Circulating Carcinoembryonic Antigen Research Articles

Aldolase B-driven lactagenesis and CEACAM6 activation promote cell renewal and chemoresistance in colorectal cancer through the Warburg effect

Colorectal cancer (CRC) is a prevalent malignancy worldwide and is associated with a high mortality rate. Changes in bioenergy metabolism, such as the Warburg effect, are often observed in CRC. Aldolase B (ALDOB) has been identified as a potential regulator of these changes, but its exact role in CRC cell behavior and bioenergetic homeostasis is not fully understood. To investigate this, two cohorts of CRC patients were analyzed independently. The results showed that higher ALDOB expression was linked to unfavorable prognosis, increased circulating carcinoembryonic antigen (CEA) levels, and altered bioenergetics in CRC. Further analysis using cell-based assays demonstrated that ALDOB promoted cell proliferation, chemoresistance, and increased expression of CEA in CRC cells. The activation of pyruvate dehydrogenase kinase-1 (PDK1) by ALDOB-induced lactagenesis and secretion, which in turn mediated the effects on CEA expression. Secreted lactate was found to enhance lactate dehydrogenase B (LDHB) expression in adjacent cells and to be a crucial modulator of ALDOB-mediated phenotypes. Additionally, the effect of ALDOB on CEA expression was downstream of the bioenergetic changes mediated by secreted lactate. The study also identified CEA cell adhesion molecule-6 (CEACAM6) as a downstream effector of ALDOB that controlled CRC cell proliferation and chemoresistance. Notably, CEACAM6 activation was shown to enhance protein stability through lysine lactylation, downstream of ALDOB-mediated lactagenesis. The ALDOB/PDK1/lactate/CEACAM6 axis plays an essential role in CRC cell behavior and bioenergetic homeostasis, providing new insights into the involvement of CEACAM6 in CRC and the Warburg effect. These findings may lead to the development of new treatment strategies for CRC patients.

Abstract 2227: Multi-analyte profiling reveals relationships among circulating biomarkers in colorectal cancer

Abstract Introduction: Blood-based tests hold great promise as cancer diagnostics but until now have largely been restricted to the analysis of a single class of molecules (eg, circulating tumor DNA, platelet mRNA, circulating proteins). The ability to analyze multiple analytes simultaneously from the same biological sample may increase the sensitivity and specificity of such tests by exploiting independent information between signals. Here, we describe an experimental and analytical system that we developed and implemented for the integrated analysis of multiple analytes from a single blood sample, which revealed examples of both correlations and orthogonality among individual analytes. Methods: De-identified blood samples were obtained from healthy individuals, as well as individuals with pre-malignant conditions and stage I-IV colorectal cancer (CRC). After plasma separation, multiple types of analytes were assayed: cell-free DNA (cfDNA) content was assessed by low-coverage whole-genome sequencing (lcWGS) and whole-genome bisulfite sequencing (WGBS), cell-free microRNA (cf-miRNA) was assessed by small-RNA sequencing, and levels of circulating proteins were measured by quantitative immunoassay. Results: lcWGS of plasma cfDNA was able to identify CRC samples with high tumor fraction (>20%) on the basis of copy number variation (CNV) across the genome. High tumor fractions, while more frequent in late-stage cancer samples, were observed in some stage I and II patients. Aberrant signals in each of the three other analytes—cf-miRNA profiles discordant with those in healthy controls, genome-wide hypomethylation at LINE1 (long interspersed nuclear element 1) CpG loci, and elevated levels of circulating carcinoembryonic antigen (CEA) and cytokeratin fragment 21-1 (CYFRA 21-1) proteins—were also observed in cancer patients. Strikingly, aberrant profiles across analytes were indicative of high tumor fraction (as estimated from cfDNA CNV), rather than cancer stage. Conclusion: Our data suggest that tumor fraction is correlated with cancer stage but has a large potential range, even in early stage samples. Previous literature on blood-based screens for detection of cancer has displayed discordance in the claimed ability of different single analytes to detect early stage cancer. Tumor fraction may be able to explain the historical disagreement, as we found that aberrant profiles among cf-miRNA, cfDNA methylation, and circulating protein levels were more strongly associated with high tumor fraction than with late stage. These findings suggest that some positive “early stage” detection results may in fact be “high tumor fraction” detection results. Our results further demonstrate that assaying multiple analytes from a single sample may enable the development of classifiers that are reliable at low tumor fraction and for detecting pre-malignant or early-stage disease. Citation Format: Daniel Delubac, Eric Ariazi, Jonathan Berliner, Adam Drake, John Dulin, Riley Ennis, Erik Gafni, Kate Niehaus, Gabriel Otte, Jennifer Pecson, Girish Putcha, Corey Schaninger, Aarushi Sharma, Mike Singer, Abraham Tzou, Jill Waters, David Weinberg, Brandon White, Imran S. Haque. Multi-analyte profiling reveals relationships among circulating biomarkers in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2227.

