**Acute Myelogenous Leukemia (AML)**

**Indications**

Genomic Sequential Analysis Panel will be considered reasonable and necessary
in the evaluation of blood or bone marrow samples in the following clinical
circumstances:

  * Newly diagnosed patients with AML who are undergoing induction therapy, and who are suitable candidates for post-induction transplantation or consolidation therapy at the time of testing, and meet one of the following cytogenetic criteria:
    * normal karyotype
    * core binding factor
  * Previously diagnosed patients with AML, who have not responded to induction chemotherapy, or who have progressed following induction. The patient must be a candidate for transplantation at the time of the testing.
  * Patients with AML, who have responded to treatment, either chemotherapy or transplantation, with evidence of relapse.  
  

**Myelodysplastic Syndromes (MDS)**

**Indications**

Genomic Sequential Analysis Panel will be considered reasonable and necessary
in the evaluation of blood or bone marrow samples in the following clinical
circumstances:

  * Patients with clinical signs or symptoms of myelodysplastic syndromes (MDS) or myelodysplastic/myeloproliferative overlap syndromes (MDS/MPN), in whom clinical, laboratory, and pathologic assessment are nondiagnostic.
  * Newly diagnosed MDS or MDS/MPN patients either
    * stratified by the IPSS or IPSS-R as intermediate risk, or
    * in MDS with ringed sideroblasts/RARS. 

  **Limitations**

  * Repeat Genomic Sequential Analysis Panel testing is not reasonable and necessary in MDS after initial diagnosis and risk stratification.



**Myeloproliferative Neoplasms (MPN)**

**Indications and Limitations of Coverage**

Genomic Sequential Analysis Panel will be considered reasonable and necessary
in the evaluation of blood or bone marrow samples in the following
circumstances:

  *   **Diagnosis:** Clinical signs or symptoms of myeloproliferative neoplasm (MPN) or myelodysplastic/myeloproliferative overlap syndromes (MDS/MPN) when
    * clinical, laboratory, and pathologic assessment are nondiagnostic; and
    * CML excluded (BCR-ABL1 negative)1,2
  * **Risk Stratification:** Newly diagnosed PMF not already classified as high-risk by Dynamic International Prognostic Scoring System (DIPSS) Plus1,3,4
  * **Monitoring:** Higher-risk MF (INT-1, INT-2, High-Risk) with progression on therapy1



**Acute Myelogenous Leukemia (AML)**

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy
characterized by the clonal expansion of myeloid blasts, primarily in the
peripheral blood and bone marrow. The American Cancer Society estimates that
approximately 60,000 new cases of leukemia will be diagnosed in 2016, with
one-third classified as acute myelogenous leukemia (AML). It accounts for the
most annual deaths from leukemia in the United States. The median age of
diagnosis is 67, with 54% diagnosed at 65 years or older (and approximately
one third diagnosed at 75 years of age or older). Moreover, AML lies at one
end of a spectrum of neoplastic myeloid diseases that includes myelodysplastic
syndromes (MDS), which often progress to AML, and which are even more common
in patients of advanced age, with an incidence of approximately 1/5000
patients over the age of 70.  
  
AML is an aggressive disease that requires immediate diagnosis and treatment,
with an average 5 yr survival rate of 28%, depending on a number of clinical
and biologic variables, including acquired genetic alterations within the
leukemic cells. Early treatment of AML generally consists of high-dose
cytotoxic chemotherapy to induce remission, followed by consolidation (i.e.,
post-remission) chemotherapy and/or bone marrow transplantation.

Steadily accumulating genomic evidence shows that certain acquired genetic
alterations within the leukemic cells are strong predictors of prognosis in
AML and, accordingly, are essential factors in the decision whether a patient
should undergo bone marrow transplantation (1-4). These alterations have been
set aside as determinants of independent diagnostic categories in WHO AML
guidelines, and as essential for AML management in NCCN guidelines (5,6).  
  
Importantly, the indication for molecular biomarkers in AML is somewhat
different from other cancers, such as non-small cell lung cancer, in that the
markers themselves are often not the direct targets of treatment. In AML,
these molecular genetic biomarkers are incorporated into a risk-based
treatment stratification that determines whether or not to recommend
transplantation.  
  
