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1.  Claudin Expression in High Grade Invasive Ductal Carcinoma of the Breast: Correlation with the Molecular Subtype 
Claudin proteins are a major component of the tight junctions. Dysregulation of claudin protein expression has been described in a number of malignancies. Gene expression profiling has stratified breast cancers into distinct molecular subtypes: luminal, HER2+ and basal-like. Recently, a novel claudin-low molecular subtype has been described. In this study we correlated the expression patterns of claudins with the molecular subtypes of breast cancer. On the basis of immunohistochemical expression 226 grade 3 invasive ductal carcinomas were stratified into 65 luminal (ER+), 65 HER2 positive (HER2+), 86 basal-like, including 14 metaplastic carcinomas (ER−, HER2−, CK5/6 and /or EGFR+), and 10 unclassified. Tissue microarrays were analyzed for expression of claudins 1, 3, 4, 7 and 8 by immunohistochemistry and scored semiquantitatively. High levels of expression were detected in 17% of all cases for claudin 1, 32% claudin 3, 41% claudin 4, 44% claudin 7, and 40% claudin 8. Luminal cancers exhibited increased claudins 7 and 8; basal-like tumors demonstrated increased claudins 1 and 4 expression. Low expression of all five claudins was detected in 30 of 226 cases (13%) and this group was designated “claudin-low”. The majority of the claudin-low subgroup were basal-like cancers (23 of 30, 77%). In contrast, only 1 of 30 (3%) claudin-low tumors were of the luminal phenotype and 6 of 30 cases (20%) were HER2+ (P<0.001). Within the basal-like subgroup, 64% of the metaplastic and 19% of the non-metaplastic tumors were claudin-low. The claudin-low group was strongly associated with disease recurrence (P=0.0093). In conclusion, this study is the first to comprehensively examine the differential expression of claudins 1, 3, 4, 7 and 8 in the molecular subtypes of high grade breast cancer. Claudin-low subtype is a frequent phenomenon in metaplastic and basal-like breast cancer and appears to be a strong predictor of disease recurrence.
doi:10.1038/modpathol.2012.187
PMCID: PMC4000969  PMID: 23222490
Breast; Carcinoma; Claudin
2.  Chromophobe hepatocellular carcinoma with abrupt anaplasia: a proposal for a new subtype of hepatocellular carcinoma with unique morphological and molecular features 
Hepatocellular carcinomas exhibit heterogeneous morphologies by routine light microscopy. Although some morphologies represent insignificant variations in growth patterns, others may represent unrecognized subtypes of hepatocellular carcinoma. Identification of these subtypes could lead to separation of hepatocellular carcinomas into discrete groups with unique underlying genetic changes, prognosis, or therapeutic responses. In order to identify potential subtypes, two pathologists independently screened a cohort of 219 unselected hepatocellular carcinoma resection specimens and divided cases into potential subtypes. One of these promising candidate subtypes was further evaluated using histological and molecular techniques. This subtype was characterized by a unique and consistent set of histological features: smooth chromophobic cytoplasm, abrupt focal nuclear anaplasia (small clusters of tumor cells with marked nuclear anaplasia in a background of tumor cells with bland nuclear cytology), and scattered microscopic pseudocysts—we designate this variant as ‘chromophobe hepatocellular carcinoma with abrupt anaplasia’. Thirteen cases were identified (6% of all hepatocellular carcinomas), including 6 men and 7 women with an average age of 61 years. Six cases occurred in cirrhotic livers. Serum AFP was elevated in 6 out of 10 cases. There were a variety of underlying liver diseases, but cases were enrichment for chronic hepatitis B, P = 0.006. Interestingly, at the molecular level, this variant was strongly associated with the alternative lengthening of telomere (ALT) phenotype by telomere FISH. ALT is a telomerase-independent mechanism of telomere maintenance and is found in approximately 8% of unselected hepatocellular carcinomas. In contrast, 11/12 (92%) of the cases of chromophobe hepatocellular carcinoma with abrupt anaplasia were ALT-positive. In summary, we propose that chromophobe hepatocellular carcinoma with abrupt anaplasia represents a new subtype of hepatocellular carcinoma with unique morphological and molecular features.
