**History/Background and/or General Information**

**I. Lynch Syndrome (LS)**

This local coverage determination limits Lynch syndrome (LS) genetic testing
to a stepped approach for Microsatellite Instability and Immunohistochemistry
(MSI/IHC) screening, _BRAF_ gene mutation, _MLH1_ gene promoter
hypermethylation and targeted mismatch repair (MMR) germ-line gene testing to
patients suspected of having LS.

Most colorectal cancer is caused by non hereditary somatic mutations.
Individuals with LS (aka Hereditary nonpolyposis colorectal cancer (HNPCC) are
predisposed to cancer due to having inherited or de novo germ-line mutations
in DNA repair genes, that result in an accelerated accumulation of somatic
mutations. LS, the most common hereditary cause of colorectal cancer, accounts
for 2-3% of all colorectal cancers, followed by familial adenomatous polyposis
(FAP) which accounts for <1% of colorectal malignancies and MUTYH-associated
polyposis (MAP) whose frequency of occurrence is very rare.

LS is an autosomal dominant familial cancer syndrome caused by mutations in
multiple susceptibility genes (e.g., _MLH1, MSH2, MSH6, PMS2, EPCAM)_ , and is
associated with an increased lifetime risk for colorectal cancer (CRC) and
other malignancies within the tumor spectrum including at least endometrial,
ovarian, gastric, small bowel, urothelial, hepatobiliary tract, sebaceous and
pancreatic cancers. Current literature suggests LS annually affects 28,000
individuals. In individuals with LS, the lifetime risk of colon cancer may be
as high as 75% by the age of 70 years, with an average age onset of 45 years
in _MLH1_ and _MSH2_ mutation carriers. While the incidence of adenomas in
individuals with LS is similar to that in the general population, the high
rate of colorectal cancer is due to an acceleration of the adenoma to
carcinoma sequence.

Cancer risks associated with LS are largely derived from family studies.
Mutations in _MLH1_ and _MSH2_ account for 70-90% of families with LS. The
risk of colon and endometrial cancer is less in _MSH6_ and _PMS2_ mutation
carriers, although the cancer risk may not be lower for _MSH6_ carriers if one
takes the data out to age 80. While individuals with a single _MLH1, MSH2,
MSH6_ and _PMS2_ mutation develop cancers in mid-life, individuals with
biallelic _MLH1, MSH2, MSH6_ and _PMS2_ mutations have a distinctive phenotype
and tumor spectrum, and often develop cancer as early as the first decade of
life.

First-degree relatives of mutation carriers have a 50% probability of having
the same germ-line mutation. Despite the high penetrance of CRC and
endometrial cancer and recommendations of consideration for screening
unaffected first-degree relatives following diagnosis of an LS proband,
testing of genetic carriers who are unaffected with a Lynch related cancer is
not a benefit, and is statutorily excluded from coverage.  
  
**II. Testing Strategy for Patients with Personal History of Colorectal Cancer  
  
Step 1: Patient selection**

Patients with colorectal and/or endometrial cancer suspected of LS must
undergo a comprehensive review of physical findings and a complete personal
and family history.

In 1989, the Amsterdam criteria defined what is known as Hereditary Non-
polyposis Colon Cancer Syndrome, and in 1999, the criteria were revised to
include extra-colonic tumors (Table 1). Today we know there are two distinct
groups comprising HNPCC: those with hereditary DNA mismatch repair germ-line
mutations, known as Lynch Syndrome, and those with normal DNA mismatch repair,
known as Familial Colorectal Cancer Type X.  
  
________________________________________  
 **Table 1. Amsterdam Criteria II (ACII)**  
________________________________________  
  
There should be at least three relatives with CRC or with a Lynch syndrome-
associated cancer: endometrial, small bowel, ureter or renal pelvis cancer.

  * One relative should be a 1st-degree relative of the other two,
  * At least two successive generations should be affected,
  * At least one tumor should be diagnosed before age 50 years,
  * FAP should be excluded in the CRC case, if any,
  * Tumors should be verified by histopathological exam

Approximately 50% of families meeting the ACII criteria have a mutation in an
MMR gene. However, these criteria are very stringent and miss as many as 68%
of patients with LS.