Does the sequence of pulmonary vasculature ligation have any oncological impact during an anatomical lung resection for non-small-cell lung cancer?

A best evidence topic in thoracic surgery was written according to a structured protocol. The question addressed was 'in patients with primary lung carcinoma, does the sequence of pulmonary vasculature ligation during anatomical lung resection influence the oncological outcomes?' A total of 48 papers were found using the reported search, of which 7 represented the best evidence to answer the question. The authors, journal, date and country of publication, patient group studied, study type, relevant outcomes and results of these papers are tabulated. Among six prospective studies included, five of them randomized patients to either pulmonary vein or artery occlusion first during anatomical lung resection, while one study was retrospective. Two reports did not find any difference between pulmonary vein and artery occlusion first during long-term follow-up in terms of either disease recurrence (51 vs 53%, P = 0.7), or 5-year overall survival (54 vs 50%, P = 0.82). One report did not find any difference with regard to circulating tumour cells either after thoracotomy (5.0 vs 3.9, P = 0.4), or after the completion of lobectomy (38.0 vs 70.0, P = 0.23). One report found a higher expression of CD44v6 (P = 0.008) and CK19 (P = 0.05) in patients undergoing pulmonary arterial occlusion first. One report found that pulmonary vein occlusion before that of the pulmonary arterial branches has a favourable outcome on circulating carcino-embryonic antigen (CEA) mRNA in the peripheral blood, while another one did not find a significant difference in circulating levels of CEA mRNA (P = 0.075) and CK19 mRNA (P = 0.086) with either method. Another study reported no correlation between circulating pin1 mRNA levels in peripheral blood after the completion of the resection and the sequence of ligation of pulmonary vessels (9.95 ± 0.91 vs 14.71 ± 1.64, P > 0.05). Based on the two studies assessing the long-term outcome of patients with primary lung cancer undergoing anatomical curative resection, the sequence of ligation of pulmonary vessels does not seem to influence the oncological outcomes or survival. However, the other studies focusing on the influence of these techniques on circulating tumour cells or their molecular products report conflicting results the clinical consequences of which cannot be predicted.

Regulation of carcinoembryonic antigen release from colorectal cancer cells

Clinical and experimental evidence suggest that circulating carcinoembryonic antigen (CEA) released from tumor cells has an instrumental role in colorectal cancer-liver metastasis. However, the precise mechanism of the regulation of the CEA release from cancer cells is not known. We investigated if the rate of CEA and another GPI-anchored protein, alkaline phosphatase (AP) release is correlated with cellular glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) expression. We also evaluated the effects of phosphatidic acid (PA), a compound known to inhibit GPI-PLD activity, on the CEA and AP release from colon cancer cells. The expression of CEA, GPI-PLD, and AP in five colon carcinoma cells (LS180, Caco2, SW742, SW1116, and HT29/219) was verified by immunoblot and real-time RT-PCR analysis. The amounts of CEA and AP released into cell culture media were determined using ELISA and a colorimetric assay, respectively. We examined the effects of PA (20-100μM) on CEA and AP release from LS180 cells. All five cancer cell lines analyzed expressed GPI-PLD protein. While there was a positive relationship between AP release and the levels of GPI-PLD transcript expression, we found no direct correlation between CEA released from cancer cells and the GPI-PLD mRNA expression level. However, the rate of CEA release was positively associated with the level of CEA transcript expression. In comparison to controls, the release of GPI-anchored CEA and AP, but not CA19-9 was inhibited significantly by both crude and pure phosphatidic acid (by 56 and 54.5%, respectively). Using PA for inhibiting CEA release from cancer cells may have therapeutic application in preventing CRC-liver metastasis.

