Frequently Asked Questions

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BRAF Negative

1. If BRAF is negative for mutation when there is loss of MSH1/PMS2 staining, and there is no germ-line mutation found. Do you then do hypermethylation testing? Should hypermethylation testing be done before germ-line testing?
  • Germline for MLH1 first (unless they are older with no family history) and then circle back to methylation testing at the end
    I. You should do methylation testing before PMS2 sequencing
    II. The recent publication from the colon CFR recommended that PMS2 genetic testing is not indicated when loss of MLH1/PMS2 occurs http://www.ncbi.nlm.nih.gov/pubmed/23017166I
  • If Methylation comes back negative then to be on the safe side, we would follow like Lynch Syndrome and highlight that we will likely have a better understanding of what to do in the future so check back with us.
2. MLH1/PMS2 absent on IHC with some family history of cancer. Negative for BRAF mutation. Adenomas and hyperplastic polyps found during colonoscopy showing dysplasia. Hypermethylation studies, MLH1 sequencing and del/dup were all normal. If calling this Lynch then the surgeon wants to do a colectomy. What are your thoughts?
  • Biallelic somatic MLH1 mutations in the tumor. Cannot test to prove it so you have to treat them like they have LS.
    I. Sourrouille, Isabelle, et.al. “Somatic Mosaicism and Double Somatic Hits Can Lead to MSI Colorectal Tumors.” Familial Cancer 12.1 (2013): 27-33.
    II. Repeat IHC since there is so much riding on it being correct.
  • It is my understanding that most centers are treating patients with abnormal MSH2, MSH6, and/or PMS2 on IHC and no germline mutation as if they have Lynch Syndrome and their family members are screened as if they have Lynch Syndrome regardless of the family history.
3. What do you do when elderly colon cancer patients show loss of MLH1/PMS2 and BRAF negative for mutation?
  • A few labs automatically order MLH1 promoter methylation testing when MLH1 is absent, regardless of age or any other criteria.
  • One lab is doing BRAF now and the counselor then sees all the cases that are absent MLH1 and PMS2 and BRAF negative and then decides whether to do MLH1 gene testing vs. methylation first based on the patient’s age and family history.
4. A patient is suspicious for Lynch Syndrome as they had synchronous colon cancer at the age of 37 and the IHC indicated weak staining for MLH1 (only focally positive) and loss of PMS2 expression. BRAF came back negative. PMS2 testing came back negative. MLH1 testing revealed a VUS through Myriad. Ambry had not seen the VUS but indicated their software would call the VUS likely damaging. City of Hope had not seen the VUS. Both parents are negative for the MLH1 variant. Does this patient have Lynch Syndrome? How should this patient be followed? Anyone had or heard of DE NOVO Lynch Syndrome in patients?
  • Yes we have had 2 cases of de novo Lynch Syndrome. There was a CCFR study published a few years ago that estimated approximately 2% of Lynch cases were de novo. http://jmg.bmj.com/content/48/8/530.long
  • Consider non-paternity
  • It does seem like this patient has Lynch Syndrome. I would consider hypermethylation testing to make sure you are not missing a constitutional MLH1 epimutation.
5. MLH1/PMS2 absent on IHC. BRAF negative for mutation. Adenocarcinoma of the transvers colon. Little family history. MLH1 sequencing and del/dup normal. Hypermethylation studies were also normal.

The patient’s FDR should have colonoscopies starting at age 40 and then every 3 to 5 years. If the family history was more suggestive of LS that would change the recommendations. Increased screening for CRC in close relatives seems reasonable but no extracolonic screening in patients with weak personal and family history suggestive of LS.


