The Co-mutational Spectrum Determines the Therapeutic Response in Murine FGFR2 Fusion-Driven Cholangiocarcinoma.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer and a highly lethal malignancy. Chemotherapeutic options are limited, but a considerable subset of patients harbors genetic lesions for which targeted agents exist. Fibroblast growth factor receptor 2 (FGFR2) fusions belong to the most frequent and therapeutically relevant alterations in ICC, and the first FGFR inhibitor was recently approved for the treatment of patients with progressed, fusion-positive ICC. Response rates of up to 35% indicate that FGFR-targeted therapies are beneficial in many but not all patients. Thus far, no established biomarkers exist that predict resistance or response to FGFR-targeted therapies in patients with ICC. APPROACH AND RESULTS: In this study, we use an autochthonous murine model of ICC to demonstrate that FGFR2 fusions are potent drivers of malignant transformation. Furthermore, we provide preclinical evidence that the co-mutational spectrum acts not only as an accelerator of tumor development, but also modifies the response to targeted FGFR inhibitors. Using pharmacologic approaches and RNA-interference technology, we delineate that Kirsten rat sarcoma oncogene (KRAS)-activated mitogen-activated protein kinase signaling causes primary resistance to FGFR inhibitors in FGFR2 fusion-positive ICC. The translational relevance is supported by the observation that a subset of human FGFR2 fusion patients exhibits transcriptome profiles reminiscent of KRAS mutant ICC. Moreover, we demonstrate that combination therapy has the potential to overcome primary resistance and to sensitize tumors to FGFR inhibition. CONCLUSIONS: Our work highlights the importance of the co-mutational spectrum as a significant modifier of response in tumors that harbor potent oncogenic drivers. A better understanding of the genetic underpinnings of resistance will be pivotal to improve biomarker-guided patient selection and to design clinically relevant combination strategies.

Risk estimation for biliary tract cancer: Development and validation of a prognostic score.

BACKGROUND & AIMS: Biliary tract cancer is a rare tumour entity characterized by a poor prognosis. We aimed to identify prognostic factors and create a prognostic score to estimate survival. METHODS: Clinical data of the training set, consisting of 569 patients treated from 2000 to 2010 at Hannover Medical School, were analysed. A prognostic model defining three prognostic risk groups was derived from Cox regression analyses. The score was applied and validated in an independent cohort of 557 patients from four different German centres. RESULTS: Median overall survival (OS) was 14.5 months. If complete resection was performed, the patients had a significantly improved OS (23.9 months; n=242) as compared to patients with non-resectable tumours (9.1 months; n=329, P<.0001). Based on univariable and multivariable analyses of clinical data, a prognostic model was created using variables available before treatment. Those were age, metastasis, C-reactive protein (CRP), international normalized ratio (INR) and bilirubin. The prognostic score distinguished three groups with a median OS of 21.8, 8.6 and 2.6 months respectively. The validation cohort had a median OS of 20.2, 14.0 and 6.5 months respectively. The prognostic impact of the score was independent of the tumour site and of treatment procedures. CONCLUSIONS: Here, we identified prognostic factors and propose a prognostic score to estimate survival, which can be applied to all patients independent of tumour site and before initial treatment. Further validation in prospective trials is required.

Patterns and challenges of treatment sequencing in patients with hepatocellular carcinoma: Experience from a German referral center.

BACKGROUND AND AIM: Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancers. Several local and systemic therapies are available for patients with HCC depending on the stage of the disease. In clinical practice, treatment decision-making, and sequencing may be very heterogeneous. METHODS: In this study, we retrospectively analyzed treatment algorithms in 2101 patients with HCC treated from 2000 to 2015 at Hannover Medical School, Germany. RESULTS: Transarterial chemoembolization was the most common initial treatment (n = 545; 25.9%), followed by resection (n = 435, 20.7%), local-ablative procedures (n = 283, 13.5%), systemic therapies (n = 275, 13.1%), and liver transplantation (n = 52; 2.5%). Most patients were treated only once (n = 960; 59.6%). A total of 433 (26.9%) and 160 (9.9%) patients received a second line and third line treatment after recurrent or progressive disease. Patients with more than one treatment line were diagnosed at significantly earlier disease stages (P < 0.001). Using binary logistic regression, AFP ≤ 200 µg/L, albumin > 36 g/L, and small tumor size (≤50 mm) were identified as predictors of achieving more than one treatment line. Subsequent treatment stage migration to a therapy suggested for the next advanced stage occurred only in 56.9%, whereas 43.1% received treatments suggested for earlier disease stages. Only 16% of all treated patients received systemic therapy in the salvage setting. CONCLUSION: Most patients were treated only once, and only a minority of patients received systemic treatment. The high dropout rate for subsequent therapies needs to be considered within therapy decision-making. There is an urgent need for prospective studies to define the best time point when to switch patients from local to systemic therapies.

