Immunohistochemical Expression of Somatostatin Receptor Subtypes in a Panel of Neuroendocrine Neoplasias

Neuroendocrine neoplasias (NENs) are known to express somatostatin receptors (SSTRs) 1-5, which are G-protein-coupled cell membrane receptors. Somatostatin receptor imaging and therapy utilizes the SSTR expression. Synthetic somatostatin analogs with radioligands are used to detect primary tumors, metastases, and recurrent disease. Receptor analogs are also used for treating NENs. Furthermore, commercially available SSTR antibodies can be used for the immunohistochemical (IHC) detection of SSTRs. We investigated different SSTR antibody clones applying diverse IHC protocol settings to identify reliable clones and feasible protocols for NENs. A tissue microarray including NENs from 12 different primary sites were stained. Only UMB clones were able to localize SSTR on the cell membranes of NENs. SSTR2 (UMB1) emerged as the most common subtype followed by SSTR5 (UMB4) and SSTR1 (UMB7). SSTR3 (UMB5) expression was mainly cytoplasmic. Yet, SSTR4 expression was weak and located primarily in the cytoplasm. Thus, appropriate IHC protocols, including proper positive and negative controls, represent requirements for high-quality NEN diagnostics and for planning personalized therapy.Peer reviewe

MAPK inhibitors induce serine peptidase inhibitor Kazal type 1 (SPINK1) secretion in BRAF V600E-mutant colorectal adenocarcinoma

The mitogen-activated protein kinase (MAPK) pathway plays a central role in colorectal cancers (CRC). In particular, BRAF V600E-mutant tumors, which represent around 10% of CRCs, are refractory to current therapies. Overexpression and secretion of serine peptidase inhibitor Kazal type 1 (SPINK1) are observed in around 50% of CRCs, and its serum level can be used as a biomarker for poor prognosis. Utilizing a recently developed extendable blocking probe assay, we analyzed the BRAF mutation status in a CRC patient cohort (N=571) using tissue-derived RNA as the starting material. From the same RNA samples, we measured the relative SPINK1 expression levels using a quantitative real-time PCR method. Expression of mutant BRAF V600E correlated with poor prognosis, as did low expression of SPINK1 mRNA. Further, BRAF V600E correlated negatively with SPINK1 levels. In order to investigate the effect of MAPK pathway-targeted therapies on SPINK1 secretion, we conducted invitro studies using both wild-type and V600E CRC cell lines. BRAF inhibitor vemurafenib, and subsequent MAPK pathway inhibitors trametinib and SCH772984, significantly increased SPINK1 secretion in V600E CRC cell lines Colo205 and HT-29 with a concomitant decrease in trypsin-1 and -2 secretion. Notably, no SPINK1 increase or trypsin-1 decrease was observed in BRAF wild-type CRC cell line Caco-2 in response to MAPK pathway inhibitors. In further mechanistic studies, we observed that only trametinib was able to diminish completely both MEK and ERK phosphorylation in the V600E CRC cells. Furthermore, the key regulator of integrated stress response, activating transcription factor 4 (ATF-4), was downregulated both at mRNA and at protein level in response to trametinib treatment. In conclusion, these data suggest that sustained inhibition of not only MAPK pathway activation, but also ATF-4 and trypsin, might be beneficial in the therapy of BRAF V600E-mutant CRC and that SPINK1 levels may serve as an indicator of therapy response.Peer reviewe

Toll-like receptor 2 and Toll-like receptor 4 predict favorable prognosis in local pancreatic cancer

Toll-like receptors play an essential role in our innate immune system and are a focus of interest in contemporary cancer research. Thus far, Toll-like receptors have shown promising prognostic value in carcinomas of the oral cavity, colon, and ovaries, but the prognostic role of Toll-like receptors in pancreatic ductal adenocarcinoma has not been established. We set out to investigate whether Toll-like receptor expression could serve in prognostic evaluation in pancreatic ductal adenocarcinoma, as well. Our study comprised 154 consecutive stage I?III pancreatic ductal adenocarcinoma patients surgically treated at Helsinki University Hospital between 2002 and 2011. Patients who received neoadjuvant therapy were excluded. Tissue microarrays and immunohistochemistry allowed assessment of the expression of Toll-like receptor 2 and Toll-like receptor 4 in pancreatic ductal adenocarcinoma tissue, and we matched staining results against clinicopathological parameters using Fisher?s test. For survival analysis, we used the Kaplan?Meier method and the log-rank test, and the Cox regression proportional hazard model for univariate and multivariate analyses. The hazard ratios were calculated for disease-specific overall survival. Strong Toll-like receptor 2 expression was observable in 51 (34%) patients and strong Toll-like receptor 4 in 50 (33%) patients. Overall, neither marker showed any direct coeffect on survival. However, strong Toll-like receptor 2 expression predicted better survival when tumor size was less than 30?mm (hazard ratio?=?0.30; 95% confidence interval?=?0.13?0.69; p?=?0.005), and strong Toll-like receptor 4 expression predicted better survival in patients with lymph-node-negative disease (hazard ratio?=?0.21; 95% confidence interval?=?0.07?0.65; p?=?0.006). In conclusion, we found strong Toll-like receptor 2 and Toll-like receptor 4 expressions to be independent factors of better prognosis in pancreatic ductal adenocarcinoma patients with stage I?II disease.Peer reviewe

