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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.

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., (© American Society for Clinical Pathology, 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

For patients with primary lung cancer, accurate determination of the tumor type significantly influences treatment decisions. However, techniques and methods for lung cancer typing lack standardization. In particular, owing to limited tumor sample amounts and the poor quality of some samples, the classification of primary lung cancers using small preoperative biopsy specimens presents a diagnostic challenge using current tools. We previously described a microRNA-based assay (miRview squamous; Rosetta Genomics Ltd., Rehovot, Israel) that accurately differentiates between squamous and nonsquamous non-small cell lung cancer. Herein, we describe the development and validation of an assay that differentiates between the four main types of lung cancer: squamous cell carcinoma, nonsquamous non-small cell lung cancer, carcinoid, and small cell carcinoma. The assay, miRview lung (Rosetta Genomics Ltd.), is based on the expression levels of eight microRNAs, measured using a sensitive quantitative RT-PCR platform. It was validated on an independent set of 451 samples, more than half of which were preoperative cytologic samples (fine-needle aspiration and bronchial brushing and washing). The assay returned a result for more than 90% of the samples with overall accuracy of 94% (95% CI, 91% to 96%), with similar performance observed in pathologic and cytologic samples. Thus, miRview lung is a simple and reliable diagnostic assay that offers an accurate and standardized classification tool for primary lung cancer using pathologic and cytologic samples., (Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

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.

Purpose: Accurate identification of tissue of origin (ToO) for patients with carcinoma of unknown primary (CUP) may help customize therapy to the putative primary and thereby improve the clinical outcome. We prospectively studied the performance of a microRNA-based assay to identify the ToO in CUP patients., Experimental Design: Formalin-fixed paraffin-embedded (FFPE) metastatic tissue from 104 patients was reviewed and 87 of these contained sufficient tumor for testing. The assay quantitates 48 microRNAs and assigns one of 25 tumor diagnoses by using a biologically motivated binary decision tree and a K-nearest neighbors (KNN). The assay predictions were compared with clinicopathologic features and, where suitable, to therapeutic response., Results: Seventy-four of the 87 cases were processed successfully. The assay result was consistent or compatible with the clinicopathologic features in 84% of cases processed successfully (71% of all samples attempted). In 65 patients, pathology and immunohistochemistry (IHC) suggested a diagnosis or (more often) a differential diagnosis. Out of those, the assay was consistent or compatible with the clinicopathologic presentation in 55 (85%) cases. Of the 9 patients with noncontributory IHC, the assay provided a ToO prediction that was compatible with the clinical presentation in 7 cases., Conclusions: In this prospective study, the microRNA diagnosis was compatible with the clinicopathologic picture in the majority of cases. Comparative effectiveness research trials evaluating the added benefit of molecular profiling in appropriate CUP subsets are warranted. MicroRNA profiling may be particularly helpful in patients in whom the IHC profile of the metastasis is nondiagnostic or leaves a large differential diagnosis., (©2011 AACR.)

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.

The definitive identification of malignant pleural mesothelioma (MPM) has significant clinical implications, yet other malignancies often involve the lung pleura, confounding the diagnosis of MPM. In the absence of accurate markers, MPM can be difficult to distinguish from peripheral lung adenocarcinoma and metastatic epithelial cancers. MicroRNA expression is tissue-specific and highly informative for identifying tumor origin. We identified microRNA biomarkers for the differential diagnosis of MPM and developed a standardized microRNA-based assay. Formalin-fixed, paraffin-embedded samples of 33 MPM and 210 carcinomas were used for assay development. Using microarrays, we identified microRNAs differentially expressed between MPM and various carcinomas. Hsa-miR-193-3p was overexpressed in MPM, while hsa-miR-200c and hsa-miR-192 were overexpressed in peripheral lung adenocarcinoma and carcinomas that frequently metastasize to lung pleura. We developed a standardized diagnostic assay based on the expression of these microRNAs. The assay reached a sensitivity of 100% and a specificity of 94% in a blinded validation set of 68 samples from the lung and pleura. This diagnostic assay can provide a useful tool in the differential diagnosis of MPM from other malignancies in the pleura.

