Case 3 -

Poorly Differentiated and Microsatellite Unstable Adenocarcinoma Arising in a Patient with Lynch Syndrome Wendy L. Frankel The Ohio State University School of Medicine Columbus, OH

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Clinical History: 58 year old man with no personal history of malignancy or other significant medical history was noted to have two tumors at colonoscopy. The tumors were located in his transverse colon and rectosigmoid. Case 3 - Slide 1 Click to view with ImageScope Click to view with a Web-Based Viewer Case 3 - Figure 1 Low power view of the tumor with a pushing border and adjacent lymphoid aggregates. Case 3 - Figure 2 In some areas, the cells form glands and nests. Case 3 - Figure 3 In other areas, the tumor cells show a medullary pattern. The malignant cells are arranged in a somewhat trabecular pattern and contain vesicular chromatin with nucleoli. There is a lymphoid infiltrate surrounding tumor cells. Case 3 - Figure 4 High power view of an area with undifferentiated sheets of tumor cells. Case 3 - Figure 5 The tumor is immunoreactive with MLH1 and PMS2. Case 3 - Figure 6 MSH2 and MSH6 mark lymphocytes and benign epithelial cells but staining is absent in tumor cells. Case 3 - Figure 7 The algorithm used at the Ohio State University on all colorectal carcinoma (CRC) patients to identify Lynch Syndrome and microsatellite instability (from Bellizzi and Frankel, Adv Anat Pathol, 2009). Introduction: Colorectal carcinoma is one of the leading causes of cancer in the US. Most tumors (85%) arise through the chromosomal instability pathway but 15% are due to microsatellite instability. Patients with these tumors have a better prognosis than those with stage-matched microsatellite stable tumors. Additionally, they do not respond as well to some chemotherapeutic agents (5- fluorouracil). Many of these tumors are due to hypermethylation of the MLH1 promoter and are sporadic rather than germline. These tumors typically occur in older (eighth decade) people and may be associated with a serrated polyp (serrated pathway). There is a smaller subset of microsatellite unstable tumors that are due to a mutation in one of the mismatch repair genes (MLH1, MSH2, PMS2, MSH6). These tumors occur in those with Lynch syndrome and are more common in younger patients (fourth or fifth decade). These individuals have an increased risk of synchronous colorectal carcinomas and other associated malignancies such as endometrial, stomach, renal pelvis and ureter cancer. Family members also need screening. Pathological/Microscopic Findings and any Immunohistochemical or Other Studies: The colon contains polypoid tumors in the transverse colon and rectosigmoid. The rectosigmoid tumor is shown in sections. At low power, a pushing border is identified. Lymphoid aggregates are present at the edge of the tumor. The tumor is poorly differentiated and the morphology varies in different areas of the slide. In some areas, the cells are arranged as glands and nests. Other areas show a medullary pattern with tumor in a sheet-like to trabecular pattern containing cells with vesicular chromatin, nucleoli and many lymphocytes surrounding tumor cells. Undifferentiated areas exist. MLH1 and PMS2 immunohistochemical stains marked the tumor cells while MSH2 and MSH6 stained lymphocytes and benign epithelial cells but not tumor cells. Differential Diagnoses: Microsatellite unstable adenocarcinoma- Lynch Syndrome Microsatellite unstable adenocarcinoma- sporadic Microsatellite stable adenocarcinoma Final Diagnosis: Poorly differentiated and microsatellite unstable adenocarcinoma arising in a patient with Lynch Syndrome Case Discussion: Other important history in our patient includes a very significant history of carcinoma in other members of his family including endometrial, ureter, lung, colorectal, thyroid, sebaceous adenoma and brain (glioblastoma multiforme). The pathologic findings of synchronous and poorly differentiated adenocarcinoma with a pushing border, Crohn-like reaction, tumor infiltrating lymphocytes, and medullary features suggest microsatellite instability. The patient's age and family history are indicative of Lynch syndrome. The immunohistochemical findings of intact (present) staining for MLH1 and PMS2 and absent staining for MSH2 and MSH6 suggest an MSH2 mutation which was confirmed with mutational testing for MSH2 (patient consent necessary). The patient was found to have the most common mutation in MSH2, the Desai mutation. His family was screened for this mutation; it was found in 4 of 9 siblings, 1 of 2 daughters and 3 others (nieces, nephews). His family is considered to have the Muir-Torre variant due to the history of sebaceous tumors and Turcot's due to the brother with glioblastoma multiforme. The family continues to be followed closely. Review of the Literature/Treatment Options: Approximately 15% of colorectal carcinomas show microsatellite instability rather than chromosomal instability. The majority of these tumors are sporadic and are due to epigenetic silencing of the MLH1 promoter by hypermethylation. Both sporadic and hereditary