Q RT-PCR detection of chromogranin A: a new standard in the identification of neuroendocrine tumor disease

Abstract

Objective: Message and protein expression of CgA was examined to evaluate the sensitivity of a PCR-based approach in the detection of covert neuroendocrine (NE) tissue.

Summary background data: Immunohistochemical (IHC) measurement of chromogranin A (CgA) discriminates gastrointestinal (GI) carcinoids from epithelial tumors. IHC is, however, an insensitive technique to identify micrometastases or delineate subpopulations of NE cells.

Methods: CgA gene expression was examined by Q-RT PCR in GI carcinoids (small intestinal and metastases, n=17, gastric, n=5), appendiceal tumors (n=10), and adenocarcinomas (gastric, n=5, colorectal, n=6). CgA protein expression levels were quantitatively analyzed following IHC by automated quantitative analysis (AQUA) in 2 tissue microarrays (GI carcinoid and GI adenocarcinoma).

Results: CgA gene was overexpressed (P<0.001) in GI carcinoids compared with GI adenocarcinomas and normal mucosa. Elevated levels (P<0.00001) were also identified in carcinoid liver and lymph node (LN) metastases. CgA levels were higher (approximately 2-4-fold) in NE appendiceal carcinoids than in adenocarcinoids, but in GI adenocarcinomas were identical to normal mucosa. Histologically normal lymph nodes expressed detectable CgA message in 30% of cases. CgA protein levels were highest in primary GI carcinoids and in liver metastases and significantly elevated (P<0.005) compared with nonmetastatic lesions. Expression in liver and LN metastases was significantly elevated (P<0.000001) compared with normal. Analysis of mRNA by Q-RT PCR was >200-fold more sensitive than by IHC.

Conclusions: Overexpression of CgA mRNA and protein in GI carcinoids can identify metastatic cells; thus, PCR for CgA can be used to identify micrometastases not evident by light microscopy or IHC as well as define tumors of ambivalent morphologic phenotype. The use of this sensitive strategy to assess NETs and apparently normal LNs and liver may be of future utility in defining therapeutic strategy.

FIGURE 1. Message levels of Chromogranin A (CgA) determined by Q RT-PCR. Levels were corrected against expression of the housekeeping gene, GAPDH. Levels of CgA were significantly overexpressed (3-Log) in small intestinal carcinoid tumor (ST) samples compared with normal mucosa (SN) (*P < 0.000001) and in liver metastases (LVM) compared with normal liver (LVN) (^#P < 0.02). CgA was elevated in lymph node metastases (LNM) compared with normal lymph nodes (LNN) (^#P < 0.02). Data are mean ± SEM. S, small bowel; LN, lymph node; LV, liver; N, normal; T, tumor; M, metastases.

FIGURE 2. Message levels of CgA determined by Q RT-PCR in gastric carcinoid specimens. Levels of CgA were significantly overexpressed (10×) in nonmetastatic (GNM) gastric carcinoid tumors (type I/II tumors) compared with normal mucosa (GN) and in malignant (GM) gastric carcinoids (>200×) (type III/IV). Levels in the latter were significantly elevated compared with the nonmalignant tumors (^#P < 0.05, *P < 0.01, **P < 0.005). Data are mean ± SEM. G, gastric; N, normal; NM, nonmetastatic; M, metastatic.

FIGURE 3. Message levels of CgA determined by Q RT-PCR in gastric adenocarcinomas and colorectal adenocarcinomas compared with gastric carcinoids and small intestinal carcinoids. Levels of CgA were elevated in carcinoids but were not different between normal mucosa and tumor samples (*P < 0.005). Data are mean ± SEM.

FIGURE 4. Message levels of CgA determined by Q RT-PCR or IHC in appendiceal carcinoids and appendiceal adenocarcinoids compared with normal mucosa. A, Levels of CgA message were elevated in neuro endocrine appendiceal carcinoids (ANM; ∼200×) and adenocarcinoids (AM, ∼50×) compared with normal appendiceal mucosa (AN) (*P < 0.05; **P < 0.01). Data are mean ± SEM. B, On the TMA, CgA levels were higher in neuro endocrine appendiceal carcinoids (ANM; ∼4×) and adenocarcinoids (AM; 2×) compared with normal appendiceal mucosa (AN). Neuroendocrine carcinoids had significantly higher CgA levels than adenocarcinoids (*P < 0.05; **P < 0.001; ^#P < 0.0002). Data are mean ± SEM. AN, normal appendix; ANM, nonmalignant appendix; AM, malignant appendix.

FIGURE 5. Message levels of CgA in histologically positive (LNM) and histologically negative (LNN) lymph nodes. CgA was significantly overexpressed in positive lymph nodes compared with negative lymph nodes. Three histologically lymph node-negative samples had elevated CgA message. ⋄, lymph node negative sample with abnormally elevated gene expression. *P < 0.002. Data are mean ± SEM. LNM, lymph node metastases; LNN, lymph node negative.

FIGURE 6. Expression levels of CgA determined by immunohistochemistry and AQUA quantitation on YTMA60. A, Three-color image of a small bowel carcinoid demonstrating significant overlap between cytokeratin and cytoplasmic CgA staining in the carcinoid tumor (inset). Immunostaining of CgA was invariably cytoplasmic localized. Blue, nuclei (DAPI); green, tumor mask (cytokeratin–Alexa488); red, CgA (Cy5). Dual membrane staining (red and green) results in yellow. (100× magnification). B, AQUA levels of CgA were significantly overexpressed in primary malignant GI carcinoid tumors (TM), and primary nonmetastatic carcinoid tumors (TNM) compared with normal small intestinal mucosa (SN). Expression levels of CgA were elevated in lymph node metastatic tissue (LNM) and in liver metastases (LVM) compared with normal lymph nodes (LNN) and normal liver (LVN), respectively. In addition, CgA levels were elevated in liver metastases compared with primary nonmalignant tumors (*P < 0.00001; ^#P < 0.005; ^@P < 0.00005). Data are mean ± SEM. LV, liver; LN, lymph node; S, small bowel; N, normal; T, tumor; M, metastases; NM, no metastases.

FIGURE 7. Expression levels of CgA determined by immunohistochemistry and AQUA quantitation on YTMA25. A, Three-color image of a colorectal adenocarcinoma. No overlap was shown between cytokeratin and cytoplasmic CgA staining in the adenocarcinoma tumor (insets). Blue, nuclei (DAPI); green, tumor mask (cytokeratin–Alexa488); red, CgA (Cy5) (100× magnification). B, AQUA levels of CgA were not significantly elevated in gastric (GT) or colorectal adenocarcinomas (CT) or in hepatocellular carcinomas (LVT) compared with normal mucosa from each of these sites. Data are mean ± SEM. C, colorectal; G, gastric; LV, liver; N, normal; T, tumor.