Type-2 somatostatin receptor mRNA levels in breast and colon cancer determined by a quantitative RT-PCR assay based on dual label fluorogenic probe and the TaqMan™ technology

We reported previously that the expression of type 2 somatostatin receptor (sst2) was positively related to patient outcome in the childhood tumor neuroblastoma. To quantitate the expression of mRNA sst2 expression, we used a competitive RT-PCR assay. To improve the practicability of this measurement and its applicability to large groups of patients, we present here an original ‘real-time’ quantitative RT-PCR method, based on a dual-labeled fluorogenic probe and the TaqMan™ technology. By this method, we have measured sst2 mRNA expression in 24 breast cancer samples and 26 colon carcinomas as well as on the corresponding non-adjacent non-neoplastic tissue of the same patients. The proposed method has a dynamic range of 4×10 4 to 4×10 8 molecules of sst2 mRNA. The intra-assay precision of the test, evaluated as signal detection variability, was 2.4%. Accuracy, evaluated by the addition of standard RNA to unknown samples, provided a mean recovery of 98±2%. A significant correlation has been observed in a study performed in 24 neuroblastoma samples measured both with the proposed method and with a competitive RT-PCR assay ( r=0.913, p<0.001). In our preliminary clinical study, no significant differences were observed in sst2 mRNA levels between normal and tumor specimens in both colorectal (normal tissue 5.1×10 7±2.0×10 7 molecules/μg total RNA, cancer tissue 9.7×10 7±4.2×10 7) and breast tumors (normal tissue 5.5×10 8±2.0×10 8, cancer tissue 4.4×10 8±3,7×10 8). However, in colorectal cancer, sst2 mRNA values of subjects with high circulating carcinoembryonic antigen (CEA) levels (>5 ng/ml) were statistically lower (2.3×10 7±6.2×10 6 molecules/μg total RNA; p<0.05) than those of subjects with low CEA concentration (1.4×10 8±6.7×10 7). Also, the sst2 mRNA ratio between normal and tumor tissue (N/T ratio) resulted significantly inversely related to CEA levels. In breast cancer, a significant difference was found between the mean N/T ratio of negative (below 10 fmol/mg protein) and positive estrogen receptor tumors ( p<0.05). Analogous results were found selecting breast tumors on the basis of the progesterone receptor status ( p<0.05). The proposed method is accurate, precise, sensitive and less labor-intensive than the competitive RT-PCR assay. For a correct evaluation of sst2 mRNA expression, it seems very important to measure the sst2 expression both in tumor and in the non-tumoral non-adjacent tumor specimens.

Early detection of liver metastasis in patients with colorectal carcinoma by increased levels of circulating IgA- and IgM-associated secretory component.

One hundred patients operated for colorectal carcinoma were followed clinically and with serial blood samples from 5 to 8 years. Levels of secretory component (SC) associated with IgA and IgM in serum were measured and related to Dukes' stage, histological differentiation, tumour expression of SC, and circulating carcinoembryonic antigen (CEA). On the whole, elevated levels of SC in serum were found in 15 of the 20 patients who already had (n = 15), or later developed (n = 5), liver metastasis. Four of the latter 5 patients showed raised SC levels with a 5.5 months median lead time from the first positive serum sample to clinically manifest liver disease. These data are interesting in view of the promising results reported for liver resection in patients with colorectal carcinoma.

Elevated Levels of Circulating Carcinoembryonic Antigen in Hypothyroidism *

The level of circulating carcinoembryonic antigen (CEA) was measured in 148 patients with various thyroid diseases. A significantly high frequency of positive CEA was observed in hypothyroid patients with Hashimoto's disease, but the serum CEA levels were not correlated with the serum calcitonin concentrations in Hashimoto's disease. The CEA levels were inversely correlated with the serum T4 concentrations (P less than 0.001) and were positively correlated with the serum TSH concentrations (P less than 0.001), but not with the titers of serum antithyroid antibodies or the size of goiter in autoimmune thyroid disease. Moreover, the increase in CEA was significantly related to the duration of hypothyroidism (P less than 0.001). The high CEA levels in all hypothyroid patients decreased when the patients attained a euthyroid state after thyroid hormone therapy for 4-9 months. The gradual decrease in the serum CEA levels during treatment was roughly correlated with the decreases in serum cholesterol concentration and serum lactic dehydrogenase and glutamic oxalacetic transaminase activities. On Sephadex G-200 column chromatography of serum from hypothyroid patients, the CEA immunoreactivity, like purified standard CEA, was recovered in the large molecular weight fraction. These findings indicate that elevated CEA levels in hypothyroid patients do not necessarily indicate malignancy. CEA elevation in hypothyroidism may be caused by decreased degradation of CEA.