Epstein-Barr virus infection and altered control of apoptotic pathways in posttransplant lymphoproliferative disorders.

Posttransplant lymphoproliferative disorders (PTLD) represent a spectrum of lymphoid diseases complicating the clinical course of transplant recipients. Most PTLD are Epstein-Barr virus (EBV) associated with viral latency type III. Several in vitro studies have revealed an interaction between EBV latency proteins and molecules of the apoptosis pathway. Data on human PTLD regarding an association between Bcl-2 family proteins and EBV are scarce. We analyzed 60 primary PTLD for expression of 8 anti- (Bcl-2, Bcl-XL, and Mcl-1) and proapoptotic proteins (Bak and Bax), the so-called BH3-only proteins (Bad, Bid, Bim, and Puma), as well as the apoptosis effector cleaved PARP by immunohistochemistry. Bim and cleaved PARP were both significantly (p = 0.001 and p = 5.251e-6) downregulated in EBV-positive compared to EBV-negative PTLD [Bim: 6/40 (15%), cleaved PARP: 10/43 (23%), vs. Bim: 13/16 (81%), cleaved PARP: 12/17 (71%)]. Additionally, we observed a tendency toward increased Bcl-2 protein expression (p = 0.24) in EBV-positive PTLD. Hence, we provide evidence of a distinct regulation of Bcl-2 family proteins in EBV-positive versus negative PTLD. The low-expression pattern of the proapoptotic proteins Bim and cleaved PARP together with the high-expression pattern of the antiapoptotic protein Bcl-2 by trend in EBV-positive tumor cells suggests disruption of the apoptotic pathway by EBV in PTLD, promoting survival signals in the host cells.

microRNA profiling in Epstein-Barr virus-associated B-cell lymphoma.

The Epstein-Barr virus (EBV) is an oncogenic human Herpes virus found in ∼15% of diffuse large B-cell lymphoma (DLBCL). EBV encodes miRNAs and induces changes in the cellular miRNA profile of infected cells. MiRNAs are small, non-coding RNAs of ∼19-26 nt which suppress protein synthesis by inducing translational arrest or mRNA degradation. Here, we report a comprehensive miRNA-profiling study and show that hsa-miR-424, -223, -199a-3p, -199a-5p, -27b, -378, -26b, -23a, -23b were upregulated and hsa-miR-155, -20b, -221, -151-3p, -222, -29b/c, -106a were downregulated more than 2-fold due to EBV-infection of DLBCL. All known EBV miRNAs with the exception of the BHRF1 cluster as well as EBV-miR-BART15 and -20 were present. A computational analysis indicated potential targets such as c-MYB, LATS2, c-SKI and SIAH1. We show that c-MYB is targeted by miR-155 and miR-424, that the tumor suppressor SIAH1 is targeted by miR-424, and that c-SKI is potentially regulated by miR-155. Downregulation of SIAH1 protein in DLBCL was demonstrated by immunohistochemistry. The inhibition of SIAH1 is in line with the notion that EBV impedes various pro-apoptotic pathways during tumorigenesis. The down-modulation of the oncogenic c-MYB protein, although counter-intuitive, might be explained by its tight regulation in developmental processes.

Molecular diagnostics and therapies for gastrointestinal tumors: a real-world experience.