BRAF Positive

1. If synchronous tumors are found at resection do we assume that the second tumor is due to the same mechanism or if the second tumor should be screened as well?
  • Only need to stain one tumor unless there is a striking family history or if the patient is young
  • You could possibly test both tumors if they were referred specifically for genetic counseling and not just part of universal screening.
2. The patient has stage 3B colorectal cancer in the ascending colon with mucinous features. There was family history that would seem like Lynch Syndrome. Pathology report indicates MSI-H for all 5 alleles. IHC indicates loss of PMS2 staining and equivocal for MLH1. BRAF shows a mutation.Pathology report states: Although the presence of BRAF mutation is often considered to be exclusionary for Lynch Syndrome, the combined morphologic and immunophenotypic features, and finding of MSI-H, in the setting of a personal and family history of cancer, suggest that further evaluation may be warranted.
  • Three people indicated that MLH1 promoter testing would be helpful to truly rule out LS.
  • You could consider IHC on the endometrial tumor. I. Comes back normal it would be okay not to purse further Lynch testing II. Comes back with loss of MLH1 and PMS2 then I would pursue germline testing. III. Wang L, et al. BRAF mutations in colon cancer are not likely attributable to defective DNA mismatch repair. Cancer Research. 2003;63(17):5209-12.
  • I suggest IHC testing on either colon or endometrial tumors for more information. If this was a low risk population case we would stop at BRAF, but since this case has a high prior probability of being Lynch I would not stop because you found a BRAF mutation. I. With these results I would consider offering PMS2 mutation testing. We have found BRAF mutation in tumors from individuals with PMS2 mutations (one case).
  • The Ontario CCFR has several cases of MLH1 germline mutations carriers with somatic BRAF mutations in their colon cancers. I would also do IHC on her endometrial cancer.
  • I am aware of one case in the literature of an MLH1 germline mutation carrier with a BRAF positive CRC. There were also a few BRAF positive CRCs in PMS2 mutation carriers reported in the 2008 Senter paper. Senter L, et al. The clinical Phenotype of Lynch Syndrome due to germ-line PMS2 mutations. Gastroenterology; 2008
  • Can you blame your proband’s cancers on something else, like morbid obesity maybe? The mother and brother meet Chompret criteria for LFS. Maybe it’s possible her cancers are unrelated to theirs? Just a consideration if the uterine tumor studies do not provide reassurance for Lynch Syndrome.

Loss of MSH2 and/or MSH6

1. What are your thoughts on tumors that stain negative for MSH6 only and germline testing for MSH6 comes back normal without a mutation detected.
  • This could be a possible MSH2 mutation.
  • This could be a false positive in patients who received neoadjuvant chemo and radiation. Bao et al. Neoadjuvant Therapy Induces Loss of MSH6 Expression in Colorectal Carcinoma. Am J Surg Pathol 2010;34:1798–1804.
2. Straight forward Lynch case with family history. IHC shows loss of MSH2/MSH6 staining but sequencing and del/dup of both genes came back normal and no mutation was detected.
  • I would say something like this in the report. I. If the pt/fm hx meets Amsterdam, I write something like, “clinical diagnosis of Lynch Syndrome with unidentifiable mutation by current testing”. II. If the pt/fm hx does not meet Amsterdam, I write something like, “probable Lynch Syndrome with unidentifiable mutation by current testing”. I also recommend Lynch screening or consideration of Lynch screening for family members, respectively.
  • Per Hampel et al. 2008 and Senter et al. 2008, the likelihood of identifying an MSH2 mutation in a patient in this clinical scenario is 67%.
  • We would call this patient “presumptive Lynch Syndrome with no mutation identified”, and recommend Lynch Syndrome screening for all close relatives.
3. Patient with an MSH2 mutation but is now having upper GI issues. Endoscopy shows diffuse fundic gland polyps ranging from 2-4mm sessile appearing. Also he has a low grade adenoma consistent with epithelial process at his GE junctions. (10mm fold in the fundus abutting the GE junctions) Are his upper GI symptoms attributed to his MSH2 mutations?

