Serdar Balci

The high-grade (WHO G3) pancreatic neuroendocrine tumor category is morphologically and biologically heterogenous and includes both well differentiated and poorly differentiated neoplasms.

The 2010 World Health Organization (WHO) classification recommends that pancreatic neuroendocrine tumors (PanNETs) be graded on the basis of the mitotic rate and Ki67 index, with grade 2 (G2) PanNETs defined as having a mitotic rate of 2 to 20 mitotic figures/10 high-power fields or a Ki67 index of 3% to 20%. Grade 3 (G3) pancreatic neuroendocrine carcinoma (NEC) is defined as having >20 mitotic figures/10 high-power fields or a Ki67 index of >20%. However, some PanNETs show discordance between the mitotic rate and Ki67 index, usually having a Ki67 index in the G3 range but a mitotic rate suggesting G2, prompting us to examine the clinical significance of the Ki67 index in a large series of clinically well-characterized mitotic G2 PanNETs. Mitotic G2 well differentiated PanNETs, surgically resected at our institutions were reviewed. Of those, 19 cases had a Ki67>20% and were selected as the study group of grade-discordant (mitotic count G2/Ki67 index G3) PanNETs. For comparison, 53 grade-concordant (both mitotic count and Ki67 index G2) PanNETs matched for presenting stage with the discordant group as well as 43 morphologically poorly differentiated (either small cell or large cell type) pancreatic NECs were also included. The percentage of Ki67-positive neoplastic cells was quantified by manual counting of at least 500 cells on printed photographic images of “hot spots.” The mean Ki67 index for grade-concordant and grade-discordant PanNETs and poorly differentiated NECs were 8.1% (range, 3% to 20%), 40% (range, 24% to 80%), and 70% (range, 40% to 98%), respectively. Overall, patients with grade-discordant PanNETs had significantly longer survival time compared with the patients with poorly differentiated NEC (median survival of 54.1 vs. 11 mo and 5 y survival of 29.1% vs. 16.1%; P=0.002). In addition, the survival time of the patients with grade-discordant PanNETs was shorter than that of the patients with grade-concordant PanNETs (median survival of 67.8 mo and 5 y survival of 62.4%); however, the difference was not statistically significant (P=0.2). Our data support the notion that the mitotic rate and Ki67 index-based grades of PanNETs can be discordant, and when the Ki67 index indicates G3, the clinical outcome is slightly worse. More importantly, we demonstrate that well differentiated PanNETs that are G3 by Ki67 are significantly less aggressive than bona fide poorly differentiated NECs, suggesting that the current WHO G3 category is heterogenous, contains 2 distinct neoplasms, and can be further separated into well differentiated PanNET with an elevated proliferation rate and poorly differentiated NEC.

Ampullary Region Carcinomas: Definition and Site Specific Classification With Delineation of Four Clinicopathologically and Prognostically Distinct Subsets in an Analysis of 249 Cases

Ampullary (AMP) carcinomas comprise a heterogenous group of cancers lacking adequate subcategorization. In the present study, 249 strictly defined primary AMP carcinomas (ACs) identified in 1469 malignant pancreatoduodenectomy specimens were analyzed for defining features. Gross and microscopic findings were used to determine tumor epicenter and extent of preinvasive component. ACs were classified into 4 distinct subtypes based on location: (1) Intra-AMP (25%): Invasive carcinomas arising in intra-ampullary papillary-tubular neoplasms with zero to minimal, duodenal surface involvement (<25% of the tumor). These tumors were more commonly found in men, they had a relatively large overall size (mean, 2.9 cm) but had smaller invasive component (mean, 1.5 cm), and were predominantly of a lower TNM stage (85%, T1/2; and 72% N0). They carried the best prognosis among the 4 groups (3-y survival, 73%). (2) AMP-ductal (15%): These were tumors forming constrictive, sclerotic, plaque-like thickening of the walls of the common bile duct and/or pancreatic duct resulting in mucosa-covered, button-like elevations of the papilla into the duodenal lumen. There was no significant exophytic (preinvasive) growth. These were the smallest tumors (mean overall size, 1.9 cm; mean invasion size 1.7 cm), but carried the worst prognosis (3-y survival, 41%), presumably due to the pancreatobiliary histology/origin (in 86%); however, even this group had significantly better prognosis when compared with 113 ordinary pancreatic ductal adenocarcinomas (3 y, 11%; P<0.0001). (3) Peri-AMP-duodenal (5%): Massive exophytic, ulcero-fungating tumors growing into the duodenal lumen and eccentrically encasing the ampullary orifice with only minimal intra-ampullary luminal involvement. These were mostly of intestinal phenotype (75%) and some had mucinous features. Although these tumors were the largest (mean overall size 4.7 cm; and mean invasion size 3.4 cm), and had the highest incidence of lymph node metastasis (50%), they carried an intermediate prognosis (3-y survival, 69%) to that seen among a group of 55 nonampullary duodenal carcinoma controls. (4) AC—not otherwise specified (“papilla of Vater”; 55%): Ulcero-nodular tumors located at the papilla of Vater, which do not show the specific characteristics identified among the other 3 subtypes. In conclusion, ACs comprise 4 clinicopathologic subtypes that are prognostically distinct.

