Success of referral to genetic counseling after positive lynch syndrome screening test.

PURPOSE: Lynch syndrome (LS) is a hereditary condition that increases one's risk of developing colorectal, endometrial, and other extracolonic cancers. MD Anderson Cancer Center at Cooper implemented a reflex screening protocol for DNA mismatch repair (dMMR) deficiency. Those with findings suspicious for LS were referred for genetic counseling (GC). Our goal was to assess compliance with GC and factors associated with successful follow-up. METHODS: Immunohistochemistry (IHC) for the MMR proteins MSH2, MLH1, MSH6, and PMS2 was performed on all colorectal tumor resections from patients ≤70 years old and all stage II cancers. Tumors with loss of MLH1/PMS2 were subsequently tested for BRAF mutation or MLH1 promoter methylation to identify tumors with likely epigenetic inactivation of MLH1. Patients with loss of MLH1/PMS2 without BRAF mutations or with absence of MLH1 promoter methylation and those with loss of MSH2/MSH6 were referred to GC. Compliance with GC was assessed. RESULTS: Between March 2014 and August 2016, 203 tumors were tested by IHC. Fifteen (7.4%) patients had abnormal MMR protein expression patterns in the absence of BRAF mutation or MLH1 promoter methylation suggestive of possible LS. GC compliance was 35.7% overall and 85.7% in those with family history of LS-associated cancers. CONCLUSIONS: Overall, GC compliance was relatively low in our study. Interestingly, patients with a strong family history of LS-associated neoplasms were more likely to pursue GC. In the future, assessing and addressing barriers to seeking GC will provide opportunities to improve patient care through increased identification of patients with cancer predisposition syndromes.

Expression of the MAP kinase phosphatase DUSP4 is associated with microsatellite instability in colorectal cancer (CRC) and causes increased cell proliferation.

DUSP4 (MKP-2), a member of the mitogen-activated protein kinase phosphatase (MKP) family and potential tumor suppressor, negatively regulates the MAPKs (mitogen-activated protein kinases) ERK, p38 and JNK. MAPKs play a crucial role in cancer development and progression. Previously, using microarray analyses we found a conspicuously frequent overexpression of DUSP4 in colorectal cancer (CRC) with high frequent microsatellite instability (MSI-H) compared to microsatellite stable (MSS) CRC. Here we studied DUSP4 expression on mRNA level in 38 CRC (19 MSI-H and 19 MSS) compared to matched normal tissue as well as in CRC cell lines by RT-qPCR. DUSP4 was overexpressed in all 19 MSI-H tumors and in 14 MSS tumors. Median expression levels in MSI-H tumors were significantly higher than in MSS-tumors (p < 0.001). Consistently, MSI-H CRC cell lines showed 6.8-fold higher DUSP4 mRNA levels than MSS cell lines. DUSP4 expression was not regulated by promoter methylation since no methylation was found by quantitative methylation analysis of DUSP4 promoter in CRC cell lines neither in tumor samples. Furthermore, no DUSP4 mutation was found on genomic DNA level in four CRC cell lines. DUSP4 overexpression in CRC cell lines through DUSP4 transfection caused upregulated expression of MAPK targets CDC25A, CCND1, EGR1, FOS, MYC and CDKN1A in HCT116 as well as downregulation of mismatch repair gene MSH2 in SW480. Furthermore, DUSP4 overexpression led to increased proliferation in CRC cell lines. Our findings suggest that DUSP4 acts as an important regulator of cell growth within the MAPK pathway and causes enhanced cell growth in MSI-H CRC.

A Rare Case of Multifocal Asynchronous Benign Granular Cell Tumors with PIK3CA Subclonal Mutation Identified in One Tumor by Next-Generation Sequencing.

Granular cell tumor (GCT) is a benign neuroectodermal tumor typically in the dermis or subcutis, although deep soft tissues and organs are occasionally involved. Multifocal GCTs are estimated to occur as many as 10% of patients. A 40-year-old female presented with multiple GCTs asynchronously involving various body sites including gastrointestinal, gynecologic, breast, urinary, and soft tissue systems. Pathologic examinations suggested benign GCTs. TruSight Tumor 170 next-generation sequencing (NGS) analysis performed on four resected tumors revealed subclonal mutation of PIK3CA p.H1047R identified in the esophageal GCT but not in the right vulva or the two cecal GCTs, suggesting that each is a primary tumor with a distinct genetic profile, rather than metastasis. PIK3CA p.H1047R is a common mutation in many cancers. Our benign GCT case demonstrates PIK3CA mutation with a low mutant allele frequency of 7%, which may represent an evolving subclone and might confer a more aggressive behavior.