Postoperative serum CA19-9, YKL-40, CRP and IL-6 in combination with CEA as prognostic markers for recurrence and survival in colorectal cancer

Background In colorectal cancer (CRC) patients, guidelines only recommend measurement of preoperative carcinoembryonic antigen (CEA), although postoperative CEA may be more informative. However, the sensitivity of both preoperative and postoperative CEA in identifying relapse is limited. We studied whether CA19-9, YKL-40, C-reactive protein (CRP) and interleukin (IL)-6 add prognostic information combined with postoperative CEA. Material and methods This post-hoc analysis included 147 radically resected stage II (n = 38), III (n = 91) and IV (n = 18) CRC patients treated with adjuvant 5-fluorouracil (5-FU)-based therapy in the phase III LIPSYT study (ISRCTN98405441). We collected postoperative blood samples a median of 48 days after surgery. We analysed relapses, sensitivity, positive predictive value (PPV) and disease-free (DFS) and overall survival (OS) by bootstrap, Kaplan-Meier and adjusted Cox-models in the elevated vs. normal biomarker groups. Results Elevated postoperative CEA associated with impaired DFS (HR 7.23; CI(95%)3.85-13.58), impaired OS (HR 7.16; CI(95%)3.76-13.63), and more relapses (HR 7.9; CI(95%)3.4-18.2); but sensitivity for CEA in finding relapses was only 31% (CI(95%)21-48%). Normal CEA combined with an elevated YKL-40 or elevated CRP showed more relapses (HR for YKL-40 2.13 [CI(95%)1.10-4.13], HR for CRP 3.14 [CI(95%)1.21-8.16]), impaired DFS (HR 2.18 [CI(95%)1.12-4.24] or 3.23 [CI(95%)1.34-7.82]), and impaired OS (2.33 [CI(95%)1.24-4.40] or 2.68 [CI(95%)1.12-6.44]). Elevated CEA combined with a concomitantly elevated CA19-9, YKL-40, CRP or IL-6 showed a respective PPV of 100, 90, 100, and 100%. Conclusion In radically operated stage II to IV CRC patients who received adjuvant 5-FU-based chemotherapy, a postoperatively elevated CEA alone or in combination with CA19-9, YKL-40, CRP, or IL-6, or a normal CEA combined with an elevated YKL-40 or with an elevated CRP, may indicate patients at high risk of relapse.Peer reviewe

Additive clinical impact of epidermal growth factor receptor and podocalyxin-like protein expression in pancreatic and periampullary adenocarcinomas

The outcome of periampullary adenocarcinomas remains poor with few treatment options. Podocalyxin-like protein (PODXL) is an anti-adhesive protein, the high expression of which has been shown to confer a poor prognosis in numerous malignancies. A correlation and adverse prognostic synergy between PODXL and the epidermal growth factor receptor (EGFR) has been observed in colorectal cancer. Here, we investigated whether this also applies to periampullary adenocarcinomas. We analyzed the immunohistochemical expression of PODXL and EGFR in tissue microarrays with tumors from two patient cohorts; (Cohort 1, n=175) and (Cohort 2, n=189). The effect of TGF-beta -induced expression and siRNA-mediated knockdown of PODXL and EGFR, were investigated in pancreatic cancer cells (PANC-1) in vitro. We found a correlation between PODXL and EGFR in these cancers, and a synergistic adverse effect on survival. Furthermore, silencing PODXL in pancreatic cancer cells resulted in the down-regulation of EGFR, but not vice versa. Consequently, these findings suggest a functional link between PODXL and EGFR, and the potential combined utility as biomarkers possibly improving patient stratification. Further studies examining the mechanistic basis underlying these observations may open new avenues of targeted treatment options for subsets of patients affected by these particularly aggressive cancers.Peer reviewe