Moreover, AML patients often have multiple combinations of these essential
mutations, again in contrast to the mutually exclusive driver oncogene
alterations seen in solid cancers such as non-small cell lung cancer. In AML,
the clinical effect of driver mutations can be modified by the wider genomic
milieu, either additively or interactively (7). Therefore, complete assessment
of AML patients requires testing multiple biomarkers concurrently, rather than
a sequential single-biomarker approach. In this regard, panel testing is
becoming the preferred approach.  

**Myelodysplastic Syndromes (MDS)**

The myelodysplastic syndromes (MDS) represent a spectrum of clonal bone marrow
diseases, with heterogeneous presentations that typically include one or more
cytopenias, defective differentiation of blood cell progenitors into mature
functional cells, and an increased rate of progression to acute myeloid
leukemia (AML). These secondary AML cases carry a worse prognosis than de novo
AML cases. Furthermore, there are myeloid neoplasms that share overlapping
characteristics with both MDS and myeloproliferative neoplasms (MPNs), such as
chronic myelomonocytic leukemia (CMML). The World Health Organization (WHO),
has designated these diseases separately as MDS/MPNs, distinct from either MDS
or MPNs (1).  
  
According to the 2016 National Comprehensive Cancer Network (NCCN) Guidelines,
the overall incidence of MDS is approximately 5/100,000 per year, primarily in
adults. MDS is rare in patients under the age of 40, but much more common in
older patients, with incidence of 30/100,000 among ages 70-79, and 60/100,000
in patients 80 years and older (2).  
  
MDS treatment can range from surveillance/observation to high dose
chemotherapy and bone marrow transplantation, with the principal determining
factors being the patient's overall health and co-morbidities, and prognostic
categorization.  
  
MDS has historically been classified by a combination of traditional
laboratory techniques, such as demonstration of stable cytopenias by complete
blood count, microscopic examination of a bone marrow biopsy, and bone marrow
cytogenetic studies. Other than the clinical feature of the number of
cytopenias and specific cytogenetic changes found recurrently in MDS, all
other diagnostic criteria in MDS rely upon light microscopy findings. These
include the number of cell lineages (i.e., platelets, red blood cells, white
blood cells) affected by dysplasia, the percentage of immature "blast" cells,
and the presence or absence of a characteristic pattern of iron deposition in
immature red blood cells called ring sideroblasts. Low risk MDS is associated
with dysplasia affecting only one cell lineage, with or without ring
sideroblasts, and isolated large deletions involving chromosome 5 (5q-). High
risk disease is associated with dysplasia across multiple lineages, increased
blast percentages, and complex karyotype. With the exception of SF3B1
mutations (see below), no specific mutations are incorporated into the current
diagnostic criteria of MDS.

However, evidence has steadily accumulated over the first two decades of the
21st century showing that certain specific acquired genetic alterations within
the myeloid cells are strong predictors of prognosis in MDS and, in the case
of SF3B1, are necessary diagnostic markers as well (3-9). In addition, other
somatic alterations may support the diagnosis of MDS in certain contexts. MDS
can be challenging to diagnose, due to the subjective morphologic assessment
of dysplasia, and a multitude of benign reactive conditions that can manifest
as peripheral cytopenias and cytologic atypia that are especially prevalent in
the elderly population. In this regard, the demonstration of these clonal
molecular alterations in clinically and/or morphologically ambiguous cases can
help establish a diagnosis of MDS and expedite therapy earlier in the disease
course, before progression to a more overt, and life-threatening, condition.
Accordingly, a number of specific genetic alterations are included in the NCCN
guideline recommendations as necessary for the diagnosis and management of
patients with MDS.  
  
Forty-seven different gene mutations have been identified as recurring
findings in MDS, including TET2, SF3B1, ASXL1, DNMT3A, SRSF2, RUNX1, TP53,
U2AF1, EZH2, ZRSR2, STAG2, CBL, NRAS, JAK2, SETBP1, IDH1, IDH2, and ETV6.
While some are more common than others, no single gene has been reported in
more than approximately one-third of cases. Most of these are useful as
adjunctive diagnostic markers for clinically/microscopically ambiguous cases,
to help establish a more firm diagnosis and, potentially, as markers of clonal
disease that can be used to monitor for disease progression and response to
interventions.

