Pulmonary large cell carcinoma - a diagnostically and clinically controversial entity - is defined as a non-small cell carcinoma lacking morphologic differentiation as either adenocarcinoma or squamous cell carcinoma, but suspected to represent an end-stage of poor differentiation of these tumor types. Given the recent advances in immunohistochemistry to distinguish adenocarcinoma and squamous cell carcinoma, and the recent insights that several therapeutically-relevant genetic alterations are distributed differentially in these tumors, we hypothesized that immunophenotyping may stratify large cell carcinomas into subsets with distinct profiles of targetable driver mutations. We therefore analyzed 102 large cell carcinomas by immunohistochemistry for TTF-1 and ΔNp63/p40 as classifiers for adenocarcinoma and squamous cell carcinoma, respectively, and correlated the resulting subtypes with 9 therapeutically-relevant genetic alterations characteristic of adenocarcinoma (EGFR, KRAS, BRAF, MAP2K1/MEK1, NRAS, ERBB2/HER2 mutations and ALK rearrangements) or more common in squamous cell carcinoma (PIK3CA and AKT1 mutations). The immunomarkers classified large cell carcinomas as variants of adenocarcinoma (n=62; 60%), squamous cell carcinoma (n=20; 20%), or marker-null (n=20; 20%). Genetic alterations were found in 38 cases (37%), including EGFR (n=1), KRAS (n=30), BRAF (n=2), MAP2K1 (n=1), ALK (n=3) and PIK3CA (n=1). All molecular alterations characteristic of adenocarcinoma occurred in tumors with immunoprofiles of adenocarcinoma or marker-null, but not in tumors with squamous immunoprofiles (combined mutation rate 50% vs 30% vs 0%, respectively; P<0.001), whereas the sole PIK3CA mutation occurred in a tumor with squamous profile (5%). Furthermore, marker-null large cell carcinomas were associated with significantly inferior disease-free (P<0.001) and overall (P=0.001) survival. In conclusion, the majority (80%) of large cell carcinomas can be classified by immunomarkers as variants of adenocarcinoma or squamous cell carcinoma, which stratifies these tumors into subsets with a distinct distribution of driver mutations and distinct prognoses. These findings have practical implications for diagnosis, predictive molecular testing and therapy selection.
large cell carcinoma; TTF-1; ΔNp63/p40; EGFR; KRAS; ALK
Intraductal carcinoma of the prostate is a marker of aggressive disease. However, intraductal carcinoma exists on a morphologic continuum with high grade prostatic intraepithelial carcinoma (PIN) and distinguishing intraductal carcinoma from PIN is a common diagnostic dilemma with significant clinical implications. We evaluated whether immunostains for PTEN and ERG can sensitively identify intraductal carcinoma and accurately distinguish it from high grade PIN. A combined immunostain for PTEN, ERG, p63 and CK903 was developed and validated. Radical prostatectomy specimens with lesions meeting criteria for intraductal carcinoma (n=45), intraductal cribriform proliferations falling short of intraductal carcinoma (n=15), and PIN lesions (n=39) were retrospectively identified and assessed for PTEN and ERG. Cytoplasmic PTEN loss was identified in 84% (38/45) of the intraductal carcinoma and 100% (15/15) of intraductal cribriform proliferation cases. In contrast, cytoplasmic PTEN loss was never observed in PIN (0/39) (p<0.0001). Of the 53 cases of intraductal carcinoma or intraductal cribriform proliferation with cytoplasmic PTEN loss, it was homogeneously lost in 42 cases (79%). Weak, focal nuclear positivity for PTEN was retained in 31 of these 42 cases (74%). ERG expression was identified in 58% (26/45) of intraductal carcinoma and 67% (10/15) of intraductal cribriform proliferations compared to 13% (5/39) of PIN. Concordance between the PTEN/ERG status of the intraductal carcinoma lesions and the concurrent invasive carcinoma was high (>95% and p<0.0001 for each), and substantially less for PIN and the concurrent invasive tumor (83% for PTEN and 67% for ERG; p=NS for each). Cytoplasmic PTEN loss occurs in the majority of intraductal carcinoma and intraductal cribriform proliferation cases. Cytoplasmic PTEN loss was never observed in PIN (100% specificity). Our study identifies PTEN loss as a potentially useful marker to distinguish intraductal carcinoma from PIN and provides a plausible molecular explanation for why intraductal carcinoma is associated with poor prognosis.
Prostatic adenocarcinoma; intraductal carcinoma; PTEN; ERG; immunohistochemistry
Epigenetics acts as an interface between environmental / exogenous factors, cellular responses and pathological processes. Aberrant epigenetic signatures are a hallmark of complex multifactorial diseases, including non-neoplastic disorders (e.g., cardiovascular diseases, hypertension, diabetes mellitus, autoimmune diseases, and some infectious diseases) and neoplasms (e.g., leukemias, lymphomas, sarcomas, and breast, lung, prostate, liver and colorectal cancers). Epigenetic signatures (DNA methylation, mRNA and microRNA expression, etc.) may serve as biomarkers for risk stratification, early detection, and disease classification, as well as targets for therapy and chemoprevention. DNA methylation assays are widely applied to formalin-fixed paraffin-embedded archival tissue specimens as clinical pathology tests. To better understand the interplay between etiologic factors, cellular molecular characteristics, and disease evolution, the field of “Molecular Pathological Epidemiology (MPE)” has emerged as an interdisciplinary integration of “molecular pathology” and “epidemiology”, with a similar conceptual framework to systems biology and network medicine. In contrast to traditional epidemiologic research including genome-wide association studies (GWAS), MPE is founded on the unique disease principle; that is, each disease process results from unique profiles of exposomes, epigenomes, transcriptomes, proteomes, metabolomes, microbiomes, and interactomes in relation to the macro-environment and tissue microenvironment. The widespread application of epigenomics (e.g., methylome) analyses will enhance our understanding of disease heterogeneity, epigenotypes (CpG island methylator phenotype, LINE-1 hypomethylation, etc.), and host-disease interactions. MPE may represent a logical evolution of GWAS, termed “GWAS-MPE approach”. Though epigenome-wide association study attracts increasing attention, currently, it has a fundamental problem in that each cell within one individual has a unique, time-varying epigenome. This article will illustrate increasing contribution of modern pathology to broader public health sciences, which attests pivotal roles of pathologists in the new integrated MPE science towards our ultimate goal of personalized medicine and prevention.