Small oral tongue cancers (ae 4 cm in diameter) with clinically negative neck : from the 7th to the 8th edition of the American Joint Committee on Cancer

One of the main changes in the 8th edition of the American Joint Committee on Cancer (AJCC) for staging of oral cancer is the inclusion of depth of invasion (DOI) in the T category. However, cancers in different oral subsites have variable behavior, with oral tongue squamous cell carcinoma (OTSCC) being the most aggressive one even at early stage. Thus, it is necessary to evaluate the performance of this new T category in homogenous cohort of early OTSCC. Therefore, we analyzed a large cohort of patients with a small (ae4 cm) OTSCC to demonstrate the differences in T stage between the AJCC 7th and 8th editions. A total of 311 early-stage cases (AJCC 7th) of OTSCC were analyzed. We used 5 mm and 10 mm DOI for upstaging from T1 to T2 and from T2 to T3 respectively, as in the AJCC 8th. We further reclassified the cases according to our own proposal suggesting 2 mm to upstage to T2 and 4 mm to upstage to T3. According to AJCC 7th, there were no significant differences in the survival analysis. When we applied the 8th edition, many cases were upstaged to T3 and thus associated with worse disease-specific survival (HR 2.37, 95% CI 1.12-4.99) and disease-free survival (HR 2.12, 95% CI 1.09-4.08). Based on our proposal, T3 cases were associated with even worse disease-specific survival (HR 4.19, 95% CI 2.27-7.74). The 8th edition provides better survival prediction for OTSCC than the 7th and can be further optimized by lowering the DOI cutoffs.Peer reviewe

PCSK2 expression in neuroendocrine tumors points to a midgut, pulmonary, or pheochromocytoma-paraganglioma origin

Neuroendocrine tumors (NETs) are often diagnosed from the metastases of an unknown primary tumor. Specific immunohistochemical (IHC) markers indicating the location of a primary tumor are needed. The proprotein convertase subtilisin/kexin type 2 (PCSK2) is found in normal neural and neuroendocrine cells, and known to express in NETs. We investigated the tissue microarray (TMA) of 86 primary tumors from 13 different organs and 9 metastatic NETs, including primary tumor-metastasis pairs, for PCSK2 expression with polymer-based IHC. PCSK2 was strongly positive in all small intestine and appendiceal NETs, the so-called midgut NETs, in most pheochromocytomas and paragangliomas, and in some of the typical and atypical pulmonary carcinoid tumors. NETs showing strong positivity were re-evaluated in larger tumor cohorts confirming the primary observation. In the metastases, the expression of PCSK2 mirrored that of the corresponding primary tumors. We found negative or weak staining in NETs from the thymus, gastric mucosa, pancreas, rectum, thyroid, and parathyroid. PCSK2 expression did not correlate with Ki-67 in well-differentiated NETs. Our data suggest that PCSK2 positivity can indicate the location of the primary tumor. Thus, PCSK2 could function in the IHC panel determined from screening metastatic NET biopsies of unknown primary origins.Peer reviewe

Extendable blocking probe in reverse transcription for analysis of RNA variants with superior selectivity

Peer reviewe

Expression of hormone receptors in oropharyngeal squamous cell carcinoma

Hormone receptors play an important role in many types of cancers. Alongside factors associated with human papillomavirus (HPV) infection, hormonal receptors may impact the tumorigenesis of oropharyngeal cancer. This study consists of 199 consecutive oropharyngeal squamous cell carcinoma (OPSCC) patients diagnosed and treated with a curative intent. We examined androgen (AR), estrogen (ER; both alpha and beta), and progesterone receptor (PR) expressions using immunohistochemistry comparing tumor and patient characteristics. AR was expressed in 16%, PR in 27% and ER-beta in 63% of the tumors. HPV- and p16-positive tumors expressed more AR and less PR than their negative counterparts. High PR expression was associated with poor disease-specific and locoregional recurrence-free survival. AR, PR, and ER-beta are expressed in OPSCC, and AR and PR expressions are associated with HPV and p16 status. Furthermore, PR appears to have prognostic significance. This may allow us to investigate the role of anti-hormone receptors in the treatment of OPSCC.Peer reviewe