**Myeloproliferative Neoplasms (MPN)**

The myeloproliferative neoplasms (MPNs) represent a group of rare clonal bone
marrow diseases, have a median age at onset of 65-70 years, and heterogeneous
presentations that typically include overgrowth of one or more of the myeloid
cell lineages in the marrow, with increased circulation of mature forms in the
peripheral blood, and an increased rate of progression to acute myeloid
leukemia (AML). Symptomatology varies between the different diseases,
typically related to the specific proliferating cell lineages.

MPNs can be subdivided in two main categories based upon the presence or
absence of BCR-ABL1: chronic myeloid leukemia (CML) and non-CML MPNs.2 The
main non-CML MPNs, also termed classical MPNs, including essential
thrombocythemia (ET), primary myelofibrosis (PMF), and polycythemia vera (PV),
had traditionally been defined by laboratory criteria such as thrombocytosis,
marrow fibrosis, and erythrocytosis, respectively. Non-CML MPN treatment can
range from observation to targeted therapies to hematopoietic stem cell
transplantation (HCT), with the principal determining factors being the
patient's overall health and co-morbidities, presence of fibrosis or increased
blasts, molecular and prognostic categorization. Other less common diagnostic
entities within the non-CML MPNs include chronic neutrophilic leukemia (CNL)
and chronic eosinophilic leukemia (CEL).

While the definitions of the entities within MPN are fairly distinct, in
practice, phenotypic overlap is common, and definitive classification can be
challenging based solely on clinical grounds and traditional laboratory tests,
such as complete blood count (CBC) and bone marrow biopsy. At diagnosis, the
discrimination from reactive conditions is often critical, and the
demonstration of several specific clonal molecular alterations in clinically
or morphologically ambiguous cases can expedite an MPN diagnosis before
progression to a more overt, life-threatening, condition. Accordingly, one or
more MPN-restricted, driver mutations are included in practice guideline
recommendations as necessary for the diagnosis or management of patients with
MPN or MPN-like conditions, including Janus kinase 2 (JAK2), calreticulin
(CALR), and myeloproliferative leukemia virus (MPL).5-7 Driver mutations are
usually mutually exclusive.

JAK2 V617F mutations in exon 14 are the _sine qua non_ of PV, reported in >90%
of cases. Another 2-3% have missense mutations or small insertion/deletions in
a secondary hotspot of JAK2, in exon 12.8,9 The JAK2 V617F mutation is not
specific within the MPNs, however, and has also been reported in approximately
40-60% of cases of ET and PMF. The JAK2 mutation is not present, however, in
CML or in benign conditions that lead to secondary erythrocytosis and its
presence is invaluable in this differential diagnosis.

Mutations in CALR exon 9, by contrast, are encountered in approximately 20-35%
of patients with ET and PMF and are not characteristic of PV. Two alterations
predominate: a 52 bp deletion (Type 1) that is more common in PMF or post-ET
myelofibrosis, and a 5 bp insertion (Type 2) that is more common in ET,
associated with more indolent disease and lower risk of thrombosis, despite an
association with extremely high platelet counts. Beyond enabling a specific
diagnosis, the presence of a CALR mutation portends a favorable prognosis in
PMF (overall survival (OS) 17.7 years vs. 3.2 years with no mutations
("triple-negative" patients), and these patients have a lower rate of
progression to AML over 10 years (9.4% vs. 34.4%).10 CALR mutation also is
associated with improved overall survival (82% vs. 56% at 4 years) and non-
relapse mortality (7% vs. 31%) following transplantation for PMF.1 The
significance of CALR mutations in ET is dependent upon the type of mutation,
as mentioned above, and less clear.

