Heterogeneity of pancreatic cancer metastases in a single patient revealed by quantitative proteomics.

Many patients with pancreatic cancer have metastases to distant organs at the time of initial presentation. Recent studies examining the evolution of pancreatic cancer at the genetic level have shown that clonal complexity of metastatic pancreatic cancer is already initiated within primary tumors, and organ-specific metastases are derived from different subclones. However, we do not yet understand to what extent the evolution of pancreatic cancer contributes to proteomic and signaling alterations. We hypothesized that genetic heterogeneity of metastatic pancreatic cancer results in heterogeneity at the proteome level. To address this, we employed a model system in which cells isolated from three sites of metastasis (liver, lung, and peritoneum) from a single patient were compared. We used a SILAC-based accurate quantitative proteomic strategy combined with high-resolution mass spectrometry to analyze the total proteome and tyrosine phosphoproteome of each of the distal metastases. Our data revealed distinct patterns of both overall proteome expression and tyrosine kinase activities across the three different metastatic lesions. This heterogeneity was significant because it led to differential sensitivity of the neoplastic cells to small molecule inhibitors targeting various kinases and other pathways. For example, R428, a tyrosine kinase inhibitor that targets Axl receptor tyrosine kinase, was able to inhibit cells derived from lung and liver metastases much more effectively than cells from the peritoneal metastasis. Finally, we confirmed that administration of R428 in mice bearing xenografts of cells derived from the three different metastatic sites significantly diminished tumors formed from liver- and lung-metastasis-derived cell lines as compared with tumors derived from the peritoneal metastasis cell line. Overall, our data provide proof-of-principle support that personalized therapy of multiple organ metastases in a single patient should involve the administration of a combination of agents, with each agent targeted to the features of different subclones.

Frozen section evaluation of breast carcinoma sentinel lymph nodes: a retrospective review of 1,940 cases.

Many sentinel lymph node biopsies (SLNBs) are evaluated intraoperatively by frozen section, which may impact the need for further axillary dissection (AD). However, the need for AD in patients with small metastases has been recently called into question, meaning that frozen SLNB may be unnecessary. Furthermore, frozen section can compromise tissue for further study. At our institution, we grossly evaluate all SLNB and freeze half of the node. Here, we evaluate the frozen SLNB discrepancy rate using this method, focusing on cause of discrepancy and need for further surgery. We reviewed surgical pathology records for all breast cancer resections with frozen section of SLNB examined from 2003 to 2012. For cases with a frozen section discrepancy, we compiled clinicopathologic data. In total, 1,940 cases involved frozen section evaluation of SLNB. In 95 cases (4.9% of total cases, 23.8% of positive node cases), the SLNB was called negative on frozen but positive on final examination (false negatives). The majority of missed metastases are isolated tumor cells or micrometastases. A trend was observed toward fewer patients receiving completion AD after a discrepant frozen SLNB in the later years of the study. The protocol of freezing half of a SLNB is a reasonable method, with results similar to or better than other studies. The main adverse outcome is the need for separate AD; however, additional positive nodes are uncommon. The trend of fewer patients getting additional AD after a discrepant frozen SLNB suggests that clinicians may be using this information differently recently.

Synergistic heterozygosity for TGFbeta1 SNPs and BMPR2 mutations modulates the age at diagnosis and penetrance of familial pulmonary arterial hypertension.

PURPOSE: We hypothesized that functional TGFbeta1 SNPs increase TGFbeta/BMP signaling imbalance in BMPR2 mutation heterozygotes to accelerate the age at diagnosis, increase the penetrance and SMAD2 expression in familial pulmonary arterial hypertension. METHODS: Single nucleotide polymorphism genotypes of BMPR2 mutation heterozygotes, age at diagnosis, and penetrance of familial pulmonary arterial hypertension were compared and SMAD2 expression was studied in lung sections. RESULTS: BMPR2 mutation heterozygotes with least active -509 or codon 10 TGFbeta1 SNPs had later mean age at diagnosis of familial pulmonary arterial hypertension (39.5 and 43.2 years) than those with more active genotypes (31.6 and 33.1 years, P = 0.03 and 0.02, respectively). Kaplan-Meier analysis also showed that those with the less active single nucleotide polymorphisms had later age at diagnosis. BMPR2 mutation heterozygotes with nonsense-mediated decay resistant BMPR2 mutations and the least, intermediate and most active -509 TGFbeta1 SNP genotypes had penetrances of 33, 72, and 80%, respectively (P = 0.003), whereas those with 0-1, 2, or 3-4 active single nucleotide polymorphism alleles had penetrances of 33, 72, and 75% (P = 0.005). The relative expression of TGFbeta1 dependent SMAD2 was increased in lung sections of those with familial pulmonary arterial hypertension compared with controls. CONCLUSIONS: The TGFbeta1 SNPs studied modulate age at diagnosis and penetrance of familial pulmonary arterial hypertension in BMPR2 mutation heterozygotes, likely by affecting TGFbeta/BMP signaling imbalance. This modulation is an example of Synergistic Heterozygosity.

MYB Labeling by Immunohistochemistry Is More Sensitive and Specific for Breast Adenoid Cystic Carcinoma than MYB Labeling by FISH.