microsatellite unstable carcinomas have prognostic and predictive implications. Microsatellite unstable colorectal carcinomas have a better prognosis than matched stage microsatellite stable carcinomas, and tumors do not respond as well to 5-fluorouracil based chemotherapy. Some institutions are using this information to help guide treatment, particularly in lower stage tumors where the use of chemotherapy is more controversial. Lynch Syndrome is the most common hereditary form of colorectal cancer. It accounts for 1 to 5% of colorectal cancers and is due to a mutation in one of the mismatch repair genes manifesting as microsatellite instability. The syndrome is characterized by early onset (fourth or fifth decade) and frequently right sided colorectal cancer. However, tumors can occur anywhere in the colon or rectum and the age range is wide including older patients. Additionally, patients are at an increased risk for synchronous or metachronous colorectal cancers and other tumors such as endometrial, ureter, and gastric. Lynch Syndrome follows a dominant pattern of inheritance with a high penetrance. Patients and families need screening and counseling to help prevent additional carcinomas or identify them at an early stage. Lynch Syndrome is identified using history, tumor morphology and/or laboratory tests that identify mismatch repair deficiency in the tumor. Amsterdam II criteria are clinical criteria to identify families with HNPCC (hereditary nonpolyposis colorectal carcinoma) while the revised Bethesda guidelines were developed to identify those who should get microsatellite instability testing. In unselected patients both show limited sensitivities and specificities. Smaller family size and the increased use of screening colonoscopies and polypectomies also decrease sensitivity of the criteria. Of course, these clinical criteria are not useful to detect the larger group of microsatellite unstable colorectal carcinomas due to an epigenetic event. Therefore, additional testing is necessary to identify these patients. Histologic features can be useful to detect microsatellite unstable colorectal carcinomas. Some of the most common include mucinous, signet-ring cell, medullary and poorly differentiated features. Tumors also show a pushing border, a Crohn-like reaction and tumor infiltrating lymphocytes. It is important to remember that there is significant histologic phenotypic overlap between sporadic and Lynch-associated microsatellite unstable tumors. Jeremy Jass has argued that the constellation of histologic features classically associated with microsatellite unstable colorectal carcinomas largely represents sporadic rather than Lynch-associated tumors. The most useful feature to help differentiate these tumors, when present, is a serrated background suggesting methylation and the serrated pathway. Table 1 compares the clinicopathologic features in Lynch Syndrome to sporadic microsatellite unstable colorectal carcinomas. Table 1: Clinicopathologic Features of Lynch Syndrome-Associated and Sporadic Microsatellite Unstable (MSI) Colorectal Cancers Lynch Sporadic MSI Clinical Age < 50 yrs > 70 yrs Sex F = M F > > M Other Lynch associated tumors Yes No Proximal tumor a Yes Yes Histology Mucinous a Yes Yes Signet-ring cell Yes Yes Medullary Yes Yes Intraepithelial lymphocytes Yes Yes Crohn's-like reaction Yes Yes Pushing border Yes Yes Poor differentiation a Yes Yes Tumor budding Yes No Tumor heterogeneity a Yes Yes Serration/serrated precursor No Yes Immunohistochemistry Absent MLH1 and PMS2 Yes, some Yes Absent MSH2 and MSH6 Yes, some No Absent MSH6 or PMS2 Yes, few No Molecular MSI-H Yes Yes BRAF mutation No Yes MLH1 promoter methylation Rare Yes Germline mutation in mismatch repair gene Yes No a Each of these features was shown to occur significantly less frequently in "HNPCC-associated" vs. sporadic MSI CRC (Young et al, Am J Pathol, 2001). Table modified from Bellizzi and Frankel, Adv Anat Pathol, 2009. In unselected patients, mismatch repair deficiency can be identified by microsatellite instability (MSI) testing on tumor DNA or by using immunohistochemistry (IHC) to detect absent mismatch repair proteins. MSI testing is done in a molecular lab and the mismatch repair deficiency manifests as microsatellite instability. IHC is typically done by performing a panel of MLH1, MSH2, MSH6 and PMS2. Some laboratories have demonstrated efficacy using a smaller panel. The staining pattern of these proteins can vary and may be diffuse or focal but must be nuclear to be considered present. Fixation may affect staining. Absence should be virtually complete in order to consider the protein lost. The mismatch repair proteins function as heterodimers. MSH2-MSH6 and MLH1-PMS2 pairs act in concert. If the obligate partner in the pair (MSH2 or MLH1) is missing, neither protein in the pair will be expressed. Therefore, a mutation or epigenetic silencing of MLH1 will lead to loss of MLH1 and PMS2 protein, while a PMS2 mutation will only manifest as negative PMS2 staining with intact MLH1. The pattern of loss can help point towards the gene that is not being expressed by