MPL mutations are another sequence alteration seen infrequently in ET and PMF,
and predicts an increased risk of transfusion dependence in PMF.10 MPL
mutations, like JAK2 and CALR, portends a favorable prognosis across MPN, when
compared to triple negative MPN (approximately 10%) that lack mutations in any
of these three genes.1

In contrast to the favorable prognosis conferred by MPL, CALR, and JAK2, a
number of other not MPN-restricted, non-driver, mutations are considered "high
risk" for progressive disease and are associated with both a shorter OS and
leukemic-free survival in PMF, including ASXL1, TET2, EZH2, SRSF2, SF3B1,
SH2B3, U2AF1, TP53, IDH1, and IDH2.1,5,6 These co-mutated, myeloid genes
additively contribute to phenotypic variability and shifts, as well as
progression to more aggressive disease.6 Mutations in at least one of these
genes confers a shortened median survival (81 vs. 148 months),11 and the
presence of 2 or more of these mutations reduces median survival from 12.2
years to 2.6 years.12 However, a study of 570 patients with PMF demonstrated a
counterbalancing effect of combining a low-risk mutation (CALR) with a high-
risk mutation (ASXL1); the median OS was longest in CALR(+)/ASXL1(-) patients
(10.4 years), shortest in CALR(-)/ASXL1(+) patients (2.3 years), and
intermediate (5.8 years) when both are present or absent.3 In triple-negative
patients, these non-driver mutations serve as a major diagnostic criteria for
both PMF and ET. WHO and NCCN guidelines recommend analysis of mutations in
these genes to assist in establishing an MPN diagnosis in the absence of
mutations in MPL, CALR, and JAK2.1,2

Moreover, patients with progressive MPNs are candidates for several different
therapies, including hypomethylating agents, induction chemotherapy, HCT, and
clinical trials. Selecting between these options is a complex determination
that includes several factors, and NCCN recommends the analysis of mutations
in ASXL1, EZH2, IDH1/2, SRSF2, and TP53 (category 2A) to assist in risk
stratification and treatment planning in select PMF patients.1 Monitoring
response to treatment is recommended every 3-6 months with additional
molecular testing reserved for MF patients with INT-1-risk or INT-2-risk/high-
risk disease to aid in decision-making regarding allogeneic HCT.1

**Acute Myelogenous Leukemia (AML)**  
The spectrum of genetic abnormalities that are relevant in AML is broad, and
includes specific sequence variants within genes, copy number changes, and
structural variants such as chromosomal translocations. Smaller scale
mutations require a molecular diagnostics method (e.g., sequencing) for
analysis, while larger scale chromosomal abnormalities may be analyzed using
either molecular diagnostics or cytogenetics (e.g., FISH, karyotype) methods.
Molecular diagnostics and cytogenetic testing play a complementary role in
helping refine prognosis, particularly in cytogenetically intermediate risk
normal karyotype AML (NK-AML), or those with core binding factor where KIT
mutations help refine the prognosis (6,8). The following molecular genetic
biomarkers are considered necessary for diagnosis and management of AML.  
  
**Ta** **ble 1** Biomarkers that require a molecular diagnostics method
(either via panel or individually):

**Gene  ** | **Alteration  ** | **Clinical Utility  ** | **NCCN Biomarkers
Category**  
---|---|---|---  
_CEBPA_   | Mutation  | Favorable risk  | 2A  
_FLT3_ | Internal tandem duplication  | Poor risk  | 2A  
_KIT_   | Mutation  | Intermediate risk  | 2A  
_NPM1_ | Insertion mutation  | Favorable risk  | 2A  
_TP53_ | Mutations, deletions  | Poor risk  | 2A  
_RUNX1_ | Mutation  | Distinct Diagnostic Category; Poor prognosis  | *  
* WHO 2016 AML Classification |   |    
  
These variants represent essential determinants of prognosis and therapy. As
additional genetic variants are shown to similarly lead to safe and effective
therapy selection and, therefore, meet Medicare coverage guidelines,
additional genes may be added to Table 1.  
  
**Table 2** Biomarkers that can be assessed by either a molecular diagnostics
method (panel only) or by a cytogenetics method:

**Gene  ** | **Alteration  ** | **Clinical Utility  **  
---|---|---  
_PML-RARA_ | Rearrangement  | All-trans retinoic acid  
_BCR-ABL1_ | Rearrangement  | Poor risk  
_CBFB-MYH11_ | Rearrangement  | Favorable risk  
_DEK-NUP214_ | Rearrangement | Poor risk  
_MLLT3-KMT2A_ | Rearrangement  | Intermediate risk  
_Other KMT2A_ | Rearrangements  | Poor risk  
_GATA2, MECOM_ | Rearrangement  | Poor risk  
_RUNX1-RUNX1T1_ | Rearrangement  | Favorable risk  
Deletion 5, 5q  | Copy number loss  | Poor risk  
Deletion 7, 7q  | Copy number loss  | Poor risk  
Trisomy 8 | Copy number gain  | Intermediate risk  
  