Documented cases of APC mutations in addition to MMR mutations. Mayo ( http://link.springer.com/article/10.1007%2Fs10689-012-9561-3),

4. Is it true that adenocarcinoma of the colon with MSI-L ( all 4 IHC proteins present but MSI was low with only 1 marker unstable ) tumors can be associated with MSH6 germline mutations? Should we start here or MLH1/MSH2 testing?
  • There was one reported case in EC tumor where MSI-L at 1/5 MSI markers were unstable and resulting in a deleterious MSH6 mutation.
  • If this was a universal screening result it would not be pursued. If there is a strong family history might pursue by starting with a panel instead of individual genes.

Other Staining Questions

1. Anyone performing IHC for PMS2 and MSH6 only versus the 4-antibody panel?
  • Usually on endometrial cancers and sometimes used on Biopsies too
  • Stanford published utility of 2 antibody panel. http://www.ncbi.nlm.nih.gov/pubmed/21499234
  • All 4 stains is the package that the lab likes to use and will be the same price regardless. Also they can see the partner proteins to double check the calls. There have been reports of MSH6 not performing well.
2. Testing Algorithm

My understanding is that if MSH6 and PMS2 are present, you stop there.
I. If MSH6 is absent, you run MSH2 to determine if both proteins are absent or just MSH6.
II. If PMS2 is absent, you run MLH1 to determine if both proteins are absent or just MSH6.
III. Therefore, ~80% of cases would only need 2 stains and 20% of cases would end up having 3 stains but that would eliminate a lot of IHC. The testing algorithm would be the same from that point on (e.g. if MLH1 and PMS2 were absent you would reflex to BRAF and/or MLH1 methylation testing).

3. All 4 tumors for IHC show up absent on endometrial tumor. No family history. All controls worked.

Run a gene panel

4. Weak MLH1/PMS2 IHC staining on an endometrial adenocarcinoma.

You could repeat the staining or have them read somewhere else. You could also move to MLH1 hypermethylation studies.

5. Case with loss of MLH1/PMS2 staining on IHC with 30% hypermethylation?

Lab concluded that this should be treated as an abnormal result and to offer genetic testing for MLH1.


Universal Screening Questions

1. What are the common turn around times for IHC and MSI universal tumor testing? Who is communicating the abnormal results?

7 to 10 days for IHC, BRAF takes at least another week, Genetic counselors

2. For those of you doing universal IHC on CRCs, what are you doing for your public aid patients who need germline testing based on the IHC?
  • Cancer Resource Foundation – They have two different voucher systems. I. For patients whose insurance does not cover the testing, the patient can apply for a full coverage voucher that will cover the cost of the testing completely II. If the insurance will cover a portion of the cost but they will have a high out of pocket, they provide up to $500 worth of copay assistance III. The patient needs to meet NCCN criteria for testing and have an income level less than 400% of the poverty level. If you go to their website the monthly and annual income cut offs are listed by number of individuals in the family. IV. Feel free to contact Deb Lundquist, 508-630-2243. She is my go-to resource at the CRF and is so helpful with all of these issues
  • Can use vouchers from Myriad
3. Where do you send your methylation samples on endometrial tumor samples?

Mayo Clinic
ARUP


IHC Informed Consent

1. Does pre-op consent cover additional reflex testing at the time of surgery for colorectal and endometrial cancers.

Informed consent is not necessary because of the following:
I. IHC testing is phenotyping and not genotyping. There are many people with absence of MMR proteins who are not found to have a germline mutation in one of the MMR genes. We do require genetic counseling and informed consent before the genetic testing portion of the process.
II. This is done in Pathology and patients do not generally sign a consent to have pathology done on their tumors.
III. This is very similar to the situation with ER, PR, and Her-2/neu because patients with a triple negative breast cancer are more likely to have a BRCA1 mutation, however, no informed consent is required for ER, PR, or Her-2/neu because it changes prognosis and treatment as does MSI status.
IV. There is one paper from an ethicist which says informed consent is not required for MSI but it *might* be required for IHC. He doesn’t believe personally that it should be required for IHC but the editors beat him up until he stated that so this paper is not too helpful but it’s in Genetics in Medicine and the author is Richard Sharp.
V. A recent survey of multiple institutions doing universal screening found that of those doing IHC, NONE were requiring informed consent. So, if peer pressure helps, that’s another argument (Beamer et al, JCO, April 2012).