mutation or an epigenetic event. When MLH1 and PMS2 are absent (most common abnormal pattern), this is either due to Lynch Syndrome or sporadic methylation of the MLH1 promoter and additional testing is necessary. When MSH2 and MSH6 or MSH6 or PMS2 protein(s) is lost, additional testing is necessary to evaluate for the high likelihood of Lynch Syndrome. Both IHC and MSI testing have limitations and choosing the best test may be dependent upon resources and ease, cost and speed of results at different institutions. Most studies including ours have shown the MSI and IHC are comparable to identify Lynch Syndrome. MSI requires tumor and normal DNA and requires sufficient tumor percentage. IHC only requires tumor (since lymphocytes and endothelial cells can serve as positive controls), and a small number of tumor cells is usually sufficient, but poor fixation can affect results. IHC does help suggest the likely gene of interest, and therefore, may help decrease cost. Some argue this comes close to "crossing the line" of germline testing without consent. The literature has argued convincingly that IHC is not germline testing since it identifies proteins that can be altered by epigenetic events. Table 2 compares IHC for the mismatch repair proteins to MSI testing to screen colorectal carcinomas for a mismatch repair deficiency. Table 2: Comparison of Immunohistochemistry for the Mismatch Repair Proteins versus Microsatellite Instability Testing Immunohistochemistry (IHC) Microsatellite Instability (MSI) Advantage Cost Variable Variable Neither Training to perform Necessary Necessary Neither Analyte Protein DNA Neither Laboratory Most pathology labs Need molecular lab IHC # tumor cells needed Very few required Need certain % depending on marker IHC Sample Tumor only Tumor and normal IHC "Contamination" by normal may interfere No Yes IHC Suggests mismatch repair gene involved Yes No IHC Turnaround time Next day 2 to 7 days IHC "Genetic" debate a Yes No MSI Distinguishes MSI-H, MSI-L, MSS b No (MSI-L and MSS similar staining) Yes MSI Variable results due to tissue fixation Yes No MSI a the "genetic debate" questions whether IHC is a genetic test since results suggest the likely involved gene. There is no debate with MSI testing. We and most publications do not consider IHC a genetic test. b Differentiating MSI-H from MSI-L and MSS does not matter for identification of Lynch syndrome but may matter for prognosis or predictive reasons. Table modified from Bellizzi and Frankel, Adv Anat Pathol, 2009. There are multiple algorithms for testing colorectal carcinomas depending on if IHC or MSI testing is used as the initial screening test. We advocate using Braf mutational testing (some choose to use MLH1 promoter methylation testing) in those with absence of MLH1 and PMS2 proteins before embarking on MLH1 germline mutation analysis. Since this pathogenic mutation has not been described in those with Lynch syndrome, there is no need to do additional testing for Lynch syndrome on those with this mutation. The majority of mutations are accounted for by a point mutation resulting in the substitution of glutamic acid for valine at codon 600 (V600E). The algorithm used at the Ohio State University is show in Figure 7. Conclusion(s): It is important to identify microsatellite instability and Lynch syndrome for both prognostic and predictive information as well as to screen affected individuals and family members in Lynch Syndrome. Currently this information is not required in the AJCC TNM 7th edition. The CAP colorectal checklist does include histologic features such as differentiation and histologic type (mucinous, medullary or signet ring cell among others). Other features suggestive of MSI including peritumoral lymphocytic response (Crohn-like reaction), intratumoral lymphocytic response (tumor-infiltrating lymphocytes), and tumor subtyping (including percent mucinous component) are listed as optional. Other optional items include ancillary testing such as MSI, immunohistochemical stains for the mismatch repair proteins and Braf testing. Many labs are doing MSI, Braf and immunohistochemical testing upon request. It is becoming more common for reflexive testing for MSI or immunohistochemistry in all colorectal carcinomas or at least in those with suggestive patient age and morphologic features. The best algorithm for testing is laboratory dependent. Communication with the surgeons and gastroenterologists is vital in whatever algorithm is chosen. References: Alexander J, Watanabe T, Wu TT, Rashid A, Li S, Hamilton SR. Histopathological identification of colon cancer with microsatellite instability. Am J Pathol 2001; 158(2):527-535. Bellizzi AM and Frankel WL. Colorectal cancer due to deficiency in DNA mismatch repair function: a review (invited paper). Adv Anat Pathol 2009; 16(6):405-417. DaCosta Byfield SA, Syngal S. Clinical guidelines versus universal molecular testing: Are we ready to choose an optimal strategy for Lynch syndrome identification? Am J Gastroenterol 2008; 103:2837-2840. Domingo E, Niessen RC, Oliveira C, et al. 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