**Myelodysplastic Syndromes (MDS)**

Five of these genes (TP53, EZH2, ETV6, RUNX1, ASXL1) have established
independent value as prognostic determinants, and a mutation in any one of
these five genes indicates more aggressive disease, worsened overall survival,
and confers a prognosis equivalent to one step worse in the Revised
International Prognostic Scoring System (IPSS-R) (3-4). Several other genes,
including DNMT3A, U2AF1, SRSF2, CBL, PRPF8, SETBP1, and KRAS, have been
associated with decreased survival, although are not formally incorporated
into treatment determinations per 2016 NCCN guidelines. Mutations in TET2 have
been associated with improved response rate to hypomethylating agents, but not
with response duration or overall survival and, therefore, alterations in this
gene are not currently recommended as sole determinants of treatment with this
class of agents. Other mutations associated with response to specific
therapies (but not yet FDA approved) include SF3B1 mutations, which are
associated with a favorable response to DNA methyl transferase inhibitors
(DNMTI), SRSF2 mutations, which are associated with a poor response to DNMTIs,
and TP53, which is associated with a poor response to lenalidomide.

Mutations in SF3B1 are associated with MDS with ringed sideroblasts
(previously designated as refractory anemia with ringed sideroblasts (RARS)),
which confers a favorable prognosis. In the revised WHO classification, this
diagnosis is established by the finding of at least 15% ringed sideroblasts by
light microscopy or by the presence of an SF3B1 mutation and at least 5%
ringed sideroblasts.  
  
The spectrum of genetic abnormalities that are relevant in MDS is broad, and
includes specific sequence variants within a large number of genes as well as
a wide range of aberrations in other genes. While it is possible to assess the
former of these with single gene assays, it is functionally impractical given
the number of hotspot variants and number of genes. The latter require more
complete coverage of the entire gene. In this regard, MDS is an appropriate
indication for multiplexed sequencing, which is typically performed by next
generation sequencing. The following molecular genetic biomarkers are
considered necessary for diagnosis and management of select MDS.

**Gene** | **Clinical Utility** | **NCCN Biomarkers Category**  
---|---|---  
     TP53 | Poor risk | 2A  
     EZH2 | Poor risk | 2A  
     ETV6 | Poor risk | 2A  
     ASXL1 | Poor risk | 2A  
     RUNX1 | Poor risk | 2A  
     SF3B1 | Low risk; diagnosis of MDS with ringed sideroblasts/RARS | 2A  
  


In total, the required nucleotide or small insertion/deletion variants occur
at numerous specific loci in six genes. These variants represent essential
determinants of prognosis and therapy. As additional genetic variants are
shown to similarly lead to safe and effective therapy selection and,
therefore, meet Medicare coverage guidelines, additional genes may be added to
the above table.

**Myeloproliferative Neoplasms (MPN)**

 The following molecular genetic biomarkers are considered necessary for
diagnosis and management of select MPNs.  

**Gene** | **Clinical Utility** | **NCCN Category**  
---|---|---  
_JAK2_ | Diagnosis, intermediate prognosis, higher risk of thrombosis | 2A  
_CALR_ | Diagnosis, improved survival, lower risk of thrombosis | 2A  
_MPL_ | Diagnosis, intermediate prognosis, lower risk of thrombosis | 2A  
Triple negative | Inferior survival | 2A  
_ASXL1_ | Diagnosis, Inferior survival, increased progression to leukemia | 2A  
_EZH2_ | Diagnosis, Inferior survival | 2A  
_IDH1/2_ | Diagnosis, Inferior leukemia-free survival | 2A  
_SRSF2_ | Diagnosis, Inferior survival, increase progression to leukemia | 2A  
_TP53_ | Diagnosis, Leukemic transformation | 2A  
  


In total, the required nucleotide or small insertion/deletion variants occur
at numerous specific loci in eight genes. These variants represent essential
determinants of prognosis and associated therapy. As additional genetic
variants are shown to similarly lead to safe and effective therapy selection
and, therefore, meet Medicare coverage guidelines, additional genes may be
added to the above table.