Characterization of natural killer and cytotoxic T-cell immune infiltrates in pancreatic ductal adenocarcinoma.

BACKGROUND AND OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor response to systemic therapies, including immunotherapy. Given the immunotherapeutic potential of natural killer (NK) cells, we evaluated intratumoral NK cell infiltrates along with cytotoxic T cells in PDAC to determine their association with patient outcomes. METHODS: We analyzed tumors from 93 PDAC patients treated from 2012 to 2020. Predictor variables included tumor-infiltrating lymphocytes (TILs), T-cell markers (CD3, CD8, CD45RO), NK marker (NKp46), and NK inhibitory marker (major histocompatibility complex class I [MHC-I]) by immunohistochemistry. Primary outcome variables were recurrence-free survival (RFS) and overall survival (OS). RESULTS: Mean TILs, CD3, and NKp46 scores were 1.3 ± 0.63, 20.6 ± 17.5, and 3.1 ± 3.9, respectively. Higher expression of CD3 and CD8 was associated with higher OS, whereas NK cell infiltration was not associated with either RFS or OS. There was a tight positive correlation between MHC-I expression and all T-cell markers, but not with NKp46. CONCLUSIONS: Overall NK cell infiltrates were low in PDAC and did not predict clinical outcomes, whereas T-cell infiltrates did. Further characterization of the immune infiltrate in PDAC, including inhibitory signals and suppressive cell types, may yield better biomarkers of prognosis and immune targeting in this refractory disease.

Precancerous neoplastic cells can move through the pancreatic ductal system.

Most adult carcinomas develop from noninvasive precursor lesions, a progression that is supported by genetic analysis. However, the evolutionary and genetic relationships among co-existing lesions are unclear. Here we analysed the somatic variants of pancreatic cancers and precursor lesions sampled from distinct regions of the same pancreas. After inferring evolutionary relationships, we found that the ancestral cell had initiated and clonally expanded to form one or more lesions, and that subsequent driver gene mutations eventually led to invasive pancreatic cancer. We estimate that this multi-step progression generally spans many years. These new data reframe the step-wise progression model of pancreatic cancer by illustrating that independent, high-grade pancreatic precursor lesions observed in a single pancreas often represent a single neoplasm that has colonized the ductal system, accumulating spatial and genetic divergence over time.

Dynamic Positron Emission Tomography/Computed Tomography Imaging Correlate of Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a severe form of nonalcoholic fatty liver disease characterized by lobular inflammation and hepatocyte injury and is a key determinant of clinical outcome.1 Liver biopsy remains the gold standard for diagnosis but is limited by risks of the procedure and interobserver variability. Although magnetic resonance imaging (MRI)-based technology may provide novel means to identify NASH,2 there remains a significant need for other modalities to diagnose NASH noninvasively. Glucose transport, an integral tissue process altered in NASH,3 is measurable with 18F-fluorodeoxyglucose positron emission tomography (FDG PET). Because unenhanced computed tomography (CT) scan can detect hepatic steatosis quite reliably,4 and PET combines unenhanced CT for attenuation correction, we hypothesized that measurement of the combination of glucose transport by PET and steatosis by CT could yield a reliable radiologic correlate of NASH.

Update on the pathology of liver neoplasms.

Many advances have developed in the pathology of liver tumors in the recent decade. Examples of these advances include the use of glutamine synthetase in the diagnosis of focal nodular hyperplasia, subtyping of hepatocellular adenomas using molecular and immunohistochemical methods, the unraveling of the fusion transcript between the DNAJB1 gene and the PRKACA gene in fibrolamellar carcinoma, and the more unified classification and terminology in intrahepatic bile duct tumors and their precursor lesions. Nevertheless, challenges still remain, e.g., the differential diagnosis between well-differentiated hepatocellular carcinoma and hepatocellular adenoma; distinction among poorly differentiated hepatocellular carcinoma, cholangiocarcinoma and metastatic neoplasm; terminology of the combined hepatocellular carcinoma-cholangiocarcinoma, etc. This review aims to address updates in the pathologic diagnosis and clinical relevance of tumors of the liver and intrahepatic bile ducts in adults and their differential diagnosis and diagnostic pitfalls.