PURPOSE: Several targeted agents demonstrated efficacy in early clinical trials for gastrointestinal (GI) cancers, but in many cases, phase-III trials and/or approval by the European Medicines Agency (EMA) are lacking. The primary focus of this study was to assess the regulatory processes associated with use and reimbursement of off-label treatment in precision oncology and to evaluate the benefit of targeted therapy in a real-world population in Germany. METHODS: Our cohort comprises 137 patients with GI cancers and is biased towards cancer entities with a high frequency of known targetable alterations, such as cholangiocarcinoma. Genetic testing was used to identify molecular targets, and therapy response was evaluated based on CT scans. RESULTS: A molecular target for precision oncology was identified in 53 patients and 43 requests for cost coverage were submitted to health insurance companies. 60% of the requests received approval after initial application and another 7% after appeal. Half of the rejected requests were denied despite ESCAT IA level evidence. The median time between initiation of molecular testing and start of therapy was 75 days. 35 patients received matched targeted therapies (n = 28) or, in the case of MSI, immunotherapy (IO) (n = 7). We observed a trend in favor of molecular therapy when compared to the immediate prior treatment. CONCLUSION: Relevant treatment options were identified by molecular testing in a significant subset of patients. When targeted therapies that lack EMA approval are considered, treatment initiation may be delayed by the duration of the molecular analysis and the regulatory processes.

Pathological processing techniques and final diagnosis of breast cancer sentinel lymph nodes.

BACKGROUND: Recommendations for intraoperative and postoperative breast sentinel lymph node (SLN) processing differ widely. Micrometastases and isolated tumor cells (ITC) have recently been proposed as prognostically and therapeutically relevant. We compared 3 SLN protocols with regard to intraoperative and postoperative diagnosis. MATERIALS AND METHODS: SLN in cohort I (270 patients) were intraoperatively assessed by stereomicroscopy. Intraoperative frozen section (IFS) was used only in stereomicroscopically suspicious SLN. In cohort II (197 patients), all SLN were examined with only 1 IFS. Final SLN workup in cohorts I and II consisted of complete step sectioning with immunohistochemistry. In cohort III (268 patients) 2 or more IFS were performed followed by 3 step sections and immunohistochemistry. RESULTS: pN1 stages were significantly higher in cohorts I and II (33.3% and 34.0% respectively) than in cohort III (24.6%). Intraoperative false negativity for the detection of metastases (pN1) ranged from 54.4% (cohort I) and 35.8% (cohort II) to 21.2% (cohort III). In contrast, ITC were detected significantly more frequently in cohort I (9.3%) and cohort II (14.7%) than in cohort III (1.9%). CONCLUSIONS: Higher rates of SLN metastases and ITC in cohort I/II compared to cohort III suggest that IFS may result in tissue loss thus increasing the risk of missing metastases. Sparse IFS but complete postoperative SLN workup with step sectioning and immunohistochemistry provides more accurate information regarding minimal disease in SLN, but often results in delayed axillary lymph node dissection. This is important for preoperative patient information and recommendations in SLN processing protocols.

Chromosomal translocations t(4;14), t(11;14) and proliferation rate stratify patients with mature plasma cell myelomas into groups with different survival probabilities: a molecular epidemiologic study on tissue microarrays.

Plasma cell myelomas (PMs) exhibit clinical and molecular heterogeneity. To date, morphology and immunohistochemistry on bone marrow trephines are of limited value to stratify patients into different prognostic categories. However, some chromosomal translocations are of prognostic and/or of predictive importance in PMs. In this study, the prognostic significance of morphology, CyclinD1 expression, proliferation index (Mib1) and presence of the translocations FGFR3/IgH [t(4;14)] and CCND1/IgH [t(11;14)] are compared in 119 patients with PM. Immunohistochemistry and fluorescence in situ hybridization analysis were carried out on a tissue microarray containing bone marrow trephines. Hundred and one PMs showed a mature morphology whereas 10 were immature. All but one PM carrying a translocation showed a mature morphology. Patients with a t(4;14) (12%) had a statistically significant shorter 1-year survival (P=0.004), whereas those with a t(11;14) (21%) had a trend towards a better clinical outcome. CyclinD1 protein expression was not significantly associated with survival. Besides the t(4;14), an immature morphology (P<0.001) and a proliferation index (Mib1) of more than 10% (P=0.002) were associated with a significantly worse outcome. A high occurrence of strong CyclinD1 protein expression in the tumor cells was predictive of either a t(11;14) or of a low level amplification of the CCND1 gene, suggesting that different molecular mechanisms may have lead to an over-expression of the CyclinD1 protein in PMs. These findings demonstrate that a high proliferation rate and translocations involving the IgH locus can stratify mature PMs into groups with distinct survival probabilities.