molecular pathologic epidemiology; genetics; omics; hypermethylation; hypomethylation; personalized therapy; unique tumor principle; CIMP; long interspersed nucleotide element
The distinction between benign and malignant cartilaginous tumors located peripherally in the bone may be a challenging task in surgical pathology. The aim of this study was to investigate inter-observer reliability in histological diagnosis of cartilaginous tumors in the setting of multiple osteochondromas and to evaluate possible histological parameters that could differentiate between osteochondroma, low- and high-grade secondary peripheral chondrosarcoma. Interobserver reliability was assessed by 12 specialized bone-tumor pathologists in a set of 38 cases. Substantial agreement on diagnosis among all the reviewers was observed (intraclass correlation coefficient = 0.78).Our study confirmed that mitotic figures and nuclear pleomorphism are hallmarks of high-grade secondary peripheral chondrosarcoma. However, despite the substantial agreement, we demonstrated that histology alone cannot distinguish osteochondroma from low-grade secondary peripheral chondrosarcoma in the setting of multiple osteochondromas, since nodularity, the presence of binucleated cells, irregular calcification, cystic/mucoid changes and necrosis were not helpful to indicate malignant transformation of an osteochondroma. On the other hand, among the concordant cases, the cartilage cap in osteochondroma was significantly less thick than in low- and high-grade secondary peripheral chondrosarcoma. Therefore, our study showed that a multidisciplinary approach integrating clinical and radio graphical features and the size of the cartilaginous cap in combination with a histological assessment are crucial to the diagnosis of cartilaginous tumors.
osteochondroma; chondrosarcoma; diagnostic criteria; cartilage tumors; bone tumors
SMARCB1 gene alterations were first described in highly malignant rhabdoid tumors of the kidney, brain (atypical teratoid/rhabdoid tumor) and soft tissue. An increasing number of tumors have now shown loss of SMARCB1 protein expression by immunohistochemistry including the majority of epithelioid sarcomas. However, investigations of SMARCB1 gene alterations in epithelioid sarcoma have produced conflicting results. The aim of this study was to evaluate SMARCB1 status using Sanger sequencing of the coding region and multiplex ligation dependent probe amplification, a rapid and sensitive method for detecting intragenic deletions and duplications, which has not been used in previous studies. Twenty-one epithelioid sarcomas of both classical and proximal type were selected for SMARCB1 gene testing and SMARCB1 immunohistochemistry. Nineteen of 21 (90%) epithelioid sarcomas were SMARCB1 negative by immunohistochemistry. Twelve of the 19 (63%) had adequate DNA recovery for evaluation. Ten of 12 (83%) tumors showed homozygous deletions of the gene. Two cases showed heterozygous deletions and polymorphisms, but no sequence mutations. These results confirm the high frequency of SMARCB1 deletions in epithelioid sarcoma and show that multiplex ligation dependent probe amplification is a reliable method for detection of deletions in these cases which can be performed on formalin-fixed, paraffin-embedded tissue. Given the high percentage of SMARCB1 alterations in epithelioid sarcoma, these findings argue against using SMARCB1 gene deletion as a tool in distinguishing them from malignant rhabdoid tumors.
Epithelioid sarcoma; malignant rhabdoid tumor; SMARCB1
The CD117 (KIT) protein is overexpressed in many human neoplasms including adenoid cystic carcinoma of salivary glands. To evaluate the function of c-kit-activating mutations in adenoid cystic carcinoma of the salivary gland, we studied 14 cases (13 primary, 1 cervical lymph node metastasis) from our institution. KIT protein expression was evaluated by immunohistochemistry using formalin-fixed paraffin-embedded tissue. Mutational analyses of c-kit extracellular (exon 9), juxtamembrane (exon 11) and tyrosine kinase domains (exons 13 and 17) were performed by polymerase chain reaction, clonal selection and DNA sequencing. All 14 cases demonstrated strong KIT expression by immunohistochemistry. Molecular analysis was successful in 8 of 14 cases, and c-kit missense point mutations were detected in seven of eight cases (88%) including seven in exon 11, two in exon 9, two in exon 13 and two in exon 17. Eight silent point mutations were detected in five cases. Two cases contained missense mutations in more than one exon. Different mutations were found in the primary tumor and the cervical lymph node metastasis of one patient. Point mutations in domains similar to those described in gastrointestinal stromal tumors were detected, including Pro551Leu and Lys558Glu (5′ end of exon 11), Leu576Phe (3′ end of exon 11), Val643Ala (exon 13) and Asn822Ser (exon 17). Additional novel point mutations in exons 9, 11, 13 and 17 were also identified. This study is the first to report c-kit gene mutations in primary adenoid cystic carcinoma of the salivary gland. Identification of such potential gain-of-function mutations in exon 11, and less frequently in exons 9, 13 and 17, suggests that KIT may be involved in the pathogenesis of adenoid cystic carcinoma of salivary glands. Our study raises a prospect of correlation of c-kit mutation and a potential treatment of adenoid cystic carcinoma with tyrosine kinase inhibitor (imatinib).