Synaptophysin-Ki67 double stain: a novel technique that improves interobserver agreement in the grading of well-differentiated gastrointestinal neuroendocrine tumors.

A common problem in the assessment of Ki67 proliferative index in well-differentiated gastrointestinal neuroendocrine tumors is distinguishing tumor from non-tumor. This is because background stromal lymphocytes, entrapped non-neoplastic glands, and the delicate vascular network characteristic of neuroendocrine tumors frequently contain a subset of proliferating cells. Furthermore, in small biopsies, crush and cautery artifact can alter the morphologic appearance of tumor cells, making the Ki67 proliferative index more difficult to assess. To address these issues, we developed a synaptophysin-Ki67 double stain using a commercially available immunohistochemistry kit, allowing simultaneous visualization of tumor and proliferating nuclei. To test this method, three gastrointestinal pathologists individually graded 50 gastrointestinal neuroendocrine tumors first using synaptophysin-Ki67 double-stained slides and then, after a washout period, using Ki67-only stained slides (along with routine hematoxylin- and eosin-stained slides). Interobserver agreement on Ki67 proliferative index was moderate using the Ki67-only stained slides (intraclass correlation 0.51, 95% confidence interval: 0.35-0.66) and improved using the synaptophysin-Ki67 double stain (intraclass correlation 0.79, 95% confidence interval: 0.69-0.86). Similarly, interobserver agreement on tumor grade was fair with Ki67-only stained slides (κ=0.39, P<0.001) and improved with the double stain (κ=0.58, P<0.001). Analysis of individual pathologists' scores revealed that fewer total number of tumor cells counted correlated with higher grade designation and appeared to contribute to grade discordance. In tumors cited as particularly challenging to assess by the pathologists, three of four tumors were grade discordant with the Ki67-only stain, whereas all four tumors were grade concordant with the synaptophysin-Ki67 stain. The synaptophysin-Ki67 double stain is the first technique to address specifically the histomorphologic challenges of evaluating Ki67 proliferative index in well-differentiated gastrointestinal neuroendocrine tumors. Although further validation is needed, this study provides evidence that the synaptophysin-Ki67 double stain can improve interobserver agreement.

Abdominal CT predictors of fibrosis in patients with chronic pancreatitis undergoing surgery.

OBJECTIVE: To determine which abdominal CT findings predict severe fibrosis and post-operative pain relief in chronic pancreatitis (CP). METHODS: Pre-operative abdominal CTs of 66 patients (mean age 52 ± 12 years, 53 % males) with painful CP who underwent the Whipple procedure (n = 32), Frey procedure (n = 32) or pancreatic head biopsy (n = 2), between 1/2003-3/2014, were evaluated. CT was evaluated for parenchymal calcifications, intraductal calculi, main pancreatic duct dilation (>5 mm), main pancreatic duct stricture, and abnormal side branch(es). The surgical histopathology was graded for fibrosis. CT findings were evaluated as predictors of severe fibrosis and post-operative pain relief using regression and area under receiver operating curve (AUC) analysis. RESULTS: Thirty-eight (58 %) patients had severe fibrosis. Parenchymal calcification(s) were an independent predictor of severe fibrosis (p = 0.03), and post-operative pain relief over a mean follow-up of 1-year (p = 0.04). Presence of >10 parenchymal calcifications had higher predictive accuracy for severe fibrosis than 1-10 parenchymal calcification(s) (AUC 0.88 vs. 0.59, p = 0.003). The predictive accuracy of >10 versus 1-10 parenchymal calcifications increased after adjusting for all other CT findings (AUC 0.89 vs. 0.63, p = 0.01). CONCLUSION: Parenchymal calcification(s) independently predict severe fibrosis and are significantly associated with post-operative pain relief in CP. The presence of >10 parenchymal calcifications is a better predictor of severe fibrosis than 1-10 parenchymal calcification(s). KEY POINTS: • Parenchymal calcifications in chronic pancreatitis independently predict post-operative pain relief • Intraductal calculi and MPD dilation are not associated with post-operative pain relief • Better patient selection for pancreatic resection surgery in painful chronic pancreatitis.