Encephalitis associated with a monoclonal protein band present in blood, urine and cerebrospinal fluid.

Introduction: Monoclonal protein bands are present mainly in blood and secondary in urine representing specific antibody produced in excess by abnormal lymphocytes or plasma cells.We describe a case of a patient with acute encephalitis associated with an unexpected finding of a monoclonal protein band present in blood, urine and in cerebrospinal fluid (CSF). Case presentation: This 50-year-old woman with no significant past medical history, with the exception of unintentional weight loss exceeding 5 kg over the last 3 months, presented to the emergency department with seizures and altered mental status, after 3 days of vomiting and headaches. Magnetic Resonance Imaging showed lesions suspicious for infectious encephalitis/meningitis and for ischemia possibly related to central nervous system (CNS) autoimmune vasculopathy/vasculitis. The patient died the following day after losing brainstem reflexes. Testing for the previously mentioned etiologies returned negative with the exception of high protein concentration and increased immunoglobulin gamma (IgG) concentration in the CSF. Protein electrophoresis, ordered in error, showed a well-defined IgG with lambda light chain monoclonal protein band running in similar positions in serum, urine and in CSF. Due to SARS-CoV-2 PCR positivity no autopsy was performed. Conclusion: The presence of this monoclonal protein band produced in the CNS suggests the diagnosis of CNS myeloma. The accelerated course in this case could be the result of the CNS myeloma or lymphoma responding to SARS-CoV-2 infection. Testing for monoclonal protein bands in CSF, in patients with pertinent clinical presentation would boost the awareness of this these diseases improving patient care.

Testing for deficient mismatch repair and microsatellite instability : A focused update.

Testing to detect mismatch repair deficiency (dMMR) and high-grade microsatellite instability (MSI-H) has become an integral part of the routine diagnostic workup for colorectal cancer (CRC). While MSI was initially considered to be a possible indicator of a hereditary disposition to cancer (Lynch syndrome, LS), today the prediction of the therapy response to immune checkpoint inhibitors (ICI) is in the foreground. Corresponding recommendations and testing algorithms are available for use in primary diagnosis (reviewed in: Rüschoff et al. 2021).Given the increasing importance for routine use and the expanding indication spectrum of ICI therapies for non-CRCs, such as endometrial, small intestinal, gastric, and biliary tract cancers, an updated review of dMMR/MSI testing is presented. The focus is on the challenges in the assessment of immunohistochemical stains and the value of PCR-based procedures, considering the expanded ICI indication spectrum. A practice-oriented flowchart for everyday diagnostic decision-making is provided that considers new data on the frequency and type of discordances between MMR-IHC and MSI-PCR findings, and the possible role of Next Generation Sequencing in clarifying them. Reference is made to the significance of systematic quality assurance measures (e.g., QuIP MSI portal and multicenter proficiency testing), including regular continued training and education.

[Testing deficient mismatch repair and microsatellite instability : A focused update. German version]. / Testung auf Mismatch-Reparatur-Defizienz und Mikrosatelliteninstabilität : Eine fokussierte Aktualisierung.

Testing to detect mismatch repair deficiency (dMMR) and high-grade microsatellite instability (MSI-H) has become an integral part of the routine diagnostic workup for colorectal cancer (CRC). While MSI was initially considered to be a possible indicator of a hereditary disposition to cancer (Lynch syndrome, LS), today the prediction of the therapy response to immune checkpoint inhibitors (ICI) is in the foreground. Corresponding recommendations and testing algorithms are available for use in primary diagnosis (reviewed in: Rüschoff et al. 2021).Given the increasing importance for routine use and the expanding indication spectrum of ICI therapies for non-CRCs, such as endometrial, small intestinal, gastric, and biliary tract cancers, an updated review of dMMR/MSI testing is presented. The focus is on the challenges in the assessment of immunohistochemical stains and the value of PCR-based procedures, considering the expanded ICI indication spectrum. A practice-oriented flowchart for everyday diagnostic decision-making is provided that considers new data on the frequency and type of discordances between MMR-IHC and MSI-PCR findings, and the possible role of Next Generation Sequencing in clarifying them. Reference is made to the significance of systematic quality assurance measures (e.g., QuIP MSI portal and multicenter proficiency testing), including regular continued training and education.