Background: Transcription factor Stat5a/b is highly critical for the viability of human prostate cancer cells in vitro and for prostate tumor growth in vivo. Stat5 is constitutively active in clinical prostate cancers but not in the normal human prostate epithelium. Moreover, Stat5a/b activation in prostate cancer is associated with high histological grade of prostate cancer. However, the molecular mechanisms underlying constitutive activation of Stat5a/b in prostate cancer are unclear. The receptor-associated tyrosine kinase Jak2 is a known key activator of Stat5a/b in prostate cancer cells in response to ligand stimulation. Recently, a single gain-of-function point mutation of JAK2 was described in myeloproliferative diseases leading to constitutive Jak2 kinase activity, subsequent Stat5a/b activation and involvement of V617F Jak2 in the pathogenesis of myeloproliferative disorders., Materials and Methods: We determined whether JAK2 undergoes the V617F activating mutation during clinical progression of human prostate cancer using a highly sensitive assay (amplification refractory mutation system) and a unique material of fresh specimens from organ-confined or castration-resistant prostate cancers., Results: The JAK2 V617F mutation was not found in any of the normal or malignant prostate samples analyzed in this study., Conclusions: Future work should focus on determining the molecular mechanisms other than V617F mutation of Jak2 resulting in continuous Stat5 activation in clinical prostate cancers.

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.

The prevalence and development of microsatellite instability (MSI) and underlying mismatch repair (MMR) deficiency in the carcinogenesis of adenocarcinomas of the papilla of Vater and their precursor lesions are not well established. We analyzed 120 ampullary adenomas (31 pure adenomas and 89 carcinoma-associated adenomas) and 170 pure adenocarcinomas for MSI, immunohistochemical expression of MMR proteins and specific histopathologic features. The most common histologic subtype was intestinal (46.5%), followed by pancreatobiliary (23.5%), poorly differentiated adenocarcinomas (12.9%), intestinal-mucinous (8.2%), and invasive papillary carcinomas (5.3%). Eight of 89 adenomas (9%) and 15/144 carcinomas (10%) showed high microsatellite instability (MSI-H), 10/89 adenomas (11%) and 5/144 carcinomas (4%) showed low microsatellite instability (MSI-L), and 71/89 adenomas (80%) and 124/144 carcinomas (86%) were microsatellite stable (MSS). MSI analysis from carcinomas contiguous with an adenomatous component (n=54) exhibited concordant results in 6/8 (75%) MSI-H and 42/46 (91.3%) MSS tumors. Of 14 carcinomas with MSI-H, 7 showed loss of MLH1 and 5/6 (83%) MLH1 promoter methylation, and 2 carcinomas showed simultaneous loss of MSH2 and MSH6. Two carcinomas and 3 adenomas with MSI-H revealed exclusive loss of MSH6. MSI-H cancers were significantly associated with intestinal mucinous subtype (P<0.001), high tumor grade (P=0.003), expansive growth pattern (P=0.044), and marked lymphoid host response (P=0.004). Patients with MSI-H carcinoma had a significantly longer overall survival (P=0.0082) than those with MSI-L or MSS tumors. Our findings indicate that the MSI-phenotype is an early event, which develops at the stage of adenoma and is reliably detectable in the precursor lesion. The MMR deficient molecular pathway of carcinogenesis is associated with a histopathologic phenotype in ampullary cancer, similar to the one that has been well described in colon cancer.