adenoid cystic carcinoma; c-kit gene mutations; KIT protein; salivary gland
While a strong etiologic relationship between human papillomavirus and a majority of oropharyngeal squamous cell carcinomas has been established, the role of human papillomavirus in non-oropharyngeal head and neck carcinomas is much less clear. Here, we investigated the prevalence and clinicopathologic significance of human papillomavirus and its reported biomarkers, CDKN2A(p16) and CDKN1A(p21), in laryngeal squamous cell carcinomas in patients treated either with primary surgery and postoperative radiation or with definitive radiation-based therapy. Nearly all of 76 tumors were keratinizing and none displayed the nonkeratinizing morphology that is typically associated with human papillomavirus infection in the oropharynx. However, CDKN2A(p16) immunohistochemistry was positive in 21 cases (28%), and CDKN1A(p21) in 34 (45%). CDKN2A(p16) and CDKN1A(p21) status strongly correlated with each other (p = 0.0038). Yet, only four cases were human papillomavirus positive by DNA in situ hybridization or by reverse transcriptase polymerase chain reaction E6/E7 mRNA [all four were CDKN2A(p16) and CDKN1A(p21) positive]. Unexpectedly, 9 additional tumors out of 20 CDKN2A(p16) positive cases harbored high-risk human papillomavirus DNA by polymerase chain reaction. For further investigation of this unexpected result, in situ hybridization for E6/E7 mRNA was performed on these 9 cases and all were negative, confirming the absence of transcriptionally active virus. Patients with CDKN1A(p21) positive tumors did have better overall survival (69% at 3 years) than those with CDKN1A(p21) negative tumors (51% at 3 years) [p = 0.045]. There was also a strong trend towards better overall survival in the CDKN2A(p16) positive group (p=0.058). Thus, it appears that the role of human papillomavirus is more complex in the larynx than in the oropharynx and that CDKN2A(p16) and CDKN1A(p21) expression may not reflect human papillomavirus driven tumors in most cases. Because of this, CDKN2A(p16) should not be used as a definitive surrogate marker of human papillomavirus driven tumors in the larynx.
Human papillomavirus; CDKN2A(p16); CDKN1A(p21); larynx; in situ hybridization; polymerase chain reaction
While cervical cancer screening relies on cervical cytology and high-risk HPV detection, histologic diagnosis, and specifically lesion grade, is the main parameter that drives clinical management of screen-positive women. Morphologically diagnosed squamous intraepithelial lesions (SIL/CIN) regress spontaneously in more than half of the cases, but identifying those likely to persist and progress is not currently possible based upon morphology. Lack of major capsid protein L1 expression has been suggested as a feature in progressive lesions, while expression of the minor capsid protein L2 has not been extensively evaluated. The goal of this study is to evaluate immunohistochemical expression of L1 and L2 in SILs in correlation with lesion grade.
One hundred fifty cervical specimens with SILs were selected based on HPV 16 or HPV 18 detection by Q-PCR. These included 89 low-grade SILs (LSIL/CIN1) and 123 high-grade SILs (75 HSIL/CIN2 and 48 HSIL/CIN3). More than one lesion/grade was identified in 53 specimens. The presence and grade of SIL was determined by a panel of pathologists. Capsid protein expression was assessed by immunohistochemistry using MAB 837 for L1 and RG-1 for L2. Lesions of different grades in the same specimen were scored separately.
Expression of capsid proteins was detected in 34/89 (40%) LSIL/CIN1, 5/75 (6%) HSIL/CIN2 and none of 48 HSIL/CIN3. L1 and L2 were co-expressed in the same area of the lesion in 22 cases. In addition, L1 alone was expressed in 6 lesions and L2 alone in 11 lesions. Among the cases with multiple lesion grades in the same specimen, none with HSIL/CIN 3 expressed capsid proteins in any portion/grade of the lesion.
HPV capsid proteins are expressed almost exclusively in LSIL/CIN1 and rarely in HSIL/CIN 2. Additional studies are warranted to examine lack of L1 and L2 expression in LSIL/CIN1 as a predictor of persistence or progression to HSIL/CIN 3, the precursor of cervical cancer.
cervical cancer precursors; biomarkers; HPV; capsid proteins; immunohistochemistry
The advent of Next-Generation sequencing technologies, which significantly increases the throughput and reduces the cost of large scale sequencing efforts, provides an unprecedented opportunity for discovery of novel gene mutations in human cancers. However, it remains a challenge to apply Next-Generation technologies to DNA extracted from formalin fixed paraffin embedded cancer specimens. We describe here the successful development of a custom DNA capture method using Next-Generation for detection of 140 driver genes in 5 formalin fixed paraffin embedded human colon cancer samples using an improved extraction process to produce high quality DNA. Isolated DNA was enriched for targeted exons and sequenced using the Illumina Next-Generation platform. An analytical pipeline using 3 software platforms to define single nucleotide variants was used to evaluate the data output. Approximately 250x average coverage was obtained with >96% of target bases having at least 30 sequence reads. Results were then compared to previously performed high throughput Sanger sequencing. Using an algorithm of needing a positive call from all 3 callers to give a positive result, 98% of the verified Sanger sequencing somatic driver gene mutations were identified by our method with a specificity of 90%. 13 insertions and deletions identified by Next-Generation were confirmed by Sanger sequencing. We also applied this technology to two components of a biphasic colon cancer which had strikingly differing histology. Remarkably, no new driver gene mutation accumulation was identified in the more undifferentiated component. Applying this method to profiling of formalin fixed paraffin embedded colon cancer tissue samples yields equivalent sensitivity and specificity for mutation detection as Sanger sequencing of matched cell lines derived from these cancers. This method directly enables high throughput comprehensive mutational profiling of colon cancer samples, and is easily extendable to enable targeted sequencing from formalin fixed paraffin embedded material for other tumor types.