A second-generation microRNA-based assay for diagnosing tumor tissue origin.

BACKGROUND: Cancers of unknown primary origin (CUP) constitute 3%-5% (50,000 to 70,000 cases) of all newly diagnosed cancers per year in the United States. Including cancers of uncertain primary origin, the total number increases to 12%-15% (180,000 to 220,000 cases) of all newly diagnosed cancers per year in the United States. Cancers of unknown/uncertain primary origins present major diagnostic and clinical challenges because the tumor tissue of origin is crucial for selecting optimal treatment. MicroRNAs are a family of noncoding, regulatory RNA genes involved in carcinogenesis. MicroRNAs that are highly stable in clinical samples and tissue specific serve as ideal biomarkers for cancer diagnosis. Our first-generation assay identified the tumor of origin based on 48 microRNAs measured on a quantitative real-time polymerase chain reaction platform and differentiated 25 tumor types. METHODS: We present here the development and validation of a second-generation assay that identifies 42 tumor types using a custom microarray. A combination of a binary decision-tree and a k-nearest-neighbor classifier was developed to identify the tumor of origin based on the expression of 64 microRNAs. RESULTS: Overall assay sensitivity (positive agreement), measured blindly on a validation set of 509 independent samples, was 85%. The sensitivity reached 90% for cases in which the assay reported a single answer (>80% of cases). A clinical validation study on 52 true CUP patients showed 88% concordance with the clinicopathological evaluation of the patients. CONCLUSION: The abilities of the assay to identify 42 tumor types with high accuracy and to maintain the same performance in samples from patients clinically diagnosed with CUP promise improved utility in the diagnosis of cancers of unknown/uncertain primary origins.

Accurate classification of metastatic brain tumors using a novel microRNA-based test.

BACKGROUND: Identification of the tissue of origin of a brain metastatic tumor is vital to its management. Carcinoma of unknown primary (CUP) is common in oncology, representing 3%-5% of all invasive malignancies. We aimed to validate a recently developed microRNA-based quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) test for identifying the tumor tissue of origin, first in a consecutive cohort of metastatic tumors of known origin and then in a cohort of CUP cases resected from the central nervous system (CNS). PATIENTS AND METHODS: One hundred two resected CNS metastatic tumors with known origin, previously classified based on the patient's clinical history and pathological data, as well as a second cohort of resected CNS tumors from 57 patients originally diagnosed as CUP were studied. A qRT-PCR diagnostic assay that measures the expression level of 48 microRNAs was used to classify the tissue of origin of these metastatic tumors. RESULTS: In this blinded study, the test predictions correctly identified the reference diagnosis of the samples of known origin, excluding samples from prostate origin, in 84% of cases. In the second CUP patient cohort, the test prediction was in agreement with the diagnosis that was later confirmed clinically or with pathological evaluation in 80% of cases. CONCLUSION: In a cohort of brain and spinal metastases, a previously developed test based on the expression of 48 microRNAs allowed accurate identification of the tumor tissue of origin in the majority of cases. The high accuracy of this test in identifying the tissue of origin of metastases of unknown primary is demonstrated for the first time and may have broad clinical application.

Intra-tumor heterogeneity of MLH1 promoter methylation revealed by deep single molecule bisulfite sequencing.