Carcinoembronic antigen and endocrine response in a patient with Sipple's syndrome following surgery.

Circulating carcinoembryonic antigen (CEA), calcitonin (CT) and adrenocorticotropic hormone (ACTH) and levels in tissue extracts of a patients with Sipple's syndrome were measured simultaneously. Biofocal medullary carcinoma of the thyroid gland (MCT), left adrenal phaeochromocytoma and ectopic para-aortal phaeochromocytoma had been removed in the first operation. Total thyroidectomy was performed thereafter. Evidence has been presented showing that (1) the first operation lowered the elevated serum levels of CEA and CT, and levels of CEA and CT in tissue extracts of MCT were considerably higher than those of tumor-uninvolved thyroid tissue and phaeochromocytomas; (2) markedly elevated levels of plasma ACTH were normalized by the first surgery and, in addition, immunoreactive ACTH and beta-melanocyte stimulating hormone (beta-MSH) were detected in the phaeochromocytomas removed. This is a documented case of the simultaneous production of CT and CEA by MCT associated with ectopic secretion of ACTH and beta-MSH from the phaeochromocytomas without manifesting clinical features of Cushing's syndrome and is based on the findings referred to above.

Carcinoembryonic antigen release from colorectal cancer cells in tissue culture

Limited data are available concerning the physicochemical and immunological characteristics of carcinoembryonic antigen spontaneously released from cancer cells in tissue culture. The Hoffman—LaRoche Z-gel assay for the detection of circulating carcinoembryonic antigen (CEA) in serum was modified for the detection of CEA in tissue culture supernatants. A number of mycoplasma free epithelial lines established from colorectal cancer specimens were evaluated for cell-bound and spontaneously released CEA. Further studies were performed using tissue culture supernatants from colon cancer line SW480, which spontaneously released microgram quantities of CEA into the cell culture supernatant. By competitive inhibition analysis this CEA appeared identical with the Roche CEA standard. Gel filtration studies on Sephadex G-200 and affinity chromatography with concanavalin A sepharose-4B revealed the supernatant CEA and the Roche standard to be similar. These data suggest that this system offers potential for further characterization of this oncofetal protein and for study of its biosynthesis and “spontaneous” release from cancer cell surface. The molecular basis of such activity on the genetic level would have significant importance in basic biological studies of malignant transformation.

Detection of circulating carcinoembryonic antigen (CEA) by counter-immunoelectrophoresis

A sensitive method is described for detecting circulating carcinoembryonic antigen (CEA) by combined use of counter-immunoelectrophoresis and intensifiers. Perchloric acid extraction of serum was followed by counter-immunoelectrophoresis against monospecific antiserum to CEA. A precipitin line formed was intensified further by electrophoretic binding of anti-γG γ-globulin and Amido Black staining. Less than 10 ng CEA/ml was detectable. Using this method, a total of 84 sera from patients with or without malignancy was examined. The results obtained were in general agreement with those reported by radioimmunoassay. The method appears to be useful as a diagnostic aid, since it requires neither labeled preparations of purified CEA nor special equipment for assay.

Elevated Carcinoembryonic Antigen Levels And Biliary Tract Obstruction

Circulating carcinoembryonic antigen (CEA) levels and their relation to liver function test values were studied in 29 jaundiced patients with benign extrahepatic biliary tract obstruction and inflammation. During the obstructive and inflammatory phase, 15 (52%) of the patients had CEA levels greater than 2.5 ng/ml. Elevated CEA levels were associated more frequently with common bile duct stones (and cholangitis) than with gallbladder stones (and cholecystitis) alone, although this difference was not statistically significant. The former often had values greater than 5.0 ng/ml. The highest values were found in two patients with liver abscesses. T'HE CEA levels returned to normal following relief of obstruction in seven of ten patients and increased in two patients who had progressive inflammation. Serum alkaline phosphatase and bilirubin levels were significantly higher in the patients with elevated CEA levels (p smaller than .05). Serum alkaline phosphatase levels showed a significant positive correlation with CEA levels (p smaller than .02). Patients with obstructive jaundice and elevated CEA levels do not necessarily have cancer.

