN A DIAGNOSIS BE MADE OTHER THAN THROUGH A GENETIC TEST?_ Comment: The diagnosis of ARMS is currently based on routine histology,

but some strongly feel that it should be supplanted by genetic studies.11, 13, 16, 18, 20, 21 The current gold standard for the diagnosis of ARMS is the combination of classic or solid

‘alveolar’ histological features and strong reactivity to myogenin by immunohistochemistry.21, 22, 23, 24, 25 New study has shown that fusion-positive ARMS may be detected by using a set of

immunohistochemical markers, AP2_β_ and P-cadherin, with a specificity of 98% and a sensitivity of 64%.26 However, at present time, cytogenetic testing is still a key ancillary test when the

tumours do not have classic ARMS histological features or strong expression of myogenin or myoD1. _3.1.2 DESCRIBE THE BURDEN OF ALTERNATIVE DIAGNOSTIC METHODS TO THE PATIENT_

Rhabdomyosarcoma is the most common soft tissue tumour in paediatric population. It has been subclassified into two major categories: ERMS and ARMS. In general, ARMS carries an unfavourable

prognosis with an aggressive clinical behaviour and a poor response to chemotherapy; thus low-stage disease requires aggressive treatment. Because of significant differences in survival and

treatment strategies, distinction between ARMS, ERMS and other small round blue cell neoplasms of childhood are of clinical importance. However, diagnosis of ARMS based solely on histology

can be very challenging, as histologic features can overlap – especially in solid ARMS and ARMS with mixed alveolar and embryonal features. Because ERMS and other paediatric small blue cell

tumours only very rarely display a _FOXO1A_ mutation, detection of a positive _FOXO1A_ mutation has great value in confirming the diagnosis of ARMS.21, 22, 23 Without cytogenetic

confirmation, a misdiagnosis or misclassification may occur if the histology or/and immunohistochemistry is atypical. Consequently, the patient may receive less optimal treatment. _3.1.3 HOW

IS THE COST EFFECTIVENESS OF ALTERNATIVE DIAGNOSTIC METHODS TO BE JUDGED?_ The cost of histological analysis (routine staining) plus immunohistochemical stains can be expensive if more than

a minimum number of immunohistochemical stains are used. Depending on the experience of the histopathologist, the number of immunohistochemical stains can range from two to twenty. _3.1.4

WILL DISEASE MANAGEMENT BE INFLUENCED BY THE RESULT OF A GENETIC TEST?_ 3.2 PREDICTIVE SETTING: THE TESTED PERSON IS CLINICALLY UNAFFECTED BUT CARRIES AN INCREASED RISK BASED ON FAMILY

HISTORY (To be answered if in 1.10 ‘B’ was marked) _3.2.1 WILL THE RESULT OF A GENETIC TEST INFLUENCE LIFESTYLE AND PREVENTION?_ If the test result is positive (please describe): If the test

is positive, patients with low-risk features will receive more aggressive therapy and may have improved FFS and lifespan. If the test result is negative (please describe): If the test is

negative but histological and immunohistochemical features indicate ARMS, patients with low stage, localised tumours currently are still not eligible for low-risk therapy and receive more

aggressive treatment than they would otherwise, but this approach has been questioned20 and may be revised in future protocols. There is currently no fusion-specific therapy.31 _3.2.2 WHICH

OPTIONS IN VIEW OF LIFESTYLE AND PREVENTION DO A PERSON AT-RISK HAVE IF NO GENETIC TEST HAS BEEN DONE (PLEASE DESCRIBE)?_ If a correct diagnosis can be made based on classic alveolar

histology and immunohistochemical stain, there will be probably no significant adverse effect on the patient's disease management. Conversely, if the patients are misclassified as ERMS,

they may receive suboptimal, less aggressive treatment and may have disease progression. 3.3 GENETIC RISK ASSESSMENT IN FAMILY MEMBERS OF A DISEASED PERSON (To be answered if in 1.10 ‘C’

was marked) _3.3.1 DOES THE RESULT OF A GENETIC TEST RESOLVE THE GENETIC SITUATION IN THAT FAMILY?_ Not applicable. _3.3.2 CAN A GENETIC TEST IN THE INDEX PATIENT SAVE GENETIC OR OTHER TESTS

IN FAMILY MEMBERS?_ Not applicable. _3.3.3 DOES A POSITIVE GENETIC TEST RESULT IN THE INDEX PATIENT ENABLE A PREDICTIVE TEST IN A FAMILY MEMBER?_ Not applicable. 3.4 PRENATAL DIAGNOSIS (To

be answered if in 1.10 ‘D’ was marked) _3.4.1 DOES A POSITIVE GENETIC TEST RESULT IN THE INDEX PATIENT ENABLE A PRENATAL DIAGNOSIS?_ Not applicable. 4. IF APPLICABLE, FURTHER CONSEQUENCES OF

TESTING Please assume that the result of a genetic test has no immediate medical consequences. Is there any evidence that a genetic test is nevertheless useful for the patient or his/her

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e587–e588. Article  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by EuroGentest, an EU-FP6 supported NoE, contract number 512148 (EuroGentest Unit 3:

‘Clinical genetics, community genetics and public health’, Workpackage 3.2). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Pathology, University of Oklahoma, Oklahoma City, OK,

USA Zhongxin Yu & David Parham * Department of Pathology, Royal Manchester Childrens Hospital, Manchester, UK Anna Kelsey * Pathology Department, University of Padova, Padova, Italy

Rita Alaggio Authors * Zhongxin Yu View author publications You can also search for this author inPubMed Google Scholar * Anna Kelsey View author publications You can also search for this

author inPubMed Google Scholar * Rita Alaggio View author publications You can also search for this author inPubMed Google Scholar * David Parham View author publications You can also search

for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Zhongxin Yu. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no conflict of interest. RIGHTS AND

PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Yu, Z., Kelsey, A., Alaggio, R. _et al._ Clinical utility gene card for: Alveolar rhabdomyosarcoma. _Eur J Hum

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