next generation sequencing; colon cancer; driver gene mutations
We studied 24 spleens with extramedullary hematopoietic proliferation, a key feature of advanced stage Philadelphia chromosome-negative myeloproliferative neoplasms, obtained from 24 patients (14 primary myelofibrosis, 7 polycythemia vera, and 3 unclassifiable). Hematoxylin and eosin, reticulin and trichrome stains and immunohistochemical stains for myeloperoxidase, glycophorin-C, CD42b, CD34, CD117, CD8, nerve growth factor receptor and smooth muscle actin were evaluated. Clinical information was correlated with the morphologic findings. Three distinct histological patterns of extramedullary hematopoietic proliferation were recognized: diffuse (12), nodular (5), and mixed-nodular and diffuse (7). The preponderant lineage was granulocytic in diffuse, trilineage in nodular and erythroid in mixed extramedullary hematopoietic proliferation. Erythropoiesis was largely intravascular, granulopoiesis was within the splenic cords and megakaryopoiesis was observed in both locations. The stromal changes paralleled the histological pattern with preservation of the splenic stromal and vascular architecture in the diffuse areas as opposed to areas of nodular extramedullary hematopoietic proliferation. The morphologic features of splenic extramedullary hematopoietic proliferation did not correlate with specific subtypes of myeloproliferative neoplasms. The mean duration of follow-up from initial diagnosis was 80 months. Fifteen of the 24 patients died of disease: 8 of 12 (67%) with diffuse, 2 of 5 (40%) with nodular and 5 of 7 (71%) with mixed growth patterns. The mean duration from diagnosis to splenectomy was shorter in patients with diffuse (83 months) as compared to those with nodular extramedullary hematopoietic proliferation (127 months). Our study demonstrates that splenic extramedullary hematopoietic proliferation in Philadelphia chromosome-negative myeloproliferative neoplasms shows distinct histological patterns that do not correlate with disease subtypes but appear to suggest a trend between the histologic patterns and clinical behavior. These results suggest a different biology of the disease in the nodular and diffuse extramedullary hematopoietic proliferation groups.
Extramedullary hematopoiesis; myeloproliferative neoplasms; spleen
The angiogenic microenvironment has been known to be a component of angioimmunoblastic T-cell lymphoma since its initial characterization. We have shown that angioimmunoblastic T-cell lymphoma endothelial cells produce vascular endothelial growth factor-A (VEGFA), and participate in lymphoma progression. In squamous cell carcinoma, endothelial BCL2 expression induces a cross-talk with tumor cells through VEGFA, a major mediator of tumoral angiogenesis. In the present study, we analyzed BCL2 and VEGFA in 30 angioimmunoblastic T-cell lymphomas, using triple immunofluorescence to identify protein co-expression in well-characterized lymphoma cells and microenvironment neoangiogenic endothelial cells. Using quantitative real-time-PCR, we assessed mRNA expression levels in laser-microdissected endothelial and lymphoma cells.
In lymphoma cells, as in endothelial cells, BCL2 and VEGFA proteins were co-expressed. BCL2 was expressed only in neoangiogenic CD34+CD105+ endothelial cells. In laser-microdissected cells, mRNA studies showed a significant relationship between BCL2 and VEGFA levels in CD34+ endothelial cells, but not in CD3+CD10+ lymphoma cells, or in CD34+ endothelial cells from lymph node hyperplasia. Further study showed that, in AITL, BCL2 mRNA levels in CD34+CD105+ neoangiogenic endothelial cells also correlated with microvessel density, International Prognostic Index, Ann Arbor stage, bone marrow involvement and elevated LDH.
BCL2 expression by CD105+ neoangiogenic endothelial cells is related to tumor progression in angioimmunoblastic T-cell lymphoma.
Adult; Aged; Aged, 80 and over; Antigens, CD; analysis; Antigens, CD34; analysis; Case-Control Studies; Chi-Square Distribution; Disease Progression; Disease-Free Survival; Endothelial Cells; chemistry; immunology; pathology; Female; Fluorescent Antibody Technique; Humans; Immunoblastic Lymphadenopathy; genetics; immunology; metabolism; mortality; pathology; therapy; Kaplan-Meier Estimate; Laser Capture Microdissection; Lymph Nodes; blood supply; chemistry; immunology; pathology; Lymphoma, T-Cell; chemistry; genetics; immunology; mortality; pathology; therapy; Male; Microvessels; chemistry; immunology; pathology; Middle Aged; Multivariate Analysis; Neovascularization, Pathologic; Paris; Proportional Hazards Models; Proto-Oncogene Proteins c-bcl-2; analysis; genetics; RNA, Messenger; analysis; Real-Time Polymerase Chain Reaction; Receptors, Cell Surface; analysis; Risk Assessment; Risk Factors; Time Factors; Treatment Outcome; Tumor Markers, Biological; analysis; genetics; Tumor Microenvironment; Vascular Endothelial Growth Factor A; analysis; genetics; Angioimmunolbastic T-Cell Lyphoma, BCL2, CD105, endothelial cell, neoangiogenesis, VEGF
Proliferation rates in diffuse large B-cell lymphoma have been associated with conflicting outcomes in the literature, more often with high proliferation associated with poor prognosis. In most studies, the proliferation rate was estimated by a pathologist using an immunohistochemical stain for the monoclonal antibody Ki-67. We hypothesized that a quantitative image analysis algorithm would give a more accurate estimate of the proliferation rate, leading to better associations with survival. Eighty-four cases of diffuse large B-cell lymphoma were selected according to World Health Organization criteria. Ki-67 percent positivity estimated by the pathologist was recorded from the original report. The same slides were then scanned using an Aperio ImageScope, and Ki-67 percent positivity was calculated using a computer-based quantitative immunohistochemistry nuclear algorithm. In addition, chart review was performed and survival time was recorded. The Ki-67 percentage estimated by the pathologist from the original report versus quantitative image analysis was significantly correlated (p<0.001) but pathologist Ki-67 percentages were significantly higher than quantitative image analysis (p=0.021). There was less agreement at lower Ki-67 percentages. Comparison of Ki-67 percent positivity versus survival did not show significant association either with pathologist estimate or quantitative image analysis. However, while not significant, there was a trend of worse survival at higher proliferation rates detected by the pathologist but not by quantitative image analysis. Interestingly, our data suggest that the Ki-67 percent positivity as assessed by the pathologist may be more closely associated with survival outcome than that identified by quantitative image analysis. This may indicate that pathologists are better at selecting appropriate areas of the slide. More cases are needed to assess whether this finding would be statistically significant. Due to the good correlation between pathologist estimate and quantitative image analysis, there is no substantial benefit to using quantitative image analysis at this point in time.