In 1997, the Bethesda guidelines were developed to identify individuals with
CRC who should be tested for MSI. In 2002, the guidelines were revised (Table
2) to clarify selection criteria for microsatellite instability (MSI) testing
and mismatch repair (MMR) protein expression by immunohistochemistry (IHC).
Screening tumors of patients meeting the Bethesda guidelines for MSI was shown
to be cost-effective with newly diagnosed CRC.

  
________________________________________  
 **Table 2 - Revised Bethesda Guidelines**  
________________________________________  
  
Meeting any of the following are sufficient for consideration of MSI/IHC
testing

  * CRC diagnosed under age 50
  * Presence of synchronous, or metachronous CRC or other Lynch-associated tumor, regardless of age
  * CRC with MSI-H histology diagnosed in an individual who is < age 60
  * CRC diagnosed with one or more 1st-degree relatives with a Lynch-related tumor, with one of the cancers diagnosed under age 50
  * CRC diagnosed in two or more 1st- or 2nd-degree relatives with a Lynch-related tumor, regardless of age

If a patient meets standards for LS testing in Step 1 (i.e., meets ACII or
Revised Bethesda guidelines), the physician should proceed to Step 2 and 3.  
  
**Step 2: Immunohistochemistry (IHC) testing for LS Screening**

The use of IHC to detect loss of DNA mismatched repair (MMR) protein
expression complements MSI to screen patients for defective MMR (DMMR),
including both sporadic dMMR and LS dMMR. IHC allows detection of loss of
protein expression for the _MLH1, MSH2, MSH6_ and _PMS2_ genes. Loss of MMR
protein expression is detected by the absence of nuclear staining in the tumor
cells and the presence of nuclear staining in lymphocytes and normal colon
crypt epithelial cells.

The MMR proteins are present as heterodimers ( _MLH1_ pairs with _PMS2_ , and
_MSH2_ pairs with _MSH6_ ). Knowledge of MMR protein expression loss patterns
allows a logical and cost effective "directed" testing appropriate for germ-
line mutation analysis. As a general rule, loss of expression of _MLH1_ or
_MSH2_ is associated with loss of their partners. For example, mutation of the
_MLH1_ gene generally leads to loss of expression of both the _MLH1_ and
_PMS2_ proteins. However, loss of _PMS2_ or _MSH6_ due to a germ-line mutation
is associated only with loss of the mutated protein. For example, mutation of
the PMS2 gene leads to loss of expression of only the _PMS2_ protein.

If IHC is done first and is abnormal, MSI testing is not warranted. Often IHC
is done first because of its rapid turn-around and minimal amount of tissue
required. If IHC demonstrates loss of protein expression for the _MLH1, MSH2,
MSH6_ and _PMS2_ genes, the following test results direct further testing:

  * _MLH1_ loss by IHC, test for _BRAF_ gene mutation (Step 4) or test for _MLH1_ promoter, (Step 5)
  * _MSH2/MS6_ loss by IHC, perform _MSH2_ germ-line testing (Step 6)

If IHC test results are normal, there remains a small chance of high levels of
microsatellite instability (MSI-H), so both IHC and MSI would be needed to
rule out LS in a clinically suspicious setting.  
  
 **Step 3: Microsatellite Instability (MSI) Analysis for LS Screening**

MSI analysis for screening LS microsatellites are short repeated segments of
DNA spread throughout the genome. Under normal conditions, the MMR gene
complex ( _MLH1, MSH2, MSH6_ and _PMS2_ genes) corrects mismatched base pairs
that occur during the final stage of DNA replication. When the MMR complex is
functioning normally, all cells show an identical pattern of microsatellite
lengths. When the MMR complex is non-functioning, due to two hits of any type,
random mutations accumulate in microsatellites, leading to differences in
microsatellite lengths (microsatellite instability, MSI). Therefore, MSI
indicates loss-of-function defects in a MMR protein, which may be due to
somatic mutations, germ-line MMR gene mutations, allelic loss, or to
epigenetic down-regulation. MSI is usually associated with absence of protein
expression of one or more of the MMR proteins ( _MLH1, MSH2, MSH6_ and _PMS2_
).