Convergent structural alterations define SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeler as a central tumor suppressive complex in pancreatic cancer.

Defining the molecular genetic alterations underlying pancreatic cancer may provide unique therapeutic insight for this deadly disease. Toward this goal, we report here an integrative DNA microarray and sequencing-based analysis of pancreatic cancer genomes. Notable among the alterations newly identified, genomic deletions, mutations, and rearrangements recurrently targeted genes encoding components of the SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex, including all three putative DNA binding subunits (ARID1A, ARID1B, and PBRM1) and both enzymatic subunits (SMARCA2 and SMARCA4). Whereas alterations of each individual SWI/SNF subunit occurred at modest-frequency, as mutational "hills" in the genomic landscape, together they affected at least one-third of all pancreatic cancers, defining SWI/SNF as a major mutational "mountain." Consistent with a tumor-suppressive role, re-expression of SMARCA4 in SMARCA4-deficient pancreatic cancer cell lines reduced cell growth and promoted senescence, whereas its overexpression in a SWI/SNF-intact line had no such effect. In addition, expression profiling analyses revealed that SWI/SNF likely antagonizes Polycomb repressive complex 2, implicating this as one possible mechanism of tumor suppression. Our findings reveal SWI/SNF to be a central tumor suppressive complex in pancreatic cancer.

PCSK2 can be Useful in a Panel Approach to Distinguish Foregut and Midgut Neuroendocrine Tumors.

A number of immunohistochemical stains have been examined for utility in establishing the site of origin for metastatic well-differentiated neuroendocrine tumors (NETs). In the gastrointestinal (GI) tract, distinguishing metastatic duodenal NETs from jejunoileal and other GI NETs is important for clinical work-up, prognosis, and therapy. A recent study indicated that prohormone convertase 2 (PCSK2 or PC2) had broad expression in small intestine and appendiceal NETs. Because the study did not include duodenal NETs, we examined PCSK2 expression in duodenal and other GI NETs.GI NETs (n = 69) and 13 corresponding lymph node metastases from stomach, duodenum, pancreas, ileum, appendix, and rectum were evaluated for the expression of PCSK2, along with ISL1, NKX2.2, CDX2, SATB2, and PAX8. Expression of each stain was evaluated using the H-score system, and differences in expression by site were evaluated by the chi square test.PCSK2 was expressed at similar frequency in duodenal (50%), pancreatic (59%), and ileal NETs (40%). PCSK2 was infrequently expressed in stomach (0%), appendiceal (8%), and rectal (25%) NETs. However, incorporating PCSK2 into a panel including ISL1, NKX2.2, CDX2, and SATB2 allowed development of an algorithm which had 87% sensitivity and 93% specificity for classification of ileal NETs; and 68% sensitivity and 98% specificity for pancreaticoduodenal NETs.In contrast to previous findings, PCSK2 does not show specificity for any particular GI site. An algorithmic approach incorporating the expression of PCSK2 with that of ISL1, NKX2.2, CDX2, and SATB2 is useful in discriminating pancreatic, duodenal, ileal, appendiceal, and rectal NETs.

A high-fat diet induces changes in mesenteric adipose tissue accelerating early-stage pancreatic carcinogenesis in mice.

Increased adiposity is a significant risk factor for pancreatic cancer development. Multiple preclinical studies have documented that high-fat, high calorie diets, rich in omega-6 fatty acids (FA) accelerate pancreatic cancer development. However, the effect of a high-fat, low sucrose diet (HFD), on pancreatic carcinogenesis remains unclear. We evaluated the impact of a HFD on early-stage pancreatic carcinogenesis in the clinically relevant KrasLSL-G12D/+; Ptf1aCre/+ (KC) genetically engineered mouse model, and characterized the role of the mesenteric adipose tissue (MAT). Cohorts of male and female KC mice were randomly assigned to a control diet (CD) or a HFD, matched for FA composition (9:1 of omega-6 FA: omega-3 FA), and fed their diets for 8 weeks. After 8 weeks on a HFD, KC mice had significantly higher body weight, fat mass, and serum leptin compared to CD-fed KC mice. Furthermore, a HFD accelerated pancreatic acinar-to-ductal metaplasia (ADM) and proliferation, associated with increased activation of ERK and STAT3, and macrophage infiltration in the pancreas, compared to CD-fed KC mice. Metabolomics analysis of the MAT revealed sex differences between diet groups. In females, a HFD altered metabolites related to FA (α-linolenic acid and linoleic acid) and amino acid metabolism (alanine, aspartate, glutamate). In males, a HFD significantly affected pathways related to alanine, aspartate, glutamate, linoleic acid, and the citric acid cycle. A HFD accelerates early pancreatic ADM through multifaceted mechanisms, including effects at the tumor and surrounding MAT. The sex-dependent changes in MAT metabolites could explain some of the sex differences in HFD-induced pancreatic ADM.