doi:10.1038/modpathol.2013.68
PMCID: PMC3974906  PMID: 23640129
ALT; hepatocellular carcinoma; telomere
3.  Relationship between PTEN, DNA Mismatch Repair, and Tumor Histotype in Endometrial Carcinoma: Retained Positive Expression of PTEN Preferentially Identifies Sporadic Non-Endometrioid Carcinomas 
Loss of PTEN (phosphatase and tensin homolog) expression and microsatellite instability are two of the more common molecular alterations in endometrial carcinoma. From the published literature, it is controversial as to whether there is a relationship between these different molecular mechanisms. Therefore, a cohort of 187 pure endometrioid and non-endometrioid endometrial carcinomas, carefully characterized as to clinical and pathological features, was examined for PTEN sequence abnormalities and the immunohistochemical expression of PTEN and the DNA mismatch repair proteins MLH1, MSH2, MSH6 and PMS2. MLH1 methylation analysis was performed when tumors had loss of MLH1 protein. Mismatch repair protein loss was more frequent in endometrioid carcinomas compared to non-endometrioid carcinomas, a difference primarily attributable to the presence of MLH1 methylation in a greater proportion of endometrioid tumors. Among the non-endometrioid group, mixed endometrioid/non-endometrioid carcinomas were the histotype that most commonly had loss of a mismatch repair protein. In endometrioid tumors, the frequency of PTEN loss measured by immunohistochemistry and mutation did not differ significantly between the mismatch repair protein intact or mismatch repair protein loss groups, suggesting that PTEN loss is independent of mismatch protein repair status in this group. However, in non-endometrioid carcinomas, both intact positive PTEN immunohistochemical expression and PTEN wild type were highly associated with retained positive expression of mismatch repair proteins in the tumor. Relevant to screening endometrial cancers for Lynch Syndrome, an initial PTEN immunohistochemistry determination may be able to replace the use of four mismatch repair immunohistochemical markers in 63% of patients with non-endometrioid endometrial carcinoma. Therefore, PTEN immunohistochemistry, in combination with tumor histotype, is a useful adjunct in the clinical evaluation of endometrial carcinomas for Lynch Syndrome.
doi:10.1038/modpathol.2013.67
PMCID: PMC3720775  PMID: 23599155
4.  KRAS mutations are associated with solid growth pattern and tumor-infiltrating leukocytes in lung adenocarcinoma 
KRAS mutations define a clinically-distinct subgroup of lung adenocarcinoma patients, characterized by smoking history, resistance to EGFR-targeted therapies, and adverse prognosis. Whether KRAS- mutated lung adenocarcinomas also have distinct histopathologic features is not well established. We tested 180 resected lung adenocarcinomas for KRAS and EGFR mutations by high-sensitivity mass spectrometry-based genotyping (Sequenom) and PCR-based sizing assays. All tumors were assessed for the proportion of standard histologic patterns (lepidic, acinar, papillary, micropapillary, solid and mucinous), several other histologic and clinical parameters, and TTF-1 expression by immunohistochemistry. Among 180 carcinomas, 63 (35%) had KRAS mutations (KRAS+), 35 (19%) had EGFR mutations (EGFR+), and 82 (46%) had neither mutation (KRAS-/EGFR-). Solid growth pattern was significantly over-represented in KRAS+ carcinomas: the mean ± standard deviation for the amount of solid pattern in KRAS+ carcinomas was 27 ± 34% compared to 3 ± 10% in EGFR+ (P<0.001) and 15 ± 27% in KRAS-/EGFR- (P=0.033) tumors. Furthermore, at least focal (>20%) solid component was more common in KRAS+ (28/63; 44%) compared to EGFR+ (2/35; 6%; P<0.001) and KRAS-/EGFR- (21/82; 26%; P=0.012) carcinomas. KRAS mutations were also over-represented in mucinous carcinomas, and were significantly associated with the presence of tumor-infiltrating leukocytes and heavier smoking history. EGFR mutations were associated with non-mucinous non-solid patterns, particularly lepidic and papillary, lack of necrosis, lack of cytologic atypia, hobnail cytology, TTF-1 expression, and never/light smoking history. In conclusion, extended molecular and clinicopathologic analysis of lung adenocarcinomas reveals a novel association of KRAS mutations with solid histology and tumor-infiltrating inflammatory cells, and expands on several previously recognized morphologic and clinical associations of KRAS and EGFR mutations. Solid growth pattern was recently shown to be a strong predictor of aggressive behavior in lung adenocarcinomas, which may underlie the unfavorable prognosis associated with KRAS mutations in these tumors.
doi:10.1038/modpathol.2013.74
PMCID: PMC3732528  PMID: 23619604
KRAS; EGFR; lung; adenocarcinoma; TTF-1
5.  Frequency of mutations and polymorphisms in borderline ovarian tumors of known cancer genes 
Borderline ovarian tumors represent an understudied subset of ovarian tumors. Most studies investigating aberrations in borderline tumors have focused on KRAS/BRAF mutations. In this study we conducted an extensive analysis of mutations and single nucleotide polymorphisms in borderline ovarian tumors.
Using the Sequenom MassARRAY platform we investigated 160 mutations/polymorphisms in 33 genes involved in cell signalling, apoptosis, angiogenesis, cell cycle regulation, and cellular senescence.
Of 52 tumors analysed, 33 were serous, 18 mucinous and 1 endometrioid. KRAS c.35G>A p.Gly12Asp mutations were detected in 8 tumors (6 serous and 2 mucinous), BRAF V600E mutations in 2 serous tumors, and PIK3CA H1047Y and PIK3CA E542K mutations in a serous and an endometrioid BOT respectively. CTNNB1 mutation was detected in a serous tumor. Potentially functional polymorphisms were found in VEGF, ABCB1, FGFR2 and PHLPP2. VEGF polymorphisms were the most common and detected at 4 loci. PHLPP2 polymorphisms were more frequent in mucinous as compared to serous tumors (p=0.04), with allelic imbalance in one case.
This study represents the largest and most comprehensive analysis of mutations and functional single nucleotide polymorphisms in borderline ovarian tumors to date. At least 25% of borderline ovarian tumors harbour somatic mutations associated with potential response to targeted therapeutics.
doi:10.1038/modpathol.2012.194
PMCID: PMC3856435  PMID: 23174937
Borderline; Ovarian; Sequenom; Tumours
6.  Minichromosome maintenance protein 7 as a potential prognostic factor for progression-free survival in high-grade serous carcinomas of the ovary 
Minichromosome maintenance protein 7 (MCM7) is involved in replicative licensing and synthesis of DNA. It was previously identified as an overexpressed gene in high-grade serous carcinomas compared with serous borderline tumors of the ovary in cDNA microarray studies. In this study, we sought to validate MCM7 expression in 342 ovarian tumors on tissue microarrays. MCM7 expression was quantified as the MCM7 labeling index, and it was independently generated by two methods: a score provided by manual review of each sample by a pathologist observer and by an automated cellular imaging system. Analyses of MCM7 scores indicated a high degree of concordance and distribution between the observer- and machine-generated MCM7 labeling indexes. MCM7 expression was significantly higher in high-grade serous carcinomas than in serous borderline tumors or other histological subtypes of ovarian cancer. For both observer- and machine-derived scores, univariate analyses indicated the significant association of a high MCM7 labeling index with better progression-free survival in high-grade serous carcinomas. These results suggest the clinical importance of MCM7 expression in high-grade serous carcinomas of the ovary and the need for further evaluation of MCM7 as a potential prognostic factor in ovarian cancer.
doi:10.1038/modpathol.2010.202
PMCID: PMC3964599  PMID: 21076460
immunohistochemistry; MCM7; ovarian cancer
7.  Extracapsular extension is a poor predictor of disease recurrence in surgically treated oropharyngeal squamous cell carcinoma 
Extracapsular extension in squamous cell carcinoma nodal metastases usually predicts worse outcome. However, there are no standard histologic grading criteria for extracapsular extension, and there have been few studies on oropharyngeal squamous cell carcinoma alone. We studied the extent of extracapsular extension utilizing a novel grading system and correlated grades with outcomes while controlling for p16 status. A cohort of surgically treated oropharyngeal squamous cell carcinoma cases were reviewed and metastases graded as 0 (within substance of node), 1 (filling subcapsular sinus with thickened capsule/pseudocapsule, but no irregular peripheral extension), 2 (≤1mm beyond capsule), 3 (>1mm beyond capsule), or 4 (no residual nodal tissue or architecture; ‘soft tissue metastasis’). There were 101 cases, for which p16 was positive in 90 (89%). Extracapsular extension grades did not correlate with nodal size (P = 0.28) or p16 status (P = 0.8). In follow up, 10 patients (10%) had disease recurrence with only 3 of 64 (5%) grade 0–3 cases and 7 of 37 (19%) with grade 4 recurring (P = 0.04). Grade 4 extracapsular extension was associated with poorer survival (P < 0.01). However, grade 4 extracapsular extension correlated with higher T-stage (P = 0.02), and in multivariate analysis, was not significantly associated with poorer overall (P = 0.14) disease-free (P = 0.2), or disease-specific survival (P = 0.09). The impact of extracapsular extension in nodal metastases is limited in oropharyngeal squamous cell carcinoma. Only extracapsular extension grade 4 associates with poorer outcomes, but not independently of T-stage and other variables.
doi:10.1038/modpathol.2011.105
PMCID: PMC3925389  PMID: 21701534
extracapsular extension; metastasis; oropharyngeal; p16; squamous cell carcinoma; survival
8.  Ovarian and endometrial endometrioid carcinomas have distinct CTNNB1 and PTEN mutation profiles 
Ovarian endometrioid carcinomas and endometrial endometrioid carcinomas share many histological and molecular alterations. These similarities are likely due to a common endometrial epithelial precursor cell of origin, with most ovarian endometrioid carcinomas arising from endometriosis. To directly compare the mutation profiles of two morphologically similar tumor types, we performed select exon capture sequencing on a panel of genes: ARID1A, PTEN, PIK3CA, KRAS, CTNNB1, PPP2R1A, TP53. We found that PTEN mutations are more frequent in low-grade endometrial endometrioid carcinomas (67.0%) compared to low-grade ovarian endometrioid carcinomas (16.6%) (p<0.0001). In contrast, CTNNB1 mutations are significantly different in low-grade ovarian endometrioid carcinomas (53.3%) compared to low-grade endometrial endometrioid carcinomas (27.5%) (p<0.0057). This difference in CTNNB1 mutation frequency may be reflective of the distinct microenvironments; the epithelial cells lining an endometriotic cyst within the ovary are exposed to a highly oxidative environment that promotes tumorigenesis. Understanding the distinct mutation patterns found in the PI3K and Wnt pathways of ovarian and endometrial endometrioid carcinomas may provide future opportunities for stratifying patients for targeted therapeutics.
doi:10.1038/modpathol.2013.107
PMCID: PMC3915240  PMID: 23765252
Endometrial; ovarian; endometrioid; carcinoma; PTEN; CTNNB1; mutations
9.  Expression of Angiopoietin-TIE System Components in Angiosarcoma 
Angiosarcoma is an aggressive malignancy of endothelial differentiation. Potential roles of the endothelial angiopoietin-tunica internal endothelial cell kinase (ANGPT-TIE) system in angiosarcoma diagnosis, pathogenesis, prognosis and treatment are undefined. To examine the expression and prognostic significance of angiopoietin-1, angiopoietin-2, TIE1 and TEK (TIE2) proteins in angiosarcoma, we immunohistochemically evaluated clinically annotated human angiosarcoma samples. Correlations of protein expression with overall survival and pathologic features were explored. The cohort included 51 patients diagnosed with angiosarcoma at age 30-86 years old (median 67). The 5-year overall survival was 45% with a median of 26 months. Moderate to strong expression of angiopoietin-1, TIE1 and TEK (TIE2) was identified in the majority of angiosarcomas and moderate to strong expression of angiopoietin-2 was observed in 42% of angiosarcomas. Increased angiopoietin-1 expression correlated with improved survival. Non-significant trends toward longer survival were also observed with increased TIE1 and TEK (TIE2) expression. Increased expression of angiopoietin-2, TIE1 and TEK (TIE2) was associated with vasoformative architecture. No differences in expression of these proteins were observed when patients were segregated by age, gender, presence or absence of metastases at diagnosis, primary tumor location, radiation association or the presence of necrosis. We conclude that components of the ANGPT-TIE system are commonly expressed in angiosarcomas. Reduced expression of these proteins is associated with non-vasoformative and clinically more aggressive lesions.
doi:10.1038/modpathol.2013.43
PMCID: PMC3706492  PMID: 23558570
Angiosarcoma; angiopoietin; sarcoma; TEK; TIE
10.  Immune response in melanoma: an in-depth analysis of the primary tumor and corresponding sentinel lymph node 
The sentinel lymph node is the initial site of metastasis. Down-regulation of anti-tumor immunity plays a role in nodal progression. Our objective was to investigate the relationship between immune modulation and sentinel lymph node positivity, correlating it with outcome in melanoma patients. Lymph node/primary tissues from melanoma patients prospectively accrued and followed at New York University Medical Center were evaluated for the presence of regulatory T-cells (Foxp3+) and dendritic cells (conventional: CD11c+, mature: CD86+) using immunohistochemistry. Primary melanoma immune cell profiles from sentinel lymph node-positive/-negative patients were compared. Logistic regression models inclusive of standard-of-care/immunologic primary tumor characteristics were constructed to predict the risk of sentinel lymph node positivity. Immunological responses in the positive sentinel lymph node were also compared to those in the negative non-sentinel node from the same nodal basin and matched negative sentinel lymph node. Decreased immune response was defined as increased regulatory T-cells or decreased dendritic cells. Associations between the expression of these immune modulators, clinicopathologic variables, and clinical outcome were evaluated using univariate/multivariate analyses. Primary tumor conventional dendritic cells and regression were protective against sentinel lymph node metastasis (odds ratio=0.714, 0.067; P=0.0099, 0.0816, respectively). Anti-tumor immunity was down-regulated in the positive sentinel lymph node with an increase in regulatory T-cells compared to the negative non-sentinel node from the same nodal basin (P=0.0005) and matched negative sentinel lymph node (P=0.0002). The positive sentinel lymph node also had decreased numbers of conventional dendritic cells compared to the negative sentinel lymph node (P<0.0001). Adding sentinel lymph node regulatory T-cell expression improved the discriminative power of a recurrence risk assessment model using clinical stage. Primary tumor regression was associated with prolonged disease-free (P=0.025) and melanoma-specific (P=0.014) survival. Our results support an assessment of local immune profiles in both the primary tumor and sentinel lymph node to help guide therapeutic decisions.
doi:10.1038/modpathol.2012.43
PMCID: PMC3882943  PMID: 22425909
melanoma; sentinel lymph node biopsy; lymphatic metastases; regulatory T-cells; dendritic cells
11.  The Effect of Prolonged Cold Ischemia Time on Estrogen Receptor Immunohistochemistry in Breast Cancer 
To facilitate accurate detection of estrogen receptor expression in breast tumors, the American Society of Clinical Oncology/College of American Pathologists recommends that cold ischemia time be kept under 1 h. However, data to address the upper threshold of cold ischemia time are limited. While it is our routine practice to keep cold ischemia time under 1 h for breast core biopsy specimens, this is difficult for surgical specimens because of the comprehensive intraoperative assessment performed at our institution. In this retrospective study, we compared estrogen receptor immunohistochemical staining results in paired breast tumor core biopsy specimens and resection specimens with cold ischemia times ranging from 64 to 357 min in 97 patients. The staining category (≥10%, positive; 1-9%, low positive; <1%, negative) between the core biopsy and resection specimens changed for 5 patients (5%). The weighted Kappa statistic for estrogen receptor staining category between the two specimen types was 0.86 (95% confidence interval, 0.74-0.99), indicating good concordance. The difference in the percentage of estrogen receptor staining between core biopsy and resection was not significantly associated with cold ischemia time (P = 0.81, Spearman correlation). Although we did not observe significant associations between the difference in estrogen receptor staining in the two specimen types and cold ischemia time after placing the patients in three groups of ‘increase’, ‘decrease’ and ‘no change’ using a difference of 25% in estrogen receptor staining percentage as the cutoff, a trend of decreased estrogen receptor staining with cold ischemia time > 2 h was detected. No statistically significant association was found between the change of estrogen receptor staining and the history of neoadjuvant chemotherapy. Our findings indicate that prolonged cold ischemia time up to 4 h (97% of our cohort) in the practice setting of our institution has minimal clinical impact on estrogen receptor immunohistochemical expression in breast tumors.
doi:10.1038/modpathol.2012.135
PMCID: PMC3881416  PMID: 22899286
estrogen receptor; breast; cold ischemia time; immunohistochemistry
12.  Secondary mutation in a coding mononucleotide tract in MSH6 causes loss of immunoexpression of MSH6 in colorectal carcinomas with MLH1/PMS2 deficiency 
Immunohistochemical staining for DNA mismatch repair proteins may be affected by various biological and technical factors. Staining variations that could potentially lead to erroneous interpretations have been recognized. A recently recognized staining variation is the significant reduction of staining for MSH6 in some colorectal carcinomas. The frequency and specific characteristics of this aberrant MSH6 staining pattern, however, have not been well analyzed. In this study of 420 colorectal carcinoma samples obtained from patients fulfilling the Revised Bethesda Guidelines, we detected 9 tumors (2%) showing extremely limited staining for MSH6 with positive staining present in <5% of the tumor cells. Our analyses showed that these tumors belonged to two distinct categories: (1) MLH1 and/or PMS2 protein-deficient carcinomas (n=5, including 1 with a pathogenic mutation in PMS2); and (2) MLH1, PMS2 and MSH2 normal but with chemotherapy or chemoradiation therapy before surgery (n=4). To test our hypothesis that somatic mutation in the coding region microsatellite of the MSH6 gene might be a potential underlying mechanism for such limited MSH6 staining, we evaluated frameshift mutation in a (C)8 tract in exon 5 of the MSH6 gene in seven tumors that had sufficient DNA for analysis, and detected mutation in four; all four tumors belonged to the MLH1/PMS2-deficient group. In conclusion, our data outline the main scenarios where significant reduction of MSH6 staining is more likely to occur in colorectal carcinoma, and suggest that somatic mutations of the coding region microsatellites of the MSH6 gene is an underlying mechanism for this staining phenomenon in MLH1/PMS2-deficient carcinomas.
doi:10.1038/modpathol.2012.138
PMCID: PMC3793326  PMID: 22918162
DNA mismatch repair; hereditary non-polyposis colorectal carcinoma; immunohistochemistry; Lynch syndrome; microsatellite instability
13.  Frequency, molecular pathology and potential clinical significance of partial chromosome 3 aberrations in uveal melanoma 
The clinical significance of partial chromosome 3 alteration in uveal melanoma is still not clear. Also, the reported frequencies vary considerably in the published literature from 0 to 48%. The aims of the following study were to identify the frequency, molecular pathology and potential clinical significance of partial chromosome 3 alteration in uveal melanoma. We studied 47 uveal melanomas with an average follow-up of 36 months. Of these, 14 had confirmed metastasis. Allelic imbalance/loss of heterozygosity was studied using microsatellite markers on chromosome 3 enriched in markers located in the previously reported smallest regions of deletion overlap. Chromosomal alterations were assessed by conventional cytogenetics or comparative genomic hybridization (CGH) in a subset of patients. Utilizing genotyping, partial chromosome 3 alteration was detected in 14/47 tumors (30%). In the 23 tumors with available cytogenetic/CGH, partial chromosome 3 alteration was detected in 8/23 (38%) and was caused by both gains (4/8) and losses (4/8) of chromosome 3 with high frequency of complex chromosome 3 aberrations detected by cytogenetics. Out of the 14 tumors with confirmed metastasis, only 1 showed partial chromosome 3 alteration and the remaining showed monosomy 3. By limiting the aggressive disease marker to monosomy 3, genotyping showed 93% sensitivity and 67% specificity for detection of aggressive uveal melanoma. In conclusion, partial chromosome 3 alterations are common in uveal melanoma and mostly caused by complex cytogenetic changes leading to partial gains and/or partial losses of chromosome 3. Partial chromosome 3 alteration is not likely to be associated with highly aggressive uveal melanoma that metastasizes within the first 3 years after treatment. Microsatellite-based genotyping of chromosome 3 is highly sensitive for detection of aggressive uveal melanoma.
doi:10.1038/modpathol.2011.51
PMCID: PMC3871992  PMID: 21499235
eye neoplasms; molecular genetics; uveal melanoma
14.  CD133 expression associated with poor prognosis in ovarian cancer 
As a putative marker for cancer stem cells in human malignant tumors, including ovarian cancer, CD133 expression may define a tumor-initiating subpopulation of cells and is associated with the clinical outcome of patients. However, at this time its clinical significance in ovarian cancer remains uncertain. The aim of this study was to clarify the clinical role of CD133 expression in human ovarian cancer. Immunohistochemical staining of CD133 expression was performed in 400 ovarian carcinoma samples using tissue microarray. The associations among CD133 expression and clinical factors (diagnosis, tumor grade, cancer stage, and clinical response to chemotherapy), overall survival and disease-free survival time were analyzed. CD133 expression was found in 31% of ovarian carcinoma samples. Fisher’s exact test and one-way analysis of variance suggested that CD133 expression was associated with high-grade serous carcinoma (P = 0.035), late-stage disease (P < 0.001), ascites level (P = 0.010), and non-response to chemotherapy (P = 0.023). CD133 expression was also associated with shorter overall survival time (P = 0.007) and shorter disease-free survival time (P < 0.001) by log-rank test. Moreover, CD133 expression was an independent predictor of shorter disease-free survival time in an unconditional logistic regression analysis with multiple covariates (P = 0.024). Our results thus show that CD133 expression is a predictor of poor clinical outcome for patients with ovarian cancer, supporting the proposed link between CD133 and cancer stem cells.
doi:10.1038/modpathol.2011.170
PMCID: PMC3855345  PMID: 22080056
CD133; immunohistochemistry; ovarian cancer; prognosis
15.  An improved prognostic model for stage T1a and T1b prostate cancer by assessments of cancer extent 
Treatment decisions on prostate cancer diagnosed by trans-urethral resection (TURP) of the prostate are difficult. The current TNM staging system for pT1 prostate cancer has not been re-evaluated for 25 years. Our objective was to optimise the predictive power of tumor extent measurements in TURP of the prostate specimens. A total of 914 patients diagnosed by TURP of the prostate between 1990 and 1996, managed conservatively were identified. The clinical end point was death from prostate cancer. Diagnostic serum prostate-specific antigen (PSA) and contemporary Gleason grading was available. Cancer extent was measured by the percentage of chips infiltrated by cancer. Death rates were compared by univariate and multivariate proportional hazards models, including baseline PSA and Gleason score. The percentage of positive chips was highly predictive of prostate cancer death when assessed as a continuous variable or as a grouped variable on the basis of and including the quintiles, quartiles, tertiles and median groups. In the univariate model, the most informative variable was a four group-split (≤ 10%, >10–25%, > 25–75% and > 75%); (HR = 2.08, 95% CI = 1.8–2.4, P < 0.0001). The same was true in a multivariate model (ΔX2 (1 d.f.) = 15.0, P = 0.0001). The current cutoff used by TNM (< = 5%) was sub-optimal (ΔX2 (1 d.f.) = 4.8, P = 0.023). The current TNM staging results in substantial loss of information. Staging by a four-group subdivision would substantially improve prognostication in patients with early stage disease and also may help to refine management decisions in patients who would do well with conservative treatments.
doi:10.1038/modpathol.2010.182
PMCID: PMC3853363  PMID: 20834240
conservative treatment; prostate cancer; stage; trans-urethral resection of prostate; watchful waiting
16.  Expression of Thyroid Transcription Factor-1 in Normal Endometrium is associated with Risk of Endometrial Cancer Development 
Thyroid transcription factor-1 (TTF-1) is a DNA-binding protein that is mainly expressed in thyroid and lung tissue, but has also been found in gynecologic tissue. Recent studies have suggested that TTF-1 has tumor suppressor function in lung adenocarcinoma models. In the current study, we examined whether expression of TTF-1 in benign endometrium and endometrial hyperplasia might impact on the risk of developing endometrial cancer. Formalin fixed paraffin embedded endometrial tissues obtained from 535 cases were used to construct an endometrial tissue microarray. One-hundred fifty of 207 patients had multiple serial endometrial specimens including 46 patients who progressed to endometrial cancer. The tissue microarray included a range of histopathologies including benign endometrium (n=231), simple hyperplasia (n=105), complex hyperplasia (n=36), simple atypical hyperplasia (n=10), complex atypical hyperplasia (n=44), and endometrial carcinoma (n=109). Expression of TTF-1 by immunohistochemistry in benign endometrium and endometrial hyperplasia was correlated with progression to cancer and clinical features known to be associated with increased risk of developing endometrial cancer. Carcinoma specimens showed a significantly greater expression of TTF-1 compared to benign endometrium and non-atypical hyperplasia (p=0.0007 and p=0.05). Presence of TTF-1 expression in benign endometrium was associated with a significantly decreased risk of cancer development (p=0.003, hazards ratio=0.104, 95% CI 0.024-0.455). TTF-1 expression in hyperplasia did not significantly correlate with progression to cancer. The data from our study shows that TTF-1 expression in normal endometrium is associated with a reduced risk of endometrial cancer development. This observation suggests that TTF-1 might function as a tumor suppressor in endometrial tissue. TTF-1 expression in normal endometrium could potentially provide clinically useful information as a biomarker for the risk of endometrial cancer.
doi:10.1038/modpathol.2012.64
PMCID: PMC3845815  PMID: 22460811
TTF-1; normal endometrium; endometrial hyperplasia; endometrial cancer; immunohistochemistry
17.  Prostate cancer cell phenotypes based on AGR2 and CD10 expression 
The combination of expression patterns of AGR2 and CD10 by prostate cancer provided four phenotypes that correlated with clinical outcome. Based on immunophenotyping, CD10lowAGR2high, CD10highAGR2high, CD10lowAGR2low, and CD10highAGR2low were distinguished. AGR2+ tumors were associated with longer recurrence-free survival and CD10+ tumors with shorter recurrence-free survival. In high-stage cases, the CD10lowAGR2high phenotype was associated with a 9-fold higher recurrence-free survival than the CD10highAGR2low phenotype. The CD10highAGR2high and CD10lowAGR2low phenotypes were intermediate. The CD10highAGR2low phenotype was most frequent in high-grade primary tumors. Conversely, bone and other soft tissue metastases, and derivative xenografts, expressed more AGR2 and less CD10. AGR2 protein was readily detected in tumor metastases. The CD10highAGR2low phenotype in primary tumors is predictive of poor outcome; however, the CD10lowAGR2high phenotype is more common in metastases. It appears that AGR2 has a protective function in primary tumors but may have a role in the distal spread of tumor cells.
doi:10.1038/modpathol.2012.238
PMCID: PMC3638070  PMID: 23348903
Prostate cancer; AGR2; CD10; cancer cell phenotypes; patient stratification; bone and soft tissue metastases; xenografts
18.  Novel Dual Color Immunohistochemical methods for detecting ERG-PTEN and ERG-SPINK1 status in prostate carcinoma 
Identification of new molecular markers has led to the molecular classification of prostate cancer based on driving genetic lesions. The translation of these discoveries for clinical use necessitates the development of simple, reliable and rapid detection systems to screen patients for specific molecular aberrations. We developed two dual color immunohistochemistry-based assays for the simultaneous assessment of ERG-PTEN and ERG-SPINK1 in prostate cancer. A total of 232 cases from 184 localized and 48 metastatic prostate cancers were evaluated for ERG-PTEN and 284 cases from 228 localized and 56 metastatic prostate cancers were evaluated for ERG-SPINK1. Of the 232 cases evaluated for ERG-PTEN, 81 (35%) ERG positive and 77 (33%) PTEN deleted cases were identified. Of the 81 ERG positive cases, PTEN loss was confirmed in 35 (15%) cases by fluorescence in situ hybridization. PTEN status was concordant in 203 cases (Sensitivity 90%; Specificity 87% (p<0.0001) by both immunohistochemisty and FISH, however, immunohistochemisty could not distinguish between heterozygous and homozygous deletion status of PTEN. Of the 284 cases evaluated for ERG-SPINK1, 111 (39%) cases were positive for ERG. In the remaining 173 ERG negative cases; SPINK1 was positive in 26 (9 %) cases. SPINK1 expression was found to be mutually exclusive with ERG expression; however, we identified two cases, of which, one showed concomitant expression of ERG and SPINK1 in the same tumor foci and in the second case ERG and SPINK1 was seen in two independent foci of the same tumor nodule. Unlike the homogenous ERG staining in cancer tissues, heterogeneous SPINK1 staining was observed in the majority of the cases. Further studies are required to understand the molecular heterogeneity of cases with concomitant ERG-SPINK1 expression. Automated dual ERG-PTEN and ERG-SPINK1 immunohistochemisty assays are simple, reliable and portable across study sites for the simultaneous assessment of these proteins in prostate cancer.
doi:10.1038/modpathol.2012.234
PMCID: PMC3672354  PMID: 23348902
Prostate cancers; Immunohistochemistry; Fluorescent in situ hybridization; Tissue Microarray
19.  Distinct profile of driver mutations and clinical features in immunomarker-defined subsets of pulmonary large cell carcinoma 
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.
doi:10.1038/modpathol.2012.195
PMCID: PMC3594043  PMID: 23196793
large cell carcinoma; TTF-1; ΔNp63/p40; EGFR; KRAS; ALK
20.  Cytoplasmic PTEN Protein Loss Distinguishes Intraductal Carcinoma of the Prostate from High Grade Prostatic Intraepithelial Neoplasia 
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.
doi:10.1038/modpathol.2012.201
PMCID: PMC3610824  PMID: 23222491
Prostatic adenocarcinoma; intraductal carcinoma; PTEN; ERG; immunohistochemistry
21.  Molecular Pathological Epidemiology of Epigenetics: Emerging Integrative Science to Analyze Environment, Host, and Disease 
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.
doi:10.1038/modpathol.2012.214
PMCID: PMC3637979  PMID: 23307060
molecular pathologic epidemiology; genetics; omics; hypermethylation; hypomethylation; personalized therapy; unique tumor principle; CIMP; long interspersed nucleotide element
22.  Interobserver Reliability in the Histopathological Diagnosis of Cartilaginous Tumors in Patients with Multiple Osteochondromas 
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.
doi:10.1038/modpathol.2012.78
PMCID: PMC3784315  PMID: 22555180
osteochondroma; chondrosarcoma; diagnostic criteria; cartilage tumors; bone tumors
23.  Epithelioid sarcoma is associated with a high percentage of SMARCB1 deletions 
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.
doi:10.1038/modpathol.2012.175
PMCID: PMC3556344  PMID: 23060122
Epithelioid sarcoma; malignant rhabdoid tumor; SMARCB1
24.  Identification of c-kit gene mutations in primary adenoid cystic carcinoma of the salivary gland 
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).
doi:10.1038/modpathol.2009.95
PMCID: PMC3746033  PMID: 19617878
adenoid cystic carcinoma; c-kit gene mutations; KIT protein; salivary gland
25.  Detection and significance of human papillomavirus, CDKN2A (p16) and CDKN1A (p21) expression in squamous cell carcinoma of the larynx 
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.
doi:10.1038/modpathol.2012.159
PMCID: PMC3529982  PMID: 22996374
Human papillomavirus; CDKN2A(p16); CDKN1A(p21); larynx; in situ hybridization; polymerase chain reaction

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