Inactivation of the APC gene is considered the initiating event in human colorectal cancer. Modifier genes that influence the penetrance of mutations in tumor-suppressor genes hold great potential for preventing the development of cancer. The mechanism by which modifier genes alter adenoma incidence can be readily studied in mice that inherit mutations in the Apc gene. We identified a new modifier locus of ApcMin-induced intestinal tumorigenesis called Modifier of Min 2 (Mom2). The polyp-resistant Mom2R phenotype resulted from a spontaneous mutation and linkage analysis localized Mom2 to distal chromosome 18. To obtain recombinant chromosomes for use in refining the Mom2 interval, we generated congenic DBA.B6 ApcMin/+, Mom2R/+ mice. An intercross revealed that Mom2R encodes a recessive embryonic lethal mutation. We devised an exclusion strategy for mapping the Mom2 locus using embryonic lethality as a method of selection. Expression and sequence analyses of candidate genes identified a duplication of four nucleotides within exon 3 of the alpha subunit of the ATP synthase (Atp5a1) gene. Tumor analyses revealed a novel mechanism of polyp suppression by Mom2R in Min mice. Furthermore, we show that more adenomas progress to carcinomas in Min mice that carry the Mom2R mutation. The absence of loss of heterozygosity (LOH) at the Apc locus, combined with the tendency of adenomas to progress to carcinomas, indicates that the sequence of events leading to tumors in ApcMin/+ Mom2R/+ mice is consistent with the features of human tumor initiation and progression.

Growing molecular evidence shows that uterine carcinosarcomas are clonal tumors. The carcinoma component has a dominant effect in the aggressive clinical behavior of these tumors. Defective DNA mismatch repair affects up to 30% of endometrial adenocarcinomas. The frequency and importance of defective DNA mismatch repair in the histiogenesis of uterine carcinosarcomas remains controversial. We studied the pattern and frequency of defective DNA mismatch repair and TP53 alterations in the epithelial and mesenchymal components of 28 uterine carcinosarcomas. We found evidence of defective DNA mismatch repair in six cases (21%) with a concordance rate of 83% for carcinoma-sarcoma pairs (kappa=0.887, P<0.001). Lack of immunostaining for the MLH1 protein was demonstrated in both components in two of these tumors. TP53 defects were evaluated by 17p deletion analysis and p53 immunostaining. Nineteen carcinoma (68%) and 18 sarcoma (64%) components had evidence of either TP53 allelic loss or p53 overexpression. These defects proved clonal in 76% of cases (kappa=0.602, P=0.003). Our results indicate that defective DNA mismatch repair and TP53 defects are common early events in carcinosarcoma tumorigenesis. The high rate of concordance for these molecular defects between the carcinoma and sarcoma components adds to existing molecular evidence that carcinosarcomas are clonal malignancies.

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.

Background: Microsatellite instability (MSI) and expression of cell cycle-related markers may predict a favorable outcome in colorectal cancer patients. The aim of this study was to elucidate the molecular profiles of patients with rectal cancers treated with neoadjuvant chemotherapy (5-Fluorouracil and CPT-11), radiotherapy and surgery that correlate with response to therapy., Patients and Methods: Fifty-seven patients with rectal cancer were treated with the same preoperative chemotherapy regimen, radiotherapy (45 to 54 Gy) followed by surgery. The microsatellite status, the expression of the mismatch repair proteins MLH1 and MSH2 and p21WAF1/C1PI, p27, bcl-2, topoisomerase II (topo II) and Ki-67 were assessed in the pretreatment biopsies. The response to adjuvant therapy was categorized in the resected specimens as complete response (CR, no microscopic residual tumor present) and partial response (PR, tumor present)., Results: p21WAF1/C1PI, expression characterized the CR with 12 out of 30 tumors (40%) positive for this marker. None of the patients whose tumors did not express p21WAFI/C1PI (10 patients) was a CR (p=0.011). Overall, the tumors with CR also showed higher expression of bcl-2, Ki-67, topo II and p27. However, p53 was more frequently expressed in the PR tumors. Tumors with high microsatellite instability showed CR (3/5, 60%) more often than PR, whereas tumors with stable microsatellites showed PR (26/36, 80%) more often than CR (p=0.099)., Conclusion: We conclude that a molecular profile characterized by high microsatellite instability with loss of mismatch repair protein expression and p21WAF1/C1PI is predictive of an improved response to neoadjuvant treatment with 5-FU, CPT-11 and radiation therapy.

Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by inherited mutations in DNA mismatch-repair genes, most commonly MLH1 or MSH2. The role MSH6 plays in inherited cancer susceptibility is less well defined. The aim of this study was to investigate the penetrance and expressivity of MSH6 mutations in kindreds ascertained through endometrial cancer probands unselected for family history. Detailed pedigrees were constructed for six MSH6 mutation carriers. All reported cancers and precancers were confirmed, and tissues were obtained when available. Tumors were analyzed for microsatellite instability (MSI) and for expression of MSH2, MLH1, and MSH6. MSH6 mutation status was determined for 59 family members. Of these 59 individuals, 19 (32%) had confirmed cancers and precancers. There was an excess of mutation carriers among the 19 affected family members (11 [58%] of 19) compared with those among the 40 unaffecteds (8 [20%] of 40, P=.0065, odds ratio = 5.5, 95% CI = 1.66-18.19). In four of the seven tumors analyzed from mutation carriers other than the probands, MSI and/or MMR protein expression was consistent with the involvement of MSH6. Overall estimated penetrance of the MHS6 mutations was 57.7%. Of the tumors in mutation carriers, 78% were part of the extended HNPCC spectrum. This study demonstrates that MSH6 germline mutations are, indeed, associated with increased cancer risk and that the penetrance of mutations may be higher than appreciated elsewhere. A combination of MSI and immunohistochemistry analyses may be helpful in screening for MSH6 mutation carriers.

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.

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.

The role of mismatch repair (MMR) in small-cell lung cancer (SCLC) is controversial, as the phenotype of a MMR-deficiency, microsatellite instability (MSI), has been reported to range from 0 to 76%. We studied the MMR pathway in a panel of 21 SCLC cell lines and observed a highly heterogeneous pattern of MMR gene expression. A significant correlation between the mRNA and protein levels was found. We demonstrate that low hMLH1 gene expression was not linked to promoter CpG methylation. One cell line (86MI) was found to be deficient in MMR and exhibited resistance to the alkylating agent MNNG. Surprisingly, MSI was not detected in 86MI and it appears to express all the major MMR components hMSH2, hMSH6, hMLH1, hPMS2, hMSH3, hMLH3, MBD4 (MED1) and hExo1. These data are consistent with at least two possibilities: (1) A missense mutation in one of the MMR genes, which dissociates MSI from drug resistance, or (2) inactivation of a second pathway that leads to MMR-deficiency and MNNG resistance, but induces negligible levels of MSI. We conclude that MMR deficiency is largely not associated with the pathogenesis of SCLC.

Colonic adenocarcinomas are among the most common type of tumors. In this report, we present the morphologic, immunohistochemical, and microsatellite findings of 2 cases with a distinct invasive papillary component. Both tumors arose from polyps in middle-aged patients, followed an aggressive course, and showed a superficial adenomatous component. The immunohistochemical stains showed that the tumor cells were negative for p27 and p53; both tumors were microsatellite stable, that is, with no microsatellite instability in the 6 markers studied, and there was no loss of the mismatch repair proteins hMSH2 or hMLH1. These findings suggest that these tumors follow the tumor-suppressor pathway and represent an aggressive subtype of colonic adenocarcinoma., (Copyright 2002, Elsevier Science (USA). All rights reserved.)

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.

Hereditary nonpolyposis colorectal cancer syndrome (HNPCC) is an autosomal dominant condition accounting for 2-5% of all colorectal carcinomas as well as a small subset of endometrial, upper urinary tract and other gastrointestinal cancers. An assay to detect the underlying defect in HNPCC, inactivation of a DNA mismatch repair enzyme, would be useful in identifying HNPCC probands. Monoclonal antibodies against hMLH1 and hMSH2, two DNA mismatch repair proteins which account for most HNPCC cancers, are commercially available. This study sought to investigate the potential utility of these antibodies in determining the expression status of these proteins in paraffin-embedded formalin-fixed tissue and to identify key technical protocol components associated with successful staining. A set of 20 colorectal carcinoma cases of known hMLH1 and hMSH2 mutation and expression status underwent immunoperoxidase staining at multiple institutions, each of which used their own technical protocol. Staining for hMSH2 was successful in most laboratories while staining for hMLH1 proved problematic in multiple labs. However, a significant minority of laboratories demonstrated excellent results including high discriminatory power with both monoclonal antibodies. These laboratories appropriately identified hMLH1 or hMSH2 inactivation with high sensitivity and specificity. The key protocol point associated with successful staining was an antigen retrieval step involving heat treatment and either EDTA or citrate buffer. This study demonstrates the potential utility of immunohistochemistry in detecting HNPCC probands and identifies key technical components for successful staining.