Positron emission tomography combined with serum biomarkers detects fibrotic MASH.

Metabolic dysfunction-associated steatohepatitis (MASH) is a rising global disease signaling the urgent need for non-invasive tests (NITs). Recent work demonstrated that dynamic 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) imaging can identify MASH by measuring liver glucose transport rate, K1, and liver CT attenuation. By combining dynamic PET/CT with the serum-based fibrosis-4 (FIB-4) test, we were able to better distinguish clinical MASH from fibrotic subtypes, enabling determination of the core tenets of MASH: steatosis, inflammation, and fibrosis. Future studies using FDG-PET technology can further enable concomitant prediction of MASH severity and extrahepatic comorbidities such as cardiovascular disease.

Lymphoglandular Complex-Like Colorectal Carcinoma-A Series of 20 Colorectal Cases, Including Newly Reported Features of Malignant Behavior.

Distinguishing colon carcinoma that is surrounded by well-circumscribed lymphoid tissue from adenomas involving lymphoglandular complexes can be difficult. We assessed a multi-institutional international cohort of 20 colorectal carcinomas with associated prominent lymphoid infiltrates, which we referred to as lymphoglandular complex-like carcinoma (LGCC). We collected clinical and endoscopic features, including lesion size, endoscopic appearance, location, procedure, follow-up, AJCC stage, and mismatch repair status. We recorded the presence of the following histologic features: haphazard gland distribution, gland angulation, gland fusion, solid nest formation, single-cell formation, stromal desmoplasia, presence of lymphovascular invasion and perineural invasion, presence of lamina propria, cytologic atypia as low- or high-grade, presence of goblet cells in the invasive component, and the presence of a surface lesion. Most cases (9 of 13) were described endoscopically as sessile polyps with an average size of 1.56 cm. Most cases (90%) were associated with a surface lesion, of which the majority were tubular adenomas, though a subset was associated with sessile serrated lesions with dysplasia (3 of 18). All cases of LGCC demonstrated haphazard gland distribution and either gland angulation, fusion, or solid nest formation. A portion of cases demonstrated single-cell infiltration (35%) and desmoplasia (50%), and rarely lymphovascular invasion was present (5%). A subset (10%) of cases invaded beyond the submucosa. Deficient mismatch repair was present in 22% (2 of 9) of cases for which it was performed. In cases of colectomy or completion colectomy, nodal metastasis was present in 38% (3 of 8). No cases demonstrated disease recurrence or disease-specific mortality. Overall, LGCC represents an enigmatic subset of carcinomas that is important to distinguish from adenomas involving lymphoglandular complexes due to its varying prognostic outcomes.

Practical Guide, Challenges, and Pitfalls in Liver Fibrosis Staging.

Liver fibrosis staging has many challenges, including the large number of proposed staging systems, the heterogeneity of the histopathologic changes of many primary liver diseases, and the potential for slight differences in histologic interpretation to significantly affect clinical management. This review focuses first on fibrosis regression. Following this, each of the major categories of liver disease is discussed in regard to (1) appropriate fibrosis staging systems, (2) emerging concepts, (3) current clinical indications for liver biopsy, (4) clinical decisions determined by fibrosis stage, and (5) histologic challenges and pitfalls related to staging.

Hirschsprung Disease for the Practicing Surgical Pathologist.