Diagnosis and Prognosis in Colon Cancer Based on a Profile of Immune Reactivity&lt;xref ref-type="fn" rid="fn2"&gt;2&lt;/xref&gt;

An immunologic profile consisting of measurements of circulating carcinoembryonic antigen (CEA), tumor antigen-induced inhibition of monomuclear cell migration (IMM) and skin reactivity to purified protein derivative, streptokinase-streptodornase, and mumps was assessed as a diagnostic and prognostic tool in 16 patients with colon cancer. Preoperatively, 10 of 14 patients tested had elevated CEA, 12 of 12 showed tumor antigen-induced IMM, and 10 of 11 failed to react to 2 or more recall antigens. Potential surgical cure (7 patients) was accompanied by normal CEA in 4, absent tumor antigen-induced IMM in all 7, and increased skin-test reactivity in 6. Disseminated cancer (9 patients) was associated with elevated CEA in all 9, with absent IMM in all 7 and with suppressed skin-test reactivity in 6 of 9.

Variations of the carcinoembryonic antigen level in the plasma of patients with gynecologic cancers during therapy

In 63 patients with histologically proved gynecologic carcinoma, circulating carcino-embryonic antigen (CEA) was determined by radioimmunoassay before and at two intervals after treatment. Thirty-one patients of 63 had CEA values over 2.5 ng. per milliliter before treatment. In general, the CEA levels were low compared to those found in endodermal carcinoma. The percentage of elevated CEA values was slightly higher in cases of carcinoma of the cervix and corpus uteri than in those of carcinoma of the ovary. All patients with CEA levels greater than 2.5 ng. per milliter treated by complete surgical resection of tumor showed a drop of CEA levels to below 2.5 ng. per milliliter seven weeks after operation. In contrast, patients with palliative therapy showed no change in CEA values. About half of the patients treated with a complete course of internal and external radiotherapy showed a drop of CEA levels to below 2.5 ng. per milliliter, whereas the other patients showed fluctuating CEA values. No correlation between clinical status and evolution of CEA levels in these patients could be drawn at the present time. The CEA test seems to be of little value for the early diagnosis of gynecologic carcinoma but appears to be interesting for the evaluation of therapy and the follow-up of patients with diagnosed cases.

Circulating Carcinoembryonic Antigen Levels and Serum Suppression of Phytohemagglutinin- Stimulated Lymphocyte DNA Synthesis: An Inverse Correlation in the Cancer Patient2

Thirty-six patients, 23 with digestive tract cancer and 13 with breast cancer, were studied to determine the relationship between circulating carcinoembryonic antigen (CEA) levels and the suppressive activity of their sera on phytohemagglutinin-induced DNA synthesis in normal lymphocytes. Ten of 11 patients with metastases had positive serum CEA levels (>2.5 ng/cm3) , but only 2 had sera which inhibited normal lymphocyte DNA synthesis to <80% of control values. Of 25 patients with primary disease and without known metastases, 9 had positive CEA, but 14 had suppressive sera. After the primary tumor was resected, inverse changes in CEA and suppressive levels occurred. This change suggested a functional immunologic relationship between serumsuppressive activity and CEA.

Circulating carcinoembryonic antigen (CEA): relationship to clinical status of patients with inflammatory bowel disease.

Plasma levels of circulating carcinoembryonic antigen (CEA) were measured by zirconyl phosphate gel radioimmunoassay in 112 patients with chronic inflammatory bowel disease. The levels were then related to category, extent, duration, and severity of disease, as well as to the ages and surgical status of the patients. The distribution of CEA levels and their mean values were significantly raised over the levels in 33 normal control subjects, and were similar among patients with ulcerative colitis compared with those with granulomatous bowel disease. Positive values were defined as those in excess of 2.5 ng/ml. Positive assays occurred in 42% of ulcerative colitis patients, in 38% of Crohn's disease patients, and in 40% of the total group with inflammatory bowel disease. Among normal control subjects, only 3% were positive. Among inflammatory bowel disease patients, positive CEA assays occurred more frequently with more severe disease, more extensive anatomical involvement