A single tumor may contain cells with different somatic mutations. By characterizing this genetic heterogeneity within tumors, advances have been made in the prognosis, treatment and understanding of tumorigenesis. In contrast, the extent of epigenetic intra-tumor heterogeneity and how it influences tumor biology is under-explored. We have characterized epigenetic heterogeneity within individual tumors using next-generation sequencing. We used deep single molecule bisulfite sequencing and sample-specific DNA barcodes to determine the spectrum of MLH1 promoter methylation across an average of 1000 molecules in each of 33 individual samples in parallel, including endometrial cancer, matched blood and normal endometrium. This first glimpse, deep into each tumor, revealed unexpectedly heterogeneous patterns of methylation at the MLH1 promoter within a subset of endometrial tumors. This high-resolution analysis allowed us to measure the clonality of methylation in individual tumors and gain insight into the accumulation of aberrant promoter methylation on both alleles during tumorigenesis.

Implementation of Laboratory Review of Test Builds Within the Electronic Health Record Reduces Errors.

CONTEXT.­: As electronic health records (EHRs) become more ubiquitous, physicians have come to expect that laboratory data from a variety of sources will be incorporated into the EHR in a structured format. The Clinical Laboratory Improvement Amendments have standards for data transmission traditionally met by pathologist review of their own hospital laboratory information system transmissions. However, with third-party laboratory data now being sent through external (nonhospital laboratory) interfaces, ownership of this review is less clear. Lack of an expert laboratory review process prior to changes being implemented can result in mapping and interfacing errors that could lead to misinterpretation and diagnostic errors. OBJECTIVE.­: To determine the impact of retrospective and prospective laboratorian-assisted review on the volume of interface errors and new builds. DESIGN.­: A seminal event led to a restructuring of the process for review of EHR laboratory builds, using laboratory expertise. RESULTS.­: A review of 26 500 test result fields found 61 of 4282 (1.4%) unique codes that could have led to misinterpretation. These were corrected and a process for proactive review and maintenance by laboratory experts was implemented. This resulted in monthly decreases in outbound error message from 4270 to 1820 (57.4%), in new test builds from 586 to 274 (53.2%), and in new result builds from 1116 to 552 (50.5%). CONCLUSIONS.­: Regular review and maintenance of external laboratory test builds in EHRs by a laboratory review team reduces interface error messages and reduces the number of new builds required for results to file into the EHR.

Optimization of the Order Menu in the Electronic Health Record Facilitates Test Patterns Consistent With Recommendations in the Choosing Wisely Initiative.

OBJECTIVES: Thyroid and rheumatologic autoimmune testing are areas where evidence-based guidance from specialty organizations and Choosing Wisely support utilizing screening tests for autoimmune and thyroid disorders prior to more specialized testing. Adjustment of the orderable options in the electronic health record (EHR) can influence ordering patterns without requiring manual review or additional effort by the clinician. METHODS: The menu was adjusted to reflect recommendations from Choosing Wisely to favor screening tests that automatically reflex to specialized testing on primary care providers' preference lists. Effectiveness was evaluated by reviewing total orders for individual tests. RESULTS: Shifts in ordering from individual screening tests (antinuclear antibody and thyrotropin) to ones that reflexed to specialized testing were observed in parallel with significant reductions in the corresponding specialized testing. CONCLUSIONS: Optimization of the EHR laboratory ordering menu can be used to shift ordering patterns toward Choosing Wisely recommendations.

Exclusion of breast cancer as an integral tumor of hereditary nonpolyposis colorectal cancer.

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant genetic predisposition syndrome that accounts for 2-7% of all colorectal cancers. Diagnosis of HNPCC is based on family history (defined by Amsterdam or Bethesda Criteria), which often includes a history of multiple synchronous or metachronous cancers. The majority of HNPCC results from germ-line mutations in the DNA mismatch repair (MMR) genes hMSH2 and hMLH1 with rare alterations in hMSH6 and hPMS2 in atypical families. Both HNPCC and sporadic MMR-deficient tumors invariably display high microsatellite instability (MSI-H). Two types of HNPCC families can be distinguished: type I (Lynch I) with tumors exclusively located in the colon; and type II (Lynch II) with tumors found in the endometrium, stomach, ovary, and upper urinary tract in addition to the colon. A proposed association of breast cancer with type II HNPCC is controversial. To address this important clinical question, we examined MSI in a series of 27 female patients who presented with synchronous or metachronous breast plus colorectal cancer. Although MSI-H was found in 5 of 27 (18.5%) of the colon cancers, in all cases the matched breast cancer was microsatellite stable. We also examined the breast tumors from three women who were carriers of MMR gene mutations from HNPCC families. None of these three breast tumors displayed MSI nor was the expression of MMR proteins altered in these tumors. We conclude that breast cancer largely arises sporadically in HNPCC patients and is rarely associated with the HNPCC syndrome.