The effect of adjuvant chemotherapy in patients with intrahepatic cholangiocarcinoma: a matched pair analysis.

PURPOSE: The purpose of this study was to identify prognostic factors of patients with intrahepatic cholangiocarcinoma (ICC) treated with resection and to investigate the effect of adjuvant chemotherapy (CT). METHODS: Patients with ICC diagnosed between 2000 and 2015 treated at Hannover Medical School were included. Clinicopathologic characteristics were analyzed in univariate and multivariate analysis. In a matched pair survival analysis, patients with or without adjuvant CT were matched by these prognostic factors. RESULTS: Two hundred and ten patients were included. Median survival was 28.7 months, 1-, 3-, and 5-year survival rates were 72.8%, 29.6%, and 14.1%, respectively. In multivariate analysis, lymph node involvement (p = 0.006, HR 1.84), larger tumor size (p = 0.013, HR 1.79), vascular invasion (p = 0.038, HR 1.70), and prolongation of prothrombin time (p < 0.001, HR 4.20) were significantly related to poor survival. Thirty-nine patients received adjuvant CT of which 60% had lymph node involvement. Each 25 patients with and without adjuvant CT were matched to the identified prognostic factors. The median survival of patients with adjuvant CT was 33.5 months, compared to 18 months in the control group (p = 0.002). The 1-, 3-, and 5-year survival rates were 96, 36, and 12%, compared to 60, 4, and 0% in non-treated patients. CONCLUSIONS: We identified several prognostic factors for patients with ICC treated with resection. Our data support the use of adjuvant CT in patients with ICC. The results of prospective randomized controlled studies will clarify the role of adjuvant CT in the future.

Ultrasonic decalcification offers new perspectives for rapid FISH, DNA, and RT-PCR analysis in bone marrow trephines.

The requisite analyses on bone marrow biopsies are increasing: Molecular analyses such as fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), and reverse transcriptase (RT)-PCR are demanded in addition to morphology and immunohistochemistry to improve diagnostic accuracy. Moreover, analysis of certain molecular prognostic or predictive biomarkers is increasingly mandatory in the assessment of hematologic diseases. In some circumstances, only formalin fixed, bone-containing tissue is available for molecular analysis. Because various fixation and decalcification procedures can impair DNA and RNA quality, there is an urgent need for standardized decalcification protocols which allow FISH and PCR analysis. In this study we developed a routinely applicable decalcification protocol to optimize the molecular analysis method although preserving morphology and immunohistochemical results. Therefore, we compared 2 different approaches including ultrasonic decalcification versus nonultrasonic procedures and ethylenediaminetetraacetate-based reagents versus acid-based ones. In our hands, the combined use of ultrasound and ethylenediaminetetraacetate-based reagents permits successful interphase FISH, PCR, and RT-PCR analysis whereas concomitantly preserving morphology and antigeneicity.

IRF8 is associated with germinal center B-cell-like type of diffuse large B-cell lymphoma and exceptionally involved in translocation t(14;16)(q32.33;q24.1).

Chromosomal translocations involving the immunoglobulin loci represent frequent oncogenic events in B-cell lymphoma development. Although IRF8 (ICSBP-1) protein expression has been demonstrated in germinal center B-cells and related lymphomas in a single report, the IRF8 gene was not described as an immunoglobulin heavy chain (IGH) translocation partner. In a discovery-driven approach we searched for new translocation partners of IGH in diffuse large B-cell lymphoma (DLBCL) by long distance inverse polymerase chain reaction (LDI-PCR) and Sanger sequencing. A t(14;16)(q32.33;q24.1) IGH/IRF8 was detected in a CD5+de novo DLBCL, confirmed by translocation specific PCR and fluorescence in situ hybridization (FISH) analysis. No further IRF8 aberration could be identified either by LDI-PCR in an additional five CD5+DLBCLs or by FISH on 78 formalin-fixed paraffin-embedded biopsies. Subsequent screening for IRF8 by immunohistochemistry revealed IRF8 expression in 18/78 (23%), correlating with a germinal center B-cell-like (GCB) type of DLBCL. This hitherto unknown translocation t(14;16)(q32.33;q24.1) is likely to represent the initiator of a multistep lymphomagenesis in a CD5+de novo DLBCL.