DNA from paraffin-embedded tumor tissue and normal tissue or peripheral blood
is used for MSI analysis. A microsatellite is considered unstable if the
distribution of the tumor fragments differs from that of the normal tissue.
Noncancerous tissue in individuals with LS does not show MSI because normal
tissue is heterozygous for the germ-line mutation.

Levels of MSI in colon tumors are classified as:

 **MSI-H** \- >30% or more of a tumor's markers are unstable;  
 **MSI-L** \- > one but < 30% of a tumor's markers are unstable;  
 **MSS** \- no loci are unstable.

MSI-L and MSS indicates the MMR mechanism is functioning adequately. Virtually
all CRC tumors from individuals with LS demonstrate MSI-H. However, MSI-H is
NOT diagnostic of LS as MSI-H can be observed in roughly 15% of sporadic
colorectal cancers. In other Lynch tumors, the % level of MSI-H is less
consistent and is inadequately studied.

As indicated above, MSI testing is not necessary if IHC demonstrates loss of
protein expression for the _MLH1, MSH2, MSH6_ and _PMS2_ genes. If IHC test
results are normal, there remains a small chance of high levels of
microsatellite instability (MSI-H), so both IHC and MSI should be performed to
rule out LS in a clinically suspicious setting such as meeting a Revised
Bethesda guideline. Additionally, some individuals with MSH6 germ-line
mutations do not manifest the MSI-H phenotype. This finding supports the
diagnostic strategy to screen suspected LS patients with CRC by both MSI and
IHC. Immunohistochemistry (IHC) can be used to identify whether the protein
products of _MLH1, MSH2, MSH6_ and _PMS2_ genes are present or absent.
Individuals with tumors that display high levels of MSI or loss of expression
of MMR proteins by IHC are then referred for targeted germ-line mutation.

Steps 4 and/or 5 apply only for tumors that are negative for _MLH1_ protein
expression by IHC.  
  
**Step 4: BRAF V600E (BRAF) Mutation Testing  **

_BRAF_ mutation testing and _MLH1_ promoter methylation studies distinguish
between sporadic dMMR and LS dMMR. This is because _BRAFM_ mutation and _MLH1
PHM_ are very seldom seen in LS. _BRAF_ mutation testing of the CRC tumor is
associated with the presence of an epigenetic alteration (i.e.,
hypermethylation of _MLH1_ ) and either finding excludes germ-line MMR gene
mutation (e.g., LS).  
  
**Step 5: MLH1 Promoter Hypermethylation (MLH1 PHM)**

The combination of _MLH1 PHM_ and a _BRAF_ mutation in tumors rules out LS and
no further molecular analysis is warranted. Tumors with _MLH1 PMH_ identify
dMMR which will most often be sporadic, but its presence does not fully rule
out LS. However, there have been rare reports of _MLH1_ hypermethylation as a
second hit in LS and there are new reports of constitutional MLH1 methylation.
As a rule, discovery of _MLH1 PHM_ indicates the tumor is not due to Lynch
syndrome.

The following combinations of _BRAF_ and _MLH1_ promoter methylation test
results direct further testing in individuals with CRCs with loss of IHC
expression of _MLH1/PMS2_ :

  * If _BRAF_ mutation is present, no further testing is medically necessary; LS is ruled out.
  * If _BRAF_ mutation is absent, _MLH1_ promoter methylation testing is indicated and directs the following testing: 
    * If _MLH1_ is hypermethylated, germline _MLH1_ is not medically necessary.
    * If the MLH1 promoter is hypermethylated and ACII if fulfilled, germ-line _MLH1_ may still be considered (2nd hit scenario). 
    * IF the _MLH1_ promoter is normally methylated, and _BRAF_ is negative for mutation then germ-line _MLH1_ testing is medically indicated. 

**Note** : There is variability in laboratory preference for _BRAF_ and MLH1
promoter testing sequence. Although _BRAF_ is generally cheaper and faster,
some labs test _MLH1_ PHM first because it is more sensitive for detection of
sporadic dMMR.