diffuse large B-cell lymphoma; quantitative image analysis; virtual microscopy; proliferation rate; Ki-67; prognosis
Glucose transporter-1 (GLUT-1) mediates the transport of glucose across the cellular membrane. Its elevated levels and/or activation have been shown to be associated with malignancy. The aim of this study was to investigate GLUT-1 expression in pulmonary neuroendocrine carcinomas. Tissue microarray-based samples of 178 neuroendocrine carcinomas, including 48 typical carcinoids, 31 atypical carcinoids, 27 large cell neuroendocrine carcinomas and 72 small cell carcinomas from different patients, were studied immunohistochemically for GLUT-1 expression. Forty-seven percent (75/161) of pulmonary neuroendocrine carcinomas were immunoreactive with GLUT-1. GLUT-1 was observed in 7% (3/46) of typical carcinoid, 21% (6/29) of atypical carcinoid, 74% (17/23) of large cell neuroendocrine carcinoma and 78% (49/63) of small cell carcinoma. GLUT-1 expression correlated with increasing patient age (P = 0.01) and with neuroendocrine differentiation/tumor type (P < 0.001), but not with gender, tumor size or stage. GLUT-1 expression was seen in a characteristic membranous pattern of staining along the luminal borders or adjacent to necrotic areas. GLUT-1 expression was associated with an increased risk of death for neuroendocrine carcinomas as a group (risk ratio = 2.519; 95% confidence interval = 1.519–4.178; P < 0.001) and carcinoids (risk ratio = 4.262; 95% confidence interval = 1.472–12.343; P = 0.01). In conclusion, GLUT-1 is expressed in approximately half of the pulmonary neuroendocrine carcinomas and shows a strong correlation with neuroendocrine differentiation/grade, but not with other clinicopathologic variables. Further studies appear plausible to elucidate the prognostic significance of GLUT-1 expression in pulmonary carcinoids.
GLUT-1; neuroendocrine carcinoma; carcinoid; survival; lung
Identification of specific somatic gene alterations is crucial for the insight into the development, progression, and clinical behavior of individual cancer types. The recently discovered recurrent ERG rearrangement in prostate cancer (PCa) might represent a PCa specific alteration that has not been systematically assessed in tumors other than PCa. Aim of this study was to assess, whether the ERG rearrangement and the distinct deletion site between TMPRSS2 and ERG, both predominantly resulting in a TMPRSS2-ERG fusion, occurs in tumors other than PCa.
We assessed 54 different tumor types (2942 samples in total) for their ERG rearrangement status by FISH. To calibrate, we analyzed 285 PCa samples for the ERG rearrangement frequency. Additionally, we interrogated a high-resolution SNP data set across 3131 cancer specimens (26 tumor types) for copy number alterations.
None of the 54 different tumor types assessed by FISH harbored an ERG rearrangement, whereas the PCa samples revealed an ERG rearrangement in 31.2%–49.5%, depending on the cohort. Furthermore, within the 26 tumor types assessed for copy number alterations by SNP, the distinct deletion site between TMPRSS2 and ERG (21q22.2-3) was detectable exclusively in PCa.
Although Ewing's sarcoma and AML have known rearrangements rarely involving ERG, we hypothesize that the ERG rearrangement as well as the distinct deletion site on 21q22.2-3 between TMPRSS2 and ERG, are PCa specific genomic alterations. These observations provide further insight into the oncogenesis of PCa and might be critical for the development of ERG rearrangement assessment as a clinical tool.
ERG rearrangement; prostate cancer; carcinoma
Benign changes ranging from atrophy and inflammation to high-grade prostatic intraepithelial neoplasia (HGPIN) are common findings on prostate core needle biopsies. Although atrophy and inflammation may be precursors of prostate cancer, only HGPIN is currently recommended to be included in surgical pathology reports. To determine whether these benign findings increase prostate cancer risk, we conducted a case–control study nested within a historical cohort of 6692 men with a benign prostate specimen collected between 1990 and 2002. The analytic sample included 574 case–control pairs comprised of cases diagnosed with prostate cancer a minimum of 1 year after cohort entry and controls matched to cases on date and age at cohort entry, race, and type of specimen. The initial benign specimen was reviewed for presence of HGPIN, atrophy (simple, lobular, and partial) and inflammation (glandular and/or stromal). HGPIN significantly increased risk for prostate cancer (odds ratio (OR) = 2.00; 95% confidence interval (CI) = 1.25–3.20). Inflammation within the stromal compartment was associated with decreased risk (OR = 0.66; CI = 0.52–0.84), and diffuse stromal inflammation of severe grade had the strongest inverse association with risk (OR = 0.21; CI = 0.07–0.62). In a model adjusted for prostate-specific antigen (PSA) level at cohort entry and inflammation, simple atrophy was associated with a 33% increased prostate cancer risk that was marginally significant (P = 0.03). Clinicians should consider patterns and extent of inflammation when managing high-risk patients with negative biopsy results. Identifying benign inflammatory processes that underlie high PSA levels would help to reduce the number of unnecessary repeated prostate biopsies.