Apoptotic enteropathy caused by antimetabolites and TNF-α antagonists.

AIMS: To investigate whether drugs others than mycophenolic acid and ipilimumab might cause graft-versus-host-like apoptotic enteropathy, the clinicopathological findings in four patients were examined who had developed watery diarrhoea and apoptotic enteropathy (three cases from colon and one case from ileal pouch) after intake of antimetabolites (methotrexate and capecitabine) and/or tumour necrosis factor-α inhibitors (etanercept and infliximab). METHODS: The clinical charts, endoscopy reports and intestinal biopsies from all endoscopies were reviewed for all patients. Biopsies were evaluated semiquantitatively for apoptosis of basal crypts, dilated damaged crypts, defined as cystically dilated crypts with flattened degenerated epithelium containing apoptotic debris and few neutrophils, and mucosal architecture. Further, the presence of intraepithelial lymphocytes, chronic inflammatory cells in the lamina propria and mucosal ulcerations was recorded and immunohistochemical analysis for human cytomegalovirus and herpes simplex virus was performed. RESULTS: Endoscopic examination revealed normal mucosa in two patients, whereas the other two showed focal ulcerations. Histological changes included increased apoptosis of basal crypts, the presence of dilated damaged crypts and architecture distortion. In all cases, a temporal association between drug intake and/or dose increase, and onset of diarrhoea, was observed, and no convincing evidence of other potentially underlying causes of colitis/enteritis was found, including infections. CONCLUSIONS: Pathologists should be aware of the expanding spectrum of drugs that can cause apoptotic enteropathy, including antimetabolites and tumour necrosis factor-α inhibitors.

Modelling of a genetically diverse evolution of Systemic Mastocytosis with Chronic Myelomonocytic Leukemia (SM-CMML) by Next Generation Sequencing.

BACKGROUND: Systemic mastocytosis (SM) is a heterogenous, clonal mast cell (MC) proliferation, rarely associated with clonal hematologic non-mast cell lineage disease (SM-AHNMD). KIT (D816V) is regarded as driver-mutation in SM-AHNMD. METHODS: DNA isolated from peripheral blood (PB) of an SM-CMML patient was investigated with targeted next generation sequencing. Variants were verified by Sanger sequencing and further characterized in the SM part of the bone marrow trephine (BMT), normal tissue, and FACS sorted PB cell subpopulations. FINDINGS: Low coverage deep-sequencing (mean 10x) on a GS 454 Junior revealed two as yet unreported SNVs (CBFA2T3 and CLTCL1), both germ-line mutations. High coverage (mean 1674x) targeted re-sequencing on an Ion Proton revealed 177 variants in coding regions. Excluding SNPs, the final list comprised 11 variants. Among these, TET2 (p.Thr1027fs, p.Cys1263Ser) and RUNX1 (p.Asn109Ser) were identified in in the peripheral blood and the SM part of BMT, but not in normal tissue. Furthermore, Sanger sequencing of PB cells revealed similar signal intensities for both TET2 mutations in FACS sorted CD34+ precursor cells and CD16+ granulocytes comparable to signals in the SM part of BMT. In contrast, RUNX1 exhibited a double intensity in CD34+ cells compared to the SM part of BMT and a homozygous variant signal in granulocytes. Both TET2 and RUNX1 mutations were not detectable in B- and T-cells. CONCLUSION: We present a heterozygous triple-mutation pattern (KIT, TET2, RUNX1) in mast cells (SM disease part) with additional LOH of RUNX1 in granulocytes (CMML disease part). These identified mutations allow a more detailed insight into a multistep pathogenesis which suggests a common tumor progenitor in SM-CMML.

MicroRNA profiling of Epstein-Barr virus-associated NK/T-cell lymphomas by deep sequencing.