In a study by Gausachs (2012), when _MLH1_ PHM testing is used in conjunction
with _BRAF_ mutation testing, the cost per additional mutation detected when
using hypermethylation analysis was lower than that of _BRAF_ and germinal
_MLH1_ mutation analysis. Somatic hypermethylation of _MLH1_ is an accurate
and cost-effective pre-screening method in the selection of patients that are
candidates for _MLH1_ germ-line analysis when LS is suspected and _MLH1_
protein expression is absent.  
  
 **Step 6: Targeted MMR ( _MLH1, MSH2, MSH6_ and _PMS2_ gene) Germ-line and
EpCAM Testing  
  
Step 6A: _MLH1_ Testing**

When IHC shows loss of both _MLH1_ and _PMS2,_ further genetic testing of
_PMS2_ is not indicated, as no cases have been reported of a _PMS2_ germ-line
mutation when IHC showed a loss of both _MLH1_ and _PMS2_. _PMS2_ mutations
have only been detected when IHC shows a loss of _PMS2_ only. If _MLH1_ gene
mutation germ-line is positively identified, then LS is diagnosed and further
testing of the patient is not medically necessary.  
  
**Step 6B: _MSH2_ Testing**

When IHC shows loss of _MSH2_ and _MSH6_ , genetic testing should start with
analysis of the _MSH2_ gene, given its frequency of germ-line mutation in LS.
If _MSH2_ germ-line mutation is identified, then LS is diagnosed, and further
testing of the patient is not medically necessary.

However, if genetic testing for germ-line mutations in _MSH2_ is negative,
analysis for deletion in the EpCAM gene should be performed (Step 7). If EpCAM
is also negative, genetic testing of _MSH6_ should be performed (Step 6C). The
presence of MSI and the loss of _MSH2/MSH6_ strongly indicate a MMR germ-line
defect.  
  
**Step 6C: _MSH6_ Testing**

When IHC shows loss of just MSH6, it suggests a germ-line mutation in _MSH6_
and genetic testing of that gene is indicated. As previously noted, _MSH6_ CRC
tumors can be MSI-H, MSI-L or MSS. This pitfall illustrates the utility of IHC
for MMR protein expression. If _MSH6_ germ-line mutation is identified, then
LS is diagnosed, and further testing of the patient is not medically
necessary.  
  
**Step 6D: _PMS2_ Testing**

If IHC shows _PMS2_ loss only, germ-line testing for _PMS2_ mutations is
indicated. No cases of a _PMS2_ germ-line mutation have been identified after
IHC showed a loss of both _MLH1_ and _PMS2_. If _PMS2_ germ-line mutation is
identified, then LS is diagnosed, and further testing of the patient is not
medically necessary.  
  
 **Step 7: _EpCAM_ Testing**

Recently, deletions in a portion of the _EpCAM_ gene were found in a subset of
families with LS with a loss of _MSH2_ by IHC. A common deletion in the 3'
region of _EpCAM_ causes somatic hypermethylation of _MSH2_ , as the 2 genes
are adjacent to one another on chromosome 2. Approximately 20% of patients
with absence of _MSH2_ and _MSH6_ protein expression by IHC, but without MSH2
or MSH6 mutation, will have germ-line deletions in _EpCAM_. Early estimates
suggest that germ-line mutations in _EpCAM_ may account for approximately 6%
of LS cases and possibly as high as 30% when IHC shows a loss of _MSH2_.  
  
**Note:** Many labs incorporate _EpCAM_ detection their _MSH2_ dup/deletion
analysis.  
  
 **Covered Indications**

**IHC and/or MSI Testing**

LS tumor screening with IHC or MSI on colorectal and/or endometrial tumors is
considered medically necessary and covered for the following indications:

  * All Individuals with colorectal cancer diagnosed at 70 years of age, and those >70 years of age who meet the revised Bethesda guidelines**, **OR  **
  * Individuals with endometrial cancer.

For coverage, the treating physician/pathologist is expected to follow the
stepped approach outlined for LS screening and targeted MMR testing in this
policy. Germ-line testing includes sequence and duplication-deletion analysis
for a given gene.  
  