atrophy; cancer risk; prostate; stromal inflammation
Studies have shown that ALDH1A1 expression in the breast is associated with worse clinical outcome. ALDH1A1 inactivates cyclophosphamide, which is an integral agent in breast cancer chemotherapy regimens. The purposes of this study were to verify these results, to correlate ALDH1A1 expression with clinical outcome in patients treated with cyclophosphamide as part of the chemotherapy (adjuvant or neoadjuvant), and to evaluate ALDH1A1 as a useful marker to predict the clinical outcome of breast cancer subsets. A total of 513 primary breast cancers were studied. Tissue microarrays of the studied cases were stained with ALDH1A1. Key clinicopathological information was obtained. Disease-free survival and overall survival were calculated. Patients with neoadjuvant therapy who had substantial residual cancer burden (RCB) were included in the study. Fisher's exact test and Kaplan–Meier methods were used for statistical analysis. ALDH1A1 was expressed in 53 (10%) patients, with a higher frequency in triple negative, followed by HER2+, and finally hormonal receptor +/HER2− (P<0.0001). Tumors with advanced stage, node-positive, or larger tumor size were correlated with ALDH1A1 expression (P=0.006, P<0.0001, and P=0.05, respectively). ALDH1A1 expression was also correlated with worse disease-free survival (P<0.006) and overall survival (P<0.01) in patients who were treated with neoadjuvant chemotherapy. In all, 8 of 22 (36%) received neoadjuvant chemotherapy and died of disease-expressed ALDH1A1 (P=0.008). Similarly, 8 of 23 (35%) who received neoadjuvant chemotherapy and had tumor recurrence expressed this marker (P=0.002). The risk of recurrence was fivefold greater than negative ALDH1A1 tumors. The risk of recurrence became 11-fold greater when cyclophosphamide but not trastuzumab was part of the regimen. Our results are consistent with previous studies. Moreover, we found that ALDH1A1 could be a useful marker to predict worse clinical outcome after chemotherapy in the neoadjuvant setting with substantial RCB. However, a larger cohort is required to verify our results.
ALDH1A1; breast cancer; neoadjuvant
Molecular testing for mutations activating the mitogen-associated protein kinase signaling pathway is being used to help diagnose thyroid carcinomas. However, the prevalence of these mutations in thyroid lymphomas has not been reported. Therefore, we studied the prevalence of BRAF, NRAS, HRAS, and KRAS mutations in 33 thyroid lymphomas and correlated the mutational status with the clinical, pathologic, cytogenetic, and immunophenotypic findings. Eleven cases were also tested for PAX8/PPARγ translocations. The lymphomas included 25 diffuse large B-cell lymphomas, 6 extranodal marginal zone lymphomas of mucosa associated lymphoid tissue type, and 2 follicular lymphomas. Seventeen diffuse large B-cell lymphomas were germinal center type, 6 non-germinal center type and 2 unclassifiable (Hans algorithm). None of the cases had an associated thyroid carcinoma. Mutations of the BRAF gene were identified in 6 (24%) diffuse large B-cell lymphomas (three D594G in germinal center diffuse large B cell lymphomas, two K601N in germinal center diffuse large B cell lymphomas, and one V600E in non-germinal center diffuse large B cell lymphomas) and of the NRAS gene in two (8%) non-germinal center diffuse large B-cell lymphomas (Q61K and Q61H). BRAF and NRAS mutations were not found in any extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue type or follicular lymphoma. HRAS and KRAS mutations were not identified in any of the cases, nor were PAX8/PPARγ translocations found. Thus, interpretation of finding a BRAF or NRAS mutation in the thyroid, particularly in preoperative thyroid aspirates, must take into account the differential diagnosis of a lymphoma. In addition to the diagnostic importance, our data also demonstrate that alteration in the mitogen-associated protein kinase pathway may play a role in the pathogenesis of some large B-cell lymphomas of the thyroid with potential therapeutic implications.
thyroid; lymphoma; BRAF; RAS; mutations
Epithelial–mesenchymal transition is an important mechanism of epithelial tumor progression, local invasion and metastasis. The E-cadherin (CDH1) repressor SLUG (SNAI2) and the basic helix–loop–helix transcription factor TWIST1 inhibit CDH1 expression in poorly differentiated malignancies as inducers of epithelial– mesenchymal transition. Epithelial–mesenchymal transition has been implicated in progression from well to poorly differentiated/anaplastic thyroid carcinoma but the expression of SNAI2 and TWIST1 proteins and their phenotypic association in human thyroid cancers has not been extensively studied. We examined the expression of SNAI2, TWIST1 and CDH1 by immunohistochemistry in a panel of well-differentiated and anaplastic thyroid cancers and by qRT-PCR in thyroid cell lines. Ten normal thyroids, 33 follicular adenomas, 56 papillary thyroid carcinomas including 28 follicular variants, 27 follicular carcinomas and 10 anaplastic thyroid carcinomas were assembled on a tissue microarray and immunostained for SNAI2, TWIST1 and CDH1. Most (8/10) anaplastic thyroid carcinomas demonstrated strong nuclear immunoreactivity for SNAI2 with associated absence of CDH1 in 6/8 cases (75%). TWIST1 was expressed in 5/10 anaplastic thyroid carcinomas with absence of CDH1 in 3/5 (60%) cases. These findings were confirmed in whole sections of all anaplastic thyroid carcinomas and in a separate validation set of 10 additional anaplastic thyroid carcinomas. All normal thyroids, follicular adenomas, papillary and follicular thyroid carcinomas were negative for SNAI2 and TWIST1 (P<0.0001) and all showed strong diffuse immunoreactivity for CDH1 (P=0.026). Expression of SNAI2, TWIST1 and CDH1 mRNA varied in a normal thyroid, papillary carcinoma and two anaplastic thyroid carcinoma cell lines tested, but the highest levels of CDH1 mRNA were detected in the normal thyroid cell line while the anaplastic thyroid carcinoma cell line demonstrated the highest levels of SNAI2 and TWIST1 mRNA. Our findings support the role of epithelial–mesenchymal transition in the development of anaplastic thyroid carcinoma.