OBJECTIVES: Hirschsprung disease (HD) is a congenital condition defined by the absence of ganglion cells in the distal-most portion of the gastrointestinal tract. Biopsies and resections for HD can be adrenaline inducing for the general surgical pathologist because specimens are infrequent; HD is 1 of only a few neuroanatomic diseases that general surgical pathologists diagnose; numerous preanalytic factors (eg, biopsy adequacy, surgeon sampling protocol, processing artifacts) can affect histologic interpretation; and most importantly, the diagnosis has high stakes. METHODS: We provide a comprehensive overview of the background, relevant clinical procedures, and pathologic assessment of HD. Grossing and frozen section protocols, an algorithmic approach to diagnosis, and histologic pearls and pitfalls are also discussed. RESULTS: Evaluation and recognition of the features of HD have evolved significantly in the past 2 decades with the discovery of the value of calretinin immunohistochemistry in the late 2000s and the recent development of straightforward and reproducible histologic criteria for identification of the HD transition zone. CONCLUSIONS: These advancements have substantially improved the pathologist's ability to reliably evaluate for HD. Nonetheless, as with any high-stakes surgical pathology specimen, clear communication with the clinical team is essential.

Hepatic safety profile of pancreatic cancer­bearing mice fed a ketogenic diet in combination with gemcitabine.

Ketogenic diets (KDs) are actively being evaluated for their potential anticancer effects. Although KDs are generally considered safe, their safety profile when combined with chemotherapy remains unknown. It is known that a KD enhances the anticancer effect of gemcitabine (2',2'-difluoro-2'-deoxycytidine) in LSL-KrasLSL-G12D/+Trp53R172H/+Pdx-1-Cre (KPC) tumor-bearing mice. In the present study, whether a KD in combination with gemcitabine affected the liver safety profile in KPC mice was evaluated. For this purpose, male and female pancreatic tumor-bearing KPC mice were allocated to a control diet (CD; % kcal: 20% fat, 65% carbohydrate, 15% protein) + gemcitabine [control plus gemcitabine group (CG)] or a KD (% kcal: 84% fat, 15% protein, 1% carbohydrate) + gemcitabine [ketogenic plus gemcitabine group (KG)] for two months. After two months of treatment, no significant differences in body weight were observed between CGs and KGs. Moreover, the KD did not significantly alter the serum protein expression levels of liver enzymes, including aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase. In addition, the KD did not alter markers of liver-lipid accumulation as well as serum cholesterol and triglyceride levels, compared with the CG-treated group. Upon histologic examination, steatosis was rare, with no notable differences between treatment groups. When examining liver fatty acid composition, KD treatment significantly increased the content of saturated fatty acids and significantly decreased levels of cis-monounsaturated fatty acids compared with the CG. Finally, the KD did not affect liver markers of inflammation and oxidative stress, nor the protein expression levels of enzymes involved in ketone bodies, such as 3-hydroxy-3-methylglutaryl-CoA lyase and hidroximetilglutaril-CoA sintasa, and glucose metabolism, such as hexokinase 2, pyruvate dehydrogenase and phosphofructokinase. In summary, a KD in combination with gemcitabine appears to be safe, with no apparent hepatotoxicity and these data support the further evaluation of a KD as an adjuvant dietary treatment for pancreatic cancer.

Loss of ATRX or DAXX expression and concomitant acquisition of the alternative lengthening of telomeres phenotype are late events in a small subset of MEN-1 syndrome pancreatic neuroendocrine tumors.

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.

Acute Appendicitis with Neuronal Hyperplasia and Swelling: A Novel Histologic Mimic of Appendiceal Goblet Cell Adenocarcinoma and Signet-Ring Cell Adenocarcinoma.

Goblet cell adenocarcinoma and signet-ring cell adenocarcinoma are well-known diagnostic pitfalls of routine appendectomy specimens. Here we present a case of acute appendicitis with prominent neuronal (ganglion cell) hyperplasia and swelling which histologically mimics goblet cell adenocarcinoma and signet-ring cell adenocarcinoma. Attention to the cytologic features of the lesional cells (absence of atypia, mitotic activity) and their close association with nerves and classic ganglion cells, along with the use of a limited panel of immunostains, ensures proper classification of this rare but striking benign process.