Breast adenoid cystic carcinoma (ACC) is a primary breast carcinoma that, like salivary gland ACC, displays the t(6;9) translocation resulting in the MYB-NFIB gene fusion and immunopositivity for MYB by immunohistochemistry (IHC). However, it is not well established whether MYB immunoreactivity or rearrangement can be used to support a diagnosis of ACC in a malignant basaloid or benign cribriform breast lesion. Whole sections of primary breast ACC (n=11), collagenous spherulosis (CS; n=7), and microglandular adenosis (MGA; n=5) and tissue microarrays containing 16 basal-like, triple-negative breast carcinomas (TNBC) were labeled for MYB by IHC and underwent MYB fluorescence in situ hybridization using a break-apart probe. Strong, diffuse nuclear MYB labeling was seen in 100% ACC compared with no cases of basal-like TNBC, CS, or MGA (P=0.0001). Any degree of nuclear MYB labeling was seen in 100% ACC compared with 54% of all other cases (P=0.007), with any labeling seen in 71% CS, 63% basal-like TNBC, and 0% MGA. MYB rearrangement was detected in 89% (8/9) of evaluable ACC compared with 4% (1/26) of all other evaluable cases (P=0.0001), with a rearrangement detected in 1 (7%; n=1/15) evaluable basal-like TNBC. Strong, diffuse nuclear labeling for MYB is more sensitive than MYB fluorescence in situ hybridization for breast ACC and can be used to support a diagnosis of ACC in a cribriform or basaloid lesion in the breast. However, weak and focal labeling should be interpreted with caution as it can be seen in other benign cribriform and malignant basaloid lesions.

Intraductal Papillary Mucinous Neoplasm of the Pancreas Arising in the Setting of an Intermixed Acinar Cell Cystadenoma of the Pancreas: Report of a Rare Case.

Background: Synchronous cystic lesions of the pancreas with different pathophysiology in the same patient are a rare occurrence.. Case Presentation: We report the incidental finding of a multicystic lesion within the pancreatic head in a morbidly obese woman during workup for bariatric surgery. The lesion contained an intraductal papillary mucinous neoplasm (IPMN) with high-grade dysplasia within an acinar cell cystadenoma (ACA). ACAs are rare tumors first described in 2002. Conclusion: To date, there have been no published reports of synchronous IPMN within an ACA. This case report intends to increase provider awareness of these lesions as well as highlight the importance of surveillance and careful histological examination of heterogeneous cystic lesions of the pancreas.

Aberrant Menin expression is an early event in pancreatic neuroendocrine tumorigenesis.

Pancreatic neuroendocrine tumors (PanNETs) are the second most common pancreatic malignancy and cause significant morbidity and mortality. Neuroendocrine microadenomas have been proposed as a potential precursor lesion for sporadic PanNETs. In this study, we applied telomere-specific fluorescent in situ hybridization (FISH) to a series of well-characterized sporadic neuroendocrine microadenomas and investigated the prevalence of alterations in known PanNET driver genes (MEN1 and ATRX/DAXX) in these same tumors using immunohistochemistry for the encoded proteins. We identified aberrant Menin expression in 14 of 19 (74%) microadenomas, suggesting that alterations in Menin, at least a subset of which was likely due to somatic mutation, are early events in pancreatic neuroendocrine tumorigenesis. In contrast, none of the microadenomas met criteria for the alternative lengthening of telomeres phenotype (ALT) based on telomere FISH, a phenotype that is strongly correlated to ATRX or DAXX mutations. Two of 13 microadenomas (15%) were noted to have very rare abnormal bright telomere foci on FISH, suggestive of early ALT, but these lesions did not show loss of ATRX or DAXX protein expression by immunohistochemistry. Overall, these data suggest that loss of Menin is an early event in pancreatic neuroendocrine tumorigenesis and that ATRX/DAXX loss and ALT are relatively late events.

Pharmacologic correction of dominant-negative GH1 deficiency causing mutations.

PURPOSE: Dominant-negative growth hormone gene (GH1) mutations cause familial isolated growth hormone deficiency type II (IGHD II), which is characterized by GH deficiency, occasional multiple anterior pituitary hormone deficiencies, and anterior pituitary hypoplasia. We have previously shown that 17.5-/22-kDa GH1 transcript ratios correlate with the severity of the IGHD II phenotype. We hypothesized that different pharmaceutical agents could affect the GH1 transcript ratio by modulating alternative splicing. METHODS: We exposed peripheral blood mononuclear cells from IGHD II patients and unaffected family members to different pharmacologic agents and then determined the 17.5-/22-kDa transcript ratios by real-time PCR. RESULTS: Dexamethasone and digoxin significantly increased the 17.5-/22-kDa transcript ratio, while sodium butyrate and 5-iodotubericidin significantly decreased the ratio. CONCLUSION: Since we have previously shown that the ratio of the 17.5-/22-kDa GH1 transcripts correlates with severity of the IGHD II phenotype, our findings here suggest that selected previously unconsidered agents could possibly reduce the severity of IGHD II, while other agents could possibly exacerbate the disease phenotype.