  • You can add a very general statement to the pre-op consent form: The tumor may also be tested to provide information about prognosis (a forecast of how the cancer might behave in the future), treatment options, and the genetic basis of the cancer
  • Since these folks had a diagnosis of cancer, they were manifesting disease and the IHC test was aiding in the diagnosis of Lynch
  • A published manuscript on a small survey about informed consent. http://www.ncbi.nlm.nih.gov/pubmed/22355048
2. Do you have a system in place so patients can opt-out of MSI/IHC testing.

Yes! We have an information sheet about our screening initiative that goes out to all CRC and endometrial surgical candidates in the pre-surgical packet. At the bottom of the form there is a telephone number for the Department of Clinical Genetics that encourages them to contact us if they have any concerns or questions about Lynch screening. So far no one has contacted us.

3. Does anyone require consent for universal screening on CRC or EC tumors for LS?

26 responded with No consent
I. MD Anderson, TX – Decided that tumor screening falls under the institutional consent to treat that everyone signs when they are treated there.
II. Department of Veterans Affairs, UT – their ethics committee decided a specific consent was not needed since it was part of the processing of a specimen that a patient has already consented to have sampled.
III. Pro Health Care, WI – Decided that tumor screening falls under the general consent to treat that everyone signs when they are treated there.

  • 2 responded with statements in Pre-op packet I. UPMC, PA added “The tumor may also be tested to provide information about prognosis (a forecast of how the cancer might behave in the future), treatment options, and the genetic basis of the cancer.” to their consent form for endometrial surgeries because the gynecologists requested it, however, the colorectal surgeons approved no consent. II. Riverside Health System in Newport New, VA has a statement on their pre-op consent
  • Intermountain Healthcare has colorectal IHC screening built into the surgical consent for all its institutions with the exception of Utah Valley Regional Medical Center, where consent is required before sending out specimens. There is not yet a standard policy for endometrial IHC.

IHC Biopsy

1. Anyone doing IHC on pre-op biopsy specimens?
  • Perform IHC on all CRC and EC pathology specimen that are processed in laboratory. Doesn’t matter if they are biopsies or surgical resections.
  • A normal IHC screen in a patient with clinical features suspicious for Lynch Syndrome needs to be re-evaluated thoughtfully
  • Concerned about losing patients to follow-up (i.e. not being able to notify those with abnormal IHC) among the biopsy specimens since these can come in from all over the place and won’t be as controlled as the surgical resection cases who have to come back for at least one post-op visit. Of course, there are a lot of pros to doing the biopsy specimens including surgical decision-making and testing the rectal cancers prior to neoadjuvant chemo and RT.
  • We recently published our experience with this at MSKCC– looked at IHC in matched biopsy and resection samples and found high (although not perfect!) concordance. http://www.ncbi.nlm.nih.gov/pubmed/21297438

 