anaplastic thyroid carcinoma; CDH1; SNAI2; thyroid carcinoma; thyroid cell lines; TWIST1
Approximately 45% of sporadic well-differentiated pancreatic neuroendocrine tumors harbor mutations in either ATRX (alpha thalassemia/mental retardation X-linked) or DAXX (death domain-associated protein). These novel tumor suppressor genes encode nuclear proteins that interact with one another and function in chromatin remodeling at telomeric and peri-centromeric regions. Mutations in these genes are associated with loss of their protein expression and correlate with the alternative lengthening of telomeres phenotype. Patients with multiple endocrine neoplasia-1 (MEN-1) syndrome, genetically defined by a germ line mutation in the MEN1 gene, are predisposed to developing pancreatic neuroendocrine tumors and thus represent a unique model for studying the timing of ATRX and DAXX inactivation in pancreatic neuroendocrine tumor development. We characterized ATRX and DAXX protein expression by immunohistochemistry and telomere status by telomere-specific fluorescence in situ hybridization in 109 well-differentiated pancreatic neuroendocrine lesions from 28 MEN-1 syndrome patients. The study consisted of 47 neuroendocrine microadenomas (<0.5 cm), 50 pancreatic neuroendocrine tumors (≥0.5 cm), and 12 pancreatic neuroendocrine tumor lymph node metastases. Expression of ATRX and DAXX was intact in all 47 microadenomas, and none showed the alternative lengthening of telomeres phenotype. ATRX and/or DAXX expression was lost in 3 of 50 (6%) pancreatic neuroendocrine tumors. In all three of these, tumor size was ≥3 cm, and loss of ATRX and/or DAXX expression correlated with the alternative lengthening of telomeres phenotype. Concurrent lymph node metastases were present for two of the three tumors, and each metastasis displayed the same changes as the primary tumor. These findings establish the existence of ATRX and DAXX defects and the alternative lengthening of telomeres phenotype in pancreatic neuroendocrine tumors in the context of MEN-1 syndrome. The observation that ATRX and DAXX defects and the alternative lengthening of telomeres phenotype occurred only in pancreatic neuroendocrine tumors measuring ≥ 3 cm and their lymph node metastases suggests that these changes are late events in pancreatic neuroendocrine tumor development.
alternative lengthening of telomeres; ATRX; DAXX; MEN-1; multiple endocrine neoplasia-1; pancreatic neuroendocrine tumor
It has been reported previously that: 1) normal breast epithelial cells that are CD24-/44+ express higher levels of stem/progenitor cell associated genes; 2) cancer cells that have undergone epithelial to mesenchymal transition display CD24-/44+ cell surface expression, a marker for breast cancer stem cells; 3) loss of E-cadherin is a preliminary step in epithelial to mesenchymal transition; and 4) vimentin is a marker of mesenchymal phenotype.
We hypothesized that stem cell subpopulations would be more frequent in metastatic than in primary tumors. Therefore we assessed by immunohistochemical analysis, tissue microarrays containing tissue from primary and associated metastatic breast cancers for expression of CD24, CD44, E-cadherin and vimentin to evaluate candidate cancer-initiating cell populations in breast cancer subtypes and metastatic lesions. The occurrence of CD24-/44+ and CD24+/44- cells did not differ in primary vs matched lymph node or distant and locoregional metastatic lesions; E-cadherin expression was decreased in primary vs lymph node metastases (P=0.018) but not decreased in distant and locoregional metastases relative to primary tumor, while vimentin, was more frequently expressed in lymph node and distant and locoregional metastases (P=0.013, P=0.004) than in matched primary cancers. Thus, the frequency of CD24-/44+ cells does not differ in metastases relative to the primary breast cancer but differs by tumor stage and subtype.
Breast cancer subtypes; CD44; CD24; Vimentin; E-cadherin; Metastases
Increased numbers of T regulatory (Treg) cells are found in B-chronic lymphocytic leukemia, but the nature and function of these Tregs remains unclear. Detailed characterization of the Tregs in chronic lymphocytic leukemia has not been performed and the degree of heterogeneity of among these cells has not been studied to date. Using 15-color flow cytometry we show that Treg cells, defined using CD4, CD25, and forkhead box P3 (FOXP3), can be divided into multiple complex subsets based on markers used for naïve, memory, and effector delineation as well as markers of Treg activation. Furthermore FOXP3+ cells can be identified among CD4+CD25− as well as CD8+CD4− populations in increased proportions in patients with chronic lymphocytic leukemia compared with healthy donors. Significantly different frequencies of naïve and effector Tregs populations are found in healthy donor controls compared with donors with chronic lymphocytic leukemia. A population of CCR7+CD39+ Tregs was significantly associated with chronic lymphocytic leukemia. This population demonstrated slightly reduced suppressive activity compared with total Tregs or Tregs of healthy donors. These data suggest that FOXP3-expressing cells, particularly in patients with chronic lymphocytic leukemia are much more complex for Treg sub-populations and transitions than previously reported. These findings demonstrate the complexity of regulation of T-cell responses in chronic lymphocytic leukemia and illustrate the use of high-dimensional analysis of cellular phenotypes in facilitating understanding of the intricacies of cellular immune responses and their dysregulation in cancer.