Frequent microsatellite instability in sporadic tumors of the upper urinary tract.

Urothelial carcinoma of the renal pelvis and ureter may develop sporadically or as a manifestation of hereditary nonpolyposis colorectal cancer. The majority of hereditary nonpolyposis colorectal cancer is caused by mutation of the human DNA mismatch repair (MMR) genes and is detected by associated microsatellite instability (MSI). Seventy-three unselected urothelial carcinomas of the ureter and/or renal pelvis were screened for MSI using the National Cancer Institute-designated reference panel (plus BAT40). Instability of at least two microsatellite markers (MSI-high) was detected in 15 samples (21%). Immunohistochemical staining of the MMR proteins (hMSH2, hMLH1, or hMSH6) was absent in 13 of 15 (87%) MSI tumors, and alteration of coding sequence microsatellites (TGFbetaRII, Bax, hMSH3, and hMSH6) was found at frequencies of 7-33% in these samples. Tumors with MSI had significantly different clinical and histopathological features including higher prevalence in female patients, low tumor stage and grade, and a papillary and frequently inverted growth pattern. Our results suggest a molecular pathway of tumorigenesis that is similar to MMR-deficient colorectal cancers and consistent with the notion that the site distributions of hereditary or sporadic MSI-high tumors may reflect tissue-specific susceptibility to lesions processed by the MMR machinery.

Microsatellite analysis of hereditary nonpolyposis colorectal cancer-associated colorectal adenomas by laser-assisted microdissection: correlation with mismatch repair protein expression provides new insights in early steps of tumorigenesis.

Although microsatellite instability (MSI) testing is a useful tool for molecular screening of hereditary nonpolyposis colorectal cancer (HNPCC) carcinomas, conflicting results have been obtained in colorectal adenomas. This might result from different techniques of tissue sampling and MSI analysis. Alternatively, some HNPCC-associated adenomas may follow a molecular route that differs from the MSI pathway. In the present study we examined the MSI status of 18 adenomas from 17 HNPCC patients by comparing manual adenoma dissection under gross visual control with laser microdissection of single adenoma crypts. After manual gross dissection, 50% (9 of 18) and 11.1% (2 of 18) of the adenomas displayed high-level (MSI-H) and low-level (MSI-L) MSI, respectively. The same set of adenomas split into 83.3% (15 of 18) MSI-H and 5.6% (1 of 18) MSI-L after laser microdissection. The expression pattern of mismatch repair (MMR) proteins showed a higher concordance rate with the MSI status in laser-dissected (94%) than gross-dissected (47%) adenomas. Whereas two adenomas remained microsatellite stable (MSS) and MMR proficient even after laser-assisted dissection, two MSI-H cases showed either rare instabilities at coding microsatellites or intratumoral heterogeneity of MSI with and without MSH2 expression. This suggests that in some adenomas development of MMR dysfunction occurs stepwise with MSI, arising before complete loss of MMR gene expression, whereas other HNPCC-associated adenomas might develop independently of MMR deficiency.

Increased risk for hereditary nonpolyposis colorectal cancer-associated synchronous and metachronous malignancies in patients with microsatellite instability-positive endometrial carcinoma lacking MLH1 promoter methylation.