The Epstein-Barr virus (EBV) is an oncogenic human Herpes virus involved in the pathogenesis of nasal NK/T-cell lymphoma. EBV encodes microRNAs (miRNAs) and induces changes in the host cellular miRNA profile. MiRNAs are short non-coding RNAs of about 19-25 nt length that regulate gene expression by post-transcriptional mechanisms and are frequently deregulated in human malignancies including cancer. The microRNA profiles of EBV-positive NK/T-cell lymphoma, non-infected T-cell lymphoma and normal thymus were established by deep sequencing of small RNA libraries. The comparison of the EBV-positive NK/T-cell vs. EBV-negative T-cell lymphoma revealed 15 up- und 16 down-regulated miRNAs. In contrast, the majority of miRNAs was repressed in the lymphomas compared to normal tissue. We also identified 10 novel miRNAs from known precursors and two so far unknown miRNAs. The sequencing results were confirmed for selected miRNAs by quantitative Real-Time PCR (qRT-PCR). We show that the proinflammatory cytokine interleukin 1 alpha (IL1A) is a target for miR-142-3p and the oncogenic BCL6 for miR-205. MiR-142-3p is down-regulated in the EBV-positive vs. EBV-negative lymphomas. MiR-205 was undetectable in EBV-negative lymphoma and strongly down-regulated in EBV-positive NK/T-cell lymphoma as compared to thymus. The targets were confirmed by reporter assays and by down-regulation of the proteins by ectopic expression of the cognate miRNAs. Taken together, our findings demonstrate the relevance of deregulated miRNAs for the post-transcriptional gene regulation in nasal NK/T-cell lymphomas.

Multiple infarcted regenerative nodules in liver cirrhosis after decompensation of cirrhosis: a case series.

INTRODUCTION: Liver cirrhosis is a common disease with many known complications. Cirrhosis represents a clinical spectrum, ranging from asymptomatic liver disease to hepatic decompensation. Manifestations of hepatic decompensation include variceal bleeding, ascites, hepatic encephalopathy, hepatorenal syndrome, hepatopulmonary syndrome, portopulmonary hypertension and hepatocellular carcinoma. There are reports about infarcted regenerative nodules in cirrhotic livers after gastrointestinal hemorrhage. CASE PRESENTATION: We report three Caucasian patients (one female and two male patients; ages: 52, 54 and 60 years) with decompensated liver cirrhosis, who showed newly infarcted regenerative nodules at necropsy. Two of them suffered from gastric variceal bleeding. Histopathology showed extensive infarction in all three cases.Hemorrhage and inflammatory changes were also observed around the infarcted regenerative nodules. CONCLUSION: These patients showed focal liver lesions, to be considered in the differential diagnosis of cirrhotic livers. Infarcted regenerative nodules may be underdiagnosed in patients with decompensation of cirrhosis. In order to differentiate these lesions from malignant tumors, serial imaging seems to be helpful. However, the main differential diagnosis should be an abscess. It is important to know the wide spectrum of image appearances of these lesions. Hypotension can lead to a reduction of portal and arterial liver flow. Since variceal bleeding or septic shock can induce hypotension - as observed in our patients - we conclude that this leads to infarction of such nodules.

D2-40 and calretinin - a tissue microarray analysis of 341 malignant mesotheliomas with emphasis on sarcomatoid differentiation.

Anti-calretinin antibodies are useful to differentiate adenocarcinomas from malignant mesotheliomas of the lung. Therefore, calretinin expression is rarely reported for sarcomatoid mesotheliomas. Anti-podoplanin antibodies (eg D2-40) react with lymphatic endothelia, Kaposi's sarcoma, lymphangioma and mesotheliomas. For the interpretation of spindle cell lesions of the pleura, knowledge of calretinin and D2-40 expression frequencies in sarcomatoid mesothelioma is desirable. To systematically investigate the sensitivity of calretinin and D2-40 antibodies in epithelioid and sarcomatoid areas of malignant mesotheliomas, a tissue microarray with 341 malignant mesotheliomas, including 112 epithelioid, 46 sarcomatoid and 183 biphasic tumors was constructed. Epithelioid and sarcomatoid differentiated tumor areas were clearly separated within the tissue microarray. Expression of calretinin and D2-40 was separately studied in epithelioid and sarcomatoid areas by immunohistochemistry. Calretinin expression was found in 91% of epithelioid and 57% of sarcomatoid tumor areas. D2-40 immunostaining was present in 66% of the epithelioid and 30% of the sarcomatoid tumor areas. A combination of calretinin and D2-40 increased the sensitivity in epithelioid tumor areas to 0.96 and in sarcomatoid tumor areas to 0.66. These data indicate that a combination of calretinin and D2-40 will improve diagnostic accuracy for spindle cell lesions of the pleura, whereas almost all epithelioid mesotheliomas are identified by calretinin alone.