Calculation of the Ki67 index in pancreatic neuroendocrine tumors: a comparative analysis of four counting methodologies

Ki67 index is now an essential part of classification of pancreatic neuroendocrine tumors. However, its adaptation into daily practice has been fraught with challenges related to counting methodology. In this study, three reviewers used four counting methodologies to calculate Ki67 index in 68 well-differentiated pancreatic neuroendocrine tumors: (1) ‘eye-ball’ estimation, which has been advocated as reliable and is widely used; (2) automated counting by image analyzer; (3) manual eye-counting (eye under a microscope without a grid); and (4) manual count of camera-captured/printed image. Pearson’s correlation (R) was used to measure pair-wise correlation among three reviewers using all four methodologies. Average level of agreement was calculated using mean of R values. The results showed that: (1) ‘eye-balling’ was least expensive and fastest (average time <1 min) but had poor reliability and reproducibility. (2) Automated count was the most expensive and least practical with major impact on turnaround time (limited by machine and personnel accessibility), and, more importantly, had inaccuracies in overcounting unwanted material. (3) Manual eye count had no additional cost, averaged 6 min, but proved impractical and poorly reproducible. (4) Camera-captured/printed image was most reliable, had highest reproducibility, but took longer than ‘eye-balling’. In conclusion, based on its comparatively low cost/benefit ratio and reproducibility, camera-captured/printed image appears to be the most practical for calculating Ki67 index. Although automated counting is generally advertised as the gold standard for index calculation, in this study it was not as accurate or cost-effective as camera-captured/printed image and was highly operator-dependent. ‘Eye-balling’ produces highly inaccurate and unreliable results, and is not recommended for routine use.

Follow-up of atypia and follicular lesions of undetermined significance in thyroid fine needle aspiration cytology.

N. Dincer, S. Balci, A. Yazgan, G. Guney, R. Ersoy, B. Cakir and G. Guler 
Follow‐up of atypia and follicular lesions of undetermined significance in thyroid fine needle aspiration cytology

Molecular Genetics of Pancreatic Neoplasms and Their Morphologic Correlates An Update on Recent Advances and Potential Diagnostic Applications

OBJECTIVES To summarize the most clinically and biologically relevant advances in molecular/genetic characteristics of various pancreatic neoplasms, with morphologic correlation. METHODS Whole-exome sequencing of numerous benign and malignant pancreatic tumors, along with the plethora of highly sensitive molecular studies now available for analyzing these tumors, provide mounting evidence to support the long-held belief that cancer is essentially a genetic disease. These genetic discoveries have not only helped to confirm the age-old, morphology-based classifications of pancreatic neoplasia but have shed new light on their mechanisms. Many of these molecular discoveries are currently being used in preoperative diagnosis. RESULTS Mutations in KRAS, P16/CDKN2A, TP53, and SMAD4/DPC4 are commonly seen in ductal neoplasia but not in nonductal tumors; ductal adenocarcinomas with SMAD4/DPC4 loss are associated with widespread metastasis and poor prognosis. GNAS and RNF43 mutations have been discovered in most intraductal pancreatic mucinous neoplasms, providing critical molecular fingerprints for their diagnosis. Mutation in DAXX/ATRX is only seen in pancreatic neuroendocrine tumors, making it a useful potential marker in distinguishing these tumors from mimics. CONCLUSIONS When combined with morphologic observations, molecular studies will increase our understanding of the pathogenesis and morphomolecular signatures associated with specific neoplasms and provide new horizons for precision medicine and targeted therapies.