PURPOSE: The aim of this study was to evaluate number and types of synchronous and metachronous malignancies in patients with endometrial carcinoma with and without microsatellite instability (MSI). EXPERIMENTAL DESIGN: From a series of 413 endometrial cancer patients, we identified 94 patients with MSI-positive (MSI+) cancers and grouped them by tumor MLH1 promoter methylation status. These 94 patients were matched by year of surgery to 94 patients with MSI-negative (MSI-) endometrial cancers from the same series. Medical records were reviewed for clinicopathologic information including rates and types of synchronous and metachronous malignancies. Hereditary nonpolyposis colorectal cancer (HNPCC)-associated second and third cancers were analyzed for MSI and MSH2, MSH6, and MLH1 expression for comparison with the corresponding endometrial cancers. RESULTS: The MSI+ and MSI- cohorts were similar with regard to age, race, grade, and histology. Twenty-eight MSI+ endometrial cancers (29.8%) were MLH1 unmethylated. Rates of synchronous and metachronous cancers were also similar in the MSI+ and MSI- groups at 20 and 23%, respectively. However, patients with MSI+ MLH1 unmethylated endometrial cancers had an excess of HNPCC-associated second and third cancers compared with those with MSI+ MLH1 methylated and MSI- endometrial cancers (18% versus 4.5%, P = 0.034, and 2.1%, P = 0.002). Six of seven second tumors from 5 patients with MSI+ MLH1 unmethylated endometrial cancers showed concordant MSI and mismatch repair protein expression status. CONCLUSIONS: Our observation that patients with MSI-positive MLH1 unmethylated endometrial carcinoma are at increased risk for HNPCC-associated synchronous and metachronous malignancies suggests inherited cancer susceptibility. These patients and their families may warrant more intense cancer surveillance.

Challenges and pitfalls in HNPCC screening by microsatellite analysis and immunohistochemistry.

Hereditary non-polyposis colorectal cancer (HNPCC) accounts for approximately 2 to 4% of the total colorectal cancer burden. For economic reasons a diagnostic "stepladder" is recommended. After evaluation of the family history, diagnostic microsatellite instability (MSI) analysis has found its place as a valuable screening tool for HNPCC. Immunohistochemical analysis can help to pinpoint the affected gene. The detection of a mutation in one of the responsible mismatch repair gene confirmed the diagnosis HNPCC. Here we demonstrate our experience of some important pitfalls that will be discussed in this study. In MSI testing, one potential source for false-negative results is intralesional heterogeneity. We demonstrate examples of a flat adenoma and a carcinoma, which required laser microdissection to correctly determine the microsatellite status. In these lesions manual microdissection, the most frequently applied method, was not sufficient. However, the number of cells obtained by using laser microdisssection can fall below a necessary minimum, which can also cause false-negative results of MSI analysis, as shown here in a mucinous carcinoma. In addition, evaluation of immunohistochemically stained tissue slides requires experience to avoid false-positive or false-negative interpretation. A case with two synchronous colorectal cancers revealed loss of MSH2 expression in one carcinoma, whereas the second carcinoma stained positively leading to a false-negative interpretation. In some cases, false-positive results can be obtained, if a perinuclear-staining pattern is interpreted as positive. In summary, there are several potential pitfalls in the molecular screening for HNPCC. Therefore the importance of correct interpretation of clinical data, immunohistochemistry, and microsatellite analysis in combination, performed by a pathologist with experience in molecular genetics is essential. In addition, laser microdissection of tumor areas that have been chosen by a pathologist is highly recommended in cases that cannot be resolved with manual microdissection.

MSI-testing in hereditary non-polyposis colorectal carcinoma (HNPCC).

Genomic instability at simple repeated sequences, termed microsatellite instability (MSI), plays an important role in the analysis of sporadic and hereditary colon cancers. In hereditary non-polyposis colorectal cancer syndrome (HNPCC) more than 90% of cases show MSI, whereas only 10-15% of sporadic colorectal cancers do so. Thus, microsatellite analysis is commonly used as the first diagnostic screening test for HNPCC. In 1997, an international collaborative workshop sponsored by the National Cancer Institute (NCI) proposed a set of guidelines for MSI-testing to improve reliability and reproducibility of the analysis as well to allow comparisons between different studies and different laboratories. In this review we assess the value of current protocols for MSI-testing and discuss some diagnostic pitfalls. Our findings support continued use of the MSI marker panel recommended in 1997. Additionally, MSI-testing should be improved by use of microdissection, which helps to identify additional patients with MSI due to enrichment of tumor cells and therefore increased sensitivity. In our view, immunohistochemical staining for mismatch repair protein expression is not a substitute for MSI-analysis but complements MSI screening and helps direct further testing. In summary, MSI-analysis is a highly sensitive and reliable screening method for HNPCC, that requires a well-equipped laboratory as well as an experienced pathologist. Integration of family history and histo-pathological features is also critical.