Molecular analysis of hereditary nonpolyposis colorectal carcinomas (HNPCC) has identified DNA mismatch repair deficiencies with resulting microsatellite instability (MSI) as a pathway of carcinogenesis that appears to be relevant for prognosis, treatment, and possibly prevention. In this study, expression of cell cycle proteins and other known prognostic markers is correlated with the microsatellite status of colorectal cancers (CRC). One hundred consecutive cases from the CRC Registry at Thomas Jefferson University were analyzed for MSI. Immunohistochemistry was performed for the mismatch repair proteins hMLH1 and hMSH2, tumor suppressor p53, apoptosis inhibitor bcl-2, cell cycle proteins p21(WAF1/CIP1), and p27 and the proliferation markers Ki-67 and topoisomerase II. High MSI (MSI-H) is significantly correlated with loss of either hMLH1 or hMSH2, presence of bcl-2, and absence of p53. p21(WAF1/CIP1) is positive in all tumors with MSI-H. Previous findings of a lower proliferation rate were confirmed with a topoisomerase II stain. Microsatellite stable (MSS) tumors generally express both MSH2 and MLH1. Other highly significant differences are positive p53 in 56% of MSS cases and negative bcl-2 in 98% of MSS cases. p27 expression is found in approximately 50% of all CRCs irrespective of the microsatellite status. MSI-H tumors follow the mutator pathway, with loss of expression of one mismatch repair protein, wild-type p53, lower proliferation, and positivity for p21(WAF1/CIP1). MSS tumors follow the suppressor pathway, characterized by p53 overexpression, higher proliferation, and absence of bcl-2 expression; p21(WAF1/CIP1) expression can be variable. These data provide a molecular basis for the clinical observation that patients with HNPCC appear to have a more favorable prognosis. HUM PATHOL 31:1506-1514., (Copyright 2000 by W.B. Saunders Company)

Aim: Colorectal cancer has been described in association with hyperplastic polyposis but the mechanism underlying this observation is unknown. The aim of this study was to characterise foci of dysplasia developing in the polyps of subjects with hyperplastic polyposis on the basis of DNA microsatellite status and expression of the DNA mismatch repair proteins hMLH1, hMSH2, and hMSH6., Materials and Methods: The material was derived from four patients with hyperplastic polyposis and between one and six synchronous colorectal cancers. Normal (four), hyperplastic (13), dysplastic (13), and malignant (11) samples were microdissected and a PCR based approach was used to identify mutations at 10 microsatellite loci, TGFbetaIIR, IGF2R, BAX, MSH3, and MSH6. Microsatellite instability-high (MSI-H) was diagnosed when 40% or more of the microsatellite loci showed mutational bandshifts. Serial sections were stained for hMLH1, hMSH2, and hMSH6., Results: DNA microsatellite instability was found in 1/13 (8%) hyperplastic samples, in 7/13 (54%) dysplastic foci, and in 8/11 (73%) cancers. None of the MSI-low (MSI-L) samples (one hyperplastic, three dysplastic, two cancers) showed loss of hMLH1 expression. All four MSI-H dysplastic foci and six MSI-H cancers showed loss of hMLH1 expression. Loss of hMLH1 in MSI-H but not in MSI-L lesions showing dysplasia or cancer was significant (p<0.001, Fisher's exact test). Loss of hMSH6 occurred in one MSI-H cancer and one MSS focus of dysplasia which also showed loss of hMLH1 staining., Conclusion: Neoplastic changes in hyperplastic polyposis may occur within a hyperplastic polyp. Neoplasia may be driven by DNA instability that is present to a low (MSI-L) or high (MSI-H) degree. MSI-H but not MSI-L dysplastic foci are associated with loss of hMLH1 expression. At least two mutator pathways drive neoplasia in hyperplastic polyposis. The role of the hyperplastic polyp in the histogenesis of sporadic DNA microsatellite unstable colorectal cancer should be examined.