Billing Questions

1. Pathology wants to use a lab that bills insurance to send our universal screening samples to. What is the cost of IHC at other institutions?
  • H1a) Two institutions have found it is easier if the hospital bills a bundled fee (DRG) for everything associated with the surgery. However this means pathology is not able to bill directly to insurance to get reimbursement for the testing.
  • Have pathology ask internal billings/reimbursement experts to look into this before making a decision about vendors.
2. Do Medicare patients meet germline-testing criteria for abnormal follow-up testing on routine endometrial IHC results?
  • Currently, an endometrial proband must meet Amsterdam criteria or have a second Lynch primary for Medicare to cover the cost of Lynch testing. The guidelines state …” This does not apply to MSI or IHC testing of non-GI primary tumors since the sensitivity and specificity of MSI/IHC testing in these tumors is poorly documented at this time.
  • Medicare would pay for germline testing if the patient had a colon or endometrial tumor with evidence of MMR deficiency. (ie MSI-H or IHC expression lost)
  • Medicare will pay for IHC ( if ordered 2 weeks after the Pt’s discharged date). They will not pay for germline testing or promoter methylation testing that is sent out of house based on IHC results alone. Have to meet Medicare guidelines. Patient has to sign an ABN or lab may not accept sample. My understanding of the Medicare situation would make me think that it’s better to order it at the time of the surgery so that it’s not really billed separately but instead it just comes out of the DRG (which is a pre-set total payment made to the hospital for all newly diagnosed endometrial cancer patients to cover their entire in-patient stay). So, this basically just cuts into your hospital profit on these cases. However, Janice just posted that her hospital is having her order it separately 2 weeks post-surgery (probably to avoid the DRG) and I would think it would then be subject to the family history guidelines but she seems to be having good reimbursement so who knows.
  • The following endometrial cancer patients do meet Medicare guidelines for genetic testing of all of the MMR genes: I. Diagnosed prior to age 50 II. Have a second primary LS cancer (meaning they meet Bethesda guidelines) III. Meet Amsterdam criteria II IV. Have a relative with a known MMR mutation
  • One way to get germline testing for Lynch Syndrome on someone who does not technically meet Medicare guidelines is to order a large cancer panel.
  • Only perform tumor testing on endometrial cancer cases diagnosed under the age of 65

Deceased Patients

1. How do you go about informing relatives of a deceased patients abnormal IHC result?
  • Inform surgeon or oncologist who can inform relatives
  • Call emergency contact listed in system
  • Send a letter to the last known address of the patient – Addressed to family of the deceased patient
  • Some hospitals scan advance directives into the electronic medical record.
2. Ordering IHC on diseased relatives tumor. How do you bill it? How do you report it out?
  • You will need to get the death certificate along with the tumor blocks and a form signed by next of kin to order MSI/IHC testing. I. Send the testing to Huntington Medical Research Institute (HMRI) because their prices are the cheapest at $650. The patient has to write a check to HMRI and send it along with the blocks. II. You cannot bill the test to a living relative’s insurance. III. The results of the testing are a new report and not an addendum from the original path report. IV. You cannot put it into the living patients EMR because it is not their record. But can document it.
  • You can send it to other labs too but the lab will directly bill the living relative. PhenoPath is easy to use. I. http://www.phenopath.com/diagnostic/clin_colorectal.htm II. To get a tumor block released you will need a signed medical release form with proof that the person signing the release from is the deceased relatives’ executor of estate.

Other Cancers

1. Are appendiceal adenocarcinomas part of the LS tumor spectrum?

They are histologically and clinically quite similar to colon primaries so it is thought that they could be. OSU did a small study on 11 patients with appendicieal primaries.
I. They were MSI negative
II. 4 of the 11 also had IHC where all the stain was present.
III. Publication by Stanley Hamilton looking at 30 appendiceal primaries. – Non were MSI-H
IV. This would suggest that appendiceal primaries do not go down the MSI pathway and are not associated with LS.
V. However, there have been reports of patients with appendiceal primaries who have LS. One had MLH1 mutation and another with MSH2 mutation. MSI/IHC helped ID these cases.
VI. These are not apart of universal tumor screening

2. Adenocarcinoma in the Duodenum where MLH1/PMS2 were absent with IHC staining. BRAF and Methylation studies have NOT been done. Sequencing of MLH1 and PMS2 were normal via Ambry lab. There was positive family history. Should BRAF or Methylation studies be done on this sample?