chronic lymphocytic leukemia; immunophenotyping; Treg
Endoscopic resection is a less invasive treatment than esophagectomy for superficial esophageal squamous cell carcinoma, but patients with lymph node metastasis need additional treatment after endoscopic resection. The purpose of this study was to establish a set of indicators to identify superficial esophageal squamous cell carcinoma patients at a high risk of metastasis. 271 superficial esophageal squamous cell carcinoma esophagectomy cases were reviewed retrospectively. The relationships between clinicopathological parameters and immunohistochemical findings (p53, Cyclin D1, EGFR and VEGF) on tissue microarrays, on the one hand, and lymph node metastasis were assessed by univariate and multivariate logistic regression analyses. Patients with intraluminal masses and ulcerated masses had a high risk of lymph node metastasis. Patients with superficial esophageal squamous cell carcinoma 1) thinner than 1200µm; 2) confined to the mucosa; 3) with submucosal invasion <250µm; 4) with submucosal invasion ≥250µm but with negative VEGF expression and well/moderately differentiated or basaloid histology; or 5) with submucosal invasion ≥250µm but with weak VEGF expression and well differentiated histology had almost no risk of lymph node metastasis. We recommend endoscopic resection for all erosive, papillary and plaque-like superficial esophageal squamous cell carcinomas where endoscopic resection is clinically feasible, and esophagectomy for all other erosive, papillary and plaque-like cases and all intraluminal masses and ulcerated tumors. No additional treatment is needed for endoscopic resection cases with superficial esophageal squamous cell carcinoma 1) thinner than 1200µm; 2) confined to the mucosa; 3) with submucosal invasion <250µm; 4) with submucosal invasion ≥250µm but with negative VEGF expression and well/moderately differentiated or basaloid histology; or 5) with submucosal invasion ≥250µm but with weak VEGF expression and well differentiated histology. These clinical and pathological criteria should enable more accurate selection of patients for these procedures.
superficial cancer; esophageal cancer; squamous cell carcinoma; endoscopic resection; lymph node metastasis
Aldo-keto reductase family 1B10 (AKR1B10) exhibits more restricted lipid substrate specificity (including farnesal, geranylgeranial, retinal and carbonyls), a n d metabolizing these lipid substrates plays a crucial role in promoting carcinogenesis. Overexpression of AKR1B10 has been identified in smoking-related carcinomas such as lung cancer. As development of pancreatic cancer is firmly linked to smoking, the aim of the present study was to examine the expression and oncogenic role of AKR1B10 in pancreatic adenocarcinoma. AKR1B10 expression was analyzed in 50 paraffin-embedded clinical pancreatic cancer samples using immunohistochemistry. Oncogenic function of AKR1B10 was examined in pancreatic carcinoma cells in vitro using western blotting and siRNA approaches, mainly on cell apoptosis and protein prenylation including KRAS protein and its downstream signals. Immunohistochemistry analysis revealed that AKR1B10 over-expressed in 70% (35/50) of pancreatic adenocarcinomas and majority of pancreatic intraepithelial neoplasia, but not in adjacent morphologically normal pancreatic tissue. Compared to a normal pancreatic ductal epithelial cell (HPDE6E7), all of six cultured pancreatic adenocarcinoma cell lines had a over-expression of AKR1B10 using immunoblotting, which correlated with increase of enzyme activity. siRNA-mediated silencing of AKR1B10 expression in pancreatic cancer cells resulted in 1) increased cell apoptosis, 2) increased non-farnesyled HDJ2 protein, and 3) decreased membrane-bound prenylated KRAS protein and its downstream signaling molecules including phosphorylated ERK and MEK and membrane-bound E-cadherin. Our findings provide first time evidence of that AKR1B10 is a unique enzyme involved in pancreatic carcinogenesis possibly via modulation of cell apoptosis and protein prenylation.
pancreatic adenocarcinoma; AKR1B10; prenylation; smoking; immunohistochemistry
Lymphoplasmacytic lymphomas and marginal zone lymphomas of nodal, extra-nodal and splenic types account for 10% of non-Hodgkin lymphomas. They are similar at the cell differentiation level, sometimes making difficult to distinguish them from other indolent non-Hodgkin lymphomas. To better characterize their genetic basis, we performed array-based comparative genomic hybridization in 101 marginal zone lymphomas (46 MALT, 35 splenic and 20 nodal marginal zone lymphomas) and 13 lymphoplasmacytic lymphomas. Overall, 90.1% exhibited copy-number abnormalities. Lymphoplasmacytic lymphomas demonstrated the most complex karyotype (median=7 copy-number abnormalities), followed by MALT (4), nodal (3.5) and splenic marginal zone lymphomas (3). A comparative analysis exposed a group of copy-number abnormalities shared by several or all the entities with few disease-specific abnormalities. Gain of chromosomes 3, 12 and 18 and loss of 6q23–q24 (TNFAIP3) were identified in all entities. Losses of 13q14.3 (MIRN15A-MIRN16-1) and 17p13.3-p12 (TP53) were found in lymphoplasmacytic and splenic marginal zone lymphomas; loss of 11q21–q22 (ATM) in nodal, splenic marginal zone and lymphoplasmacytic lymphomas; loss of 7q32.1–q33 in MALT, splenic and lymphoplasmacytic lymphomas. Abnormalities affecting the NF-kB pathway were observed in 70% of MALT and lymphoplasmacytic lymphomas and 30% of splenic and nodal marginal zone lymphomas, suggesting distinct roles of this pathway in the pathogenesis/progression of these subtypes. Elucidation of the genetic alterations contributing to the pathogenesis of these lymphomas may guide to design specific therapeutic approaches.
MZL; LPL; aCGH; copy-number abnormalities; NF-kB pathway