**MMR Germline Gene Mutation Testing Exception**

If a lab is unable to perform the stepped testing approach outlined in this
LCD, multiple germ-line gene testing will be covered only for one or more of
the following findings:

  * MSI/IHC testing yields normal IHC and MSI-H, suggesting LS 
  * If tumor is not available or determined by a pathologist to be inadequate to assess DNA MMR deficiency by MSI or IHC, then MMR germ-line testing can be conducted on blood if the individual fulfills the ACII or revised Bethesda guidelines. 
  * CRC tumor diagnosis prior to eligibility **AND** tumor sample no longer available **AND** individual meets ACII or revised Bethesda guidelines or was diagnosed with endometrial cancer before 50

If targeted gene testing is not possible, _MLH1_ and _MSH2_ testing should be
performed first, since these two genes account for the majority of germ-line
mutations. If no mutation is identified in _MLH1_ or _MSH2_ , testing of
_MSH6_ is indicated. If no mutation is identified in _MSH6_ , testing of
_PMS2_ may be considered.  
  
**Testing for Known Familial Variant**

Testing for a specific known familial variant is considered medically
necessary and covered only when the individual being tested has signs and
symptoms of a Lynch-associated cancer AND has a blood relative with the
specific disease-causing mutation for LS.  
  
 **Note:** This LCD does not imply that testing family members of a known
familial variant is not medically warranted. The scope of the benefit requires
the beneficiary to have signs and symptoms of disease. Coverage of molecular
testing for LS for carrier status or family studies is considered screening
and is statutorily excluded from coverage.  
  
 **Limitations**  
  
 _Universal Testing for CRC and Endometrial Cancer_

Universal testing of CRC and endometrial cancers by MSI/MMR protein expression
by IHC is not a benefit. The NCCN colorectal cancer screening guidelines
(V2.2013) recommends that "risk assessment be individualized and include a
careful family history ... and if a patient meets the criteria for an inherited
colorectal syndrome, further risk evaluation and counseling, as outlined in
the guidelines, is required. The guidelines indicates that "when any one of
the revised Bethesda criteria are met, the possibility of LS is suggested, and
IHC staining for the four MMR proteins and/or MSI testing on the colon tumor
of the youngest affected family member is warranted."  
  
The NCCN colon cancer treatment guidelines (V3.2013) "recommend that MMR
protein testing be performed for all patients younger than 50 years with colon
cancer, based on an increased likelihood of LS in this population. MMR testing
should also be considered in all patients with stage II disease, because stage
II MSI-H patients may have a good prognosis and stage II MSI-H patients do not
benefit from 5-FU adjuvant therapy."  
  
Similarly, although the Evaluation of Genomic Applications in Practice and
Prevention (EGAPP) Working Group found sufficient evidence in 2009 to
recommendation offering genetic testing for LS to individuals with newly
diagnosed CRC to reduce morbidity and mortality in relatives, molecular
testing for LS for identify carrier status or family studies is not a benefit.

The ordering/treating physician or pathologist is expected to obtain
sufficient clinical and family history to warrant first-line testing
(IHC/MSI), and subsequent targeted MMR germ-line testing or for germ-line
mutation exceptions (as above). The clinical/family data to support IHC/MSI
testing should be documented in the test interpretation/report and the
information should be available to the lab performing targeted testing to
assist the lab in the appropriate selection of target genes. Labs performing
MMR germ-line panels without appropriate selection of targeted genes based on
patient data, screening test (MSI/IHC) results, or exceptions are not
reasonable and necessary.

At the current time, there is insufficient data to warrant MMR testing for
prostate cancer, even though preliminary studies suggest that prostate cancer
in MMR gene mutation carriers share a molecular profile and at least one
pathological feature in common with other LS-associated tumors. Similarly the
clinical significance of MMR testing in other malignancies is not known.
Therefore, molecular testing for malignancies other than those specifically
cited in this LCD is non-covered.



As published in the CMS IOM Publication 100-08, _Medicare Program Integrity
Manual_ , Chapter 13, Section 13.5.4, an item or service may be covered by a
contractor LCD if it is reasonable and necessary under the Social Security Act
Section 1862 (a)(1)(A). Contractors shall determine and describe the
circumstances under which the item or service is considered reasonable and
necessary.