No data for Duodenum cancers. Could try MLH1 methylation testing however, BRAF would not give the data you were looking for.

3. Any information on biliary cancer where more than one FDR has been affected and died.

No evidence-based screening for these cancers or that of the gallbladder and bile duct as they are quite rare. Labs could be run yearly looking for liver function, USG evaluation of the gallbladder, and common bile duct might be reasonable. Very unclear benefits however.


IHC on Mets

1. What are your thoughts regarding IHC testing on colorectal cancer distant metastases?

We are fairly confident that tumor testing of a mets should be a good surrogate for a primary.


Endometrial Cancer

1. Testing on patients with synchronous endometrial and ovarian primaries. Is there something else besides Lynch Syndrome going on?
  • Three institutions had multiple cases that presents in a similar way and none of them have been confirmed having Lynch Syndrome or BRCA mutations.
  • MD Anderson did a series of these types of cases resulting in most of them not having Lynch Syndrome.
  • Could be some type of hormonal field effect that can cause these two synchronous primaries (endo + ov) so it is not necessarily a sign of an underlying genetic susceptibility like it usually is when we see synchronous primaries
  • One person had found a mutation in MSH6 but had a strong family history of endometrial and colon cancer.
2. Thoughts on BRAF testing when MLH1/PMS2 absent on endometrial IHC?
  • BRAF is rarely positive in sporadic endometrial cancers and is not a recommended test. If you are going to start with germline MLH1, I would reflex to hypermethylation of MLH1 if sequencing and del/dup is normal and not germline PMS2.
  • A paper recently published on a small sample size about endometrial cancer and BRAF V600 mutations indicated they did find that some mucinous endometrial cancers are associated with a BRAF mutations. He, M., et al. BRAF V600E Mutations in Endometrial Adenocarcinoma. Diagnostic Mole Path: 2013:22(1):35-40.
3. IHC shows loss of MLH1, PMS2, and MSH6 staining, it is considered and MSI-H case with hypermethylation of MLH1. The patient had 3 hyperplastic colon polyps, has had endometroid endometrial cancer. She has not had any radiation. After testing the patient for MLH1, MSH2, MSH6, EPCAM, and PMS2 there was no mutation found. Where would you go from here and what suggestions for follow-up would you tell the patient?

I would treat this case like a methylated MLH1 case (usually sporadic). There is a microsatellite in MSH6 that can become unstable in MSI-H tumors. When this happens, it somehow causes absence of MSH6 on IHC. So, when I see a weird result with MSH6 absent when it shouldn’t be, I usually work up the other missing stains and ignore the MSH6.
I. Chang, D. K., et.al. Microsatellites in the eukaryotic DNA mismatch repair genes as modulators of evolutionary mutation rate. Genome Research; 2001:11, 1145.
II. Shia, J., et al. Immunohistochemistry as first-line screening for detecting colorectal cancer patients at risk for hereditary nonpolyposis colorectal cancer syndrome: a 2-antibody panel may be as predictive as a 4-antibody panel. American Journal of Surgical Pathology; 2009:33, 1639.
III. Shia J., et al. Secondary mutation in a coding mononucleotide tract in MSH6 causes loss of immunoexpression of MSH6 in colorectal carcinomas with MLH1/PMS2 deficiency. Mod Pathol. 2013 Jan;26(1):131-8.


Support

1. A teen was recently found to have Lynch Syndrome. I am aware of Lynch Syndrome International but don’t see any sort of online chat forum on their website. Is there any other organization others have found to be useful? Is there a chat forum that I somehow can’t find?

UCSF has created Kintalk.org where people can chat on-line with each other, learn about Lynch Syndrome and share their genetic information with at-risk relatives. Additionally, there are many active Facebook groups. If your patient types in Lynch Syndrome into Facebooks search field he should be able to find these groups. Although they are private groups, anyone can get access. There is also a large community on Twitter he can connect with. Let me know if you need more info.