Androgen receptor status is a prognostic marker in non-basal triple negative breast cancers and determines novel therapeutic options.

Triple negative breast cancers are a heterogeneous group of tumors characterized by poor patient survival and lack of targeted therapeutics. Androgen receptor has been associated with triple negative breast cancer pathogenesis, but its role in the different subtypes has not been clearly defined. We examined androgen receptor protein expression by immunohistochemical analysis in 678 breast cancers, including 396 triple negative cancers. Fifty matched lymph node metastases were also examined. Association of expression status with clinical (race, survival) and pathological (basal, non-basal subtype, stage, grade) features was also evaluated. In 160 triple negative breast cancers, mRNA microarray expression profiling was performed, and differences according to androgen receptor status were analyzed. In triple negative cancers the percentage of androgen receptor positive cases was lower (24.8% vs 81.6% of non-triple negative cases), especially in African American women (16.7% vs 25.5% of cancers of white women). No significant difference in androgen receptor expression was observed in primary tumors vs matched metastatic lesions. Positive androgen receptor immunoreactivity was inversely correlated with tumor grade (p<0.01) and associated with better overall patient survival (p = 0.032) in the non-basal triple negative cancer group. In the microarray study, expression of three genes (HER4, TNFSF10, CDK6) showed significant deregulation in association with androgen receptor status; eg CDK6, a novel therapeutic target in triple negative cancers, showed significantly higher expression level in androgen receptor negative cases (p<0.01). These findings confirm the prognostic impact of androgen receptor expression in non-basal triple negative breast cancers, and suggest targeting of new androgen receptor-related molecular pathways in patients with these cancers.

Real-Time Elastography for Distinguishing Angiomyolipoma From Renal Cell Carcinoma: Preliminary Observations

OBJECTIVE The objective of this study was to determine the diagnostic performance of sonoelastography for differentiating angiomyolipomas from renal cell carcinomas. SUBJECTS AND METHODS Twenty-eight angiomyolipomas and 19 renal cell carcinomas were prospectively examined with real-time elastography. Lesions were classified according to four elastographic patterns on the basis of the distribution of the blue area (representing no strain and hardest tissue component). The elasticity patterns and the strain ratios of the angiomyolipomas and renal cell carcinomas were evaluated independently by two observers. Diagnostic performance and interobserver agreement were analyzed. RESULTS All angiomyolipomas were classified as having a high-strain elastographic pattern (blue areas in < 50% of lesion, considered type 1 or type 2) by both radiologists, whereas 18 of 19 renal cell carcinomas were classified as having a low-strain elastographic pattern (blue areas in ≥ 50% of lesion, considered type 3 or 4) by both radiologists. The respective mean strain ratios measured by two radiologists were 0.15 ± 0.06 and 0.18 ± 0.09 for the angiomyolipomas and 0.64 ± 0.15 and 0.63 ± 0.19 for the renal cell carcinomas. There were significant differences between the elasticity patterns and strain ratios for angiomyolipomas and renal cell carcinomas (p < 0.001). Interobserver agreement was excellent for elasticity patterns and strain ratios, with a weighted kappa coefficient of 0.96 and an intraclass correlation coefficient score of 0.95. CONCLUSION Our results show that real-time elastography may be useful in differentiating angiomyolipomas from renal cell carcinomas, by use of both elasticity patterns and strain ratios.

Stem cell-related markers in primary breast cancers and associated metastatic lesions.

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, whereas 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.

Whipple Made Simple For Surgical Pathologists: Orientation, Dissection, and Sampling of Pancreaticoduodenectomy Specimens For a More Practical and Accurate Evaluation of Pancreatic, Distal Common Bile Duct, and Ampullary Tumors

Pancreaticoduodenectomy (PD) specimens present a challenge for surgical pathologists because of the relative rarity of these specimens, combined with the anatomic complexity. Here, we describe our experience on the orientation, dissection, and sampling of PD specimens for a more practical and accurate evaluation of pancreatic, distal common bile duct (CBD), and ampullary tumors. For orientation of PDs, identification of the “trapezoid,” created by the vascular bed at the center, the pancreatic neck margin on the left, and the uncinate margin on the right, is of outmost importance in finding all the pertinent margins of the specimen including the CBD, which is located at the upper right edge of this trapezoid. After orientation, all the margins can be sampled. We submit the uncinate margin entirely as a perpendicular inked margin because this adipose tissue–rich area often reveals subtle satellite carcinomas that are grossly invisible, and, with this approach, the number of R1 resections has doubled in our experience. Then, to ensure proper identification of all lymph nodes (LNs), we utilize the orange-peeling approach, in which the soft tissue surrounding the pancreatic head is shaved off in 7 arbitrarily defined regions, which also serve as shaved samples of the so-called “peripancreatic soft tissue” that defines pT3 in the current American Joint Committee on Cancer TNM. With this approach, our LN count increased from 6 to 14 and LN positivity rate from 50% to 73%. In addition, in 90% of pancreatic ductal adenocarcinomas there are grossly undetected microfoci of carcinoma. For determination of the primary site and the extent of the tumor, we believe bisectioning of the pancreatic head, instead of axial (transverse) slicing, is the most revealing approach. In addition, documentation of the findings in the duodenal surface of the ampulla is crucial for ampullary carcinomas and their recent site-specific categorization into 4 categories. Therefore, we probe both the CBD and the pancreatic duct from distal to the ampulla and cut the pancreatic head to the ampulla at a plane that goes through both ducts. Then, we sample the bisected pancreatic head depending on the findings of the case. For example, for proper staging of ampullary carcinomas, it is imperative to take the sections perpendicular to the duodenal serosa at the “groove” area, as ampullary carcinomas often extend to this region. Amputative (axial) sectioning of the ampulla, although good for documentation of the peri-Oddi spread of the intra-ampullary tumors, unfortunately disallows documentation of mucosal spread of the papilla of Vater tumors (those arising from the edge of the ampulla, where the ducts transition to duodenal mucosa and extending) into the neighboring duodenum. Axial sectioning also often fails to document tumor spread to the “groove” area. In conclusion, knowledge of the gross characteristics of the anatomic hallmarks is essential for proper dissection of PD specimens. The approach described above allows practical and accurate documentation and staging of pancreas, distal CBD, and ampullary cancers.

Glut-1 Expression in Pancreatic Neoplasia: Implications in Pathogenesis, Diagnosis, and Prognosis

Objectives: GLUT-1 has been found to have an important role in the upregulation of various cellular pathways and implicated in neoplastic transformation correlating with biological behavior in malignancies. However, literature regarding the significance of GLUT-1 expression in pancreatic neoplasia has been limited and controversial. Methods: Immunohistochemical expression of GLUT-1 was tested in a variety of pancreatic neoplasia including ductal adenocarcinomas (DAs), pancreatic intraepithelial neoplasms (PanINs), intraductal papillary mucinous neoplasms (IPMNs), and serous cystadenomas. Results: There was a progressive increase in the expression of GLUT-1 from low- to higher-grade dysplastic lesions: All higher-grade PanINs/IPMNs (the ones with moderate/high-grade dysplasia) revealed noticeable GLUT-1 expression. Among the 94 DAs analyzed, there were minimal/moderate expression in 46 and significant expression in 24 DAs. However, all 4 clear-cell variants of DAs revealed significant GLUT-1 immunolabeling, as did areas of clear-cell change seen in other DAs. Moreover, all 12 serous cystadenomas expressed significant GLUT-1. GLUT-1 expression was also directly correlated with DA histological grade (P = 0.016) and tumor size (P = 0.03). Conclusions: GLUT-1 may give rise to the distinctive clear-cell appearance of these tumors by inducing the accumulation of glycogen in the cytoplasm. Additionally, because GLUT-1 expression was related to histological grade and tumor size of DA, further studies are warranted to investigate the association of GLUT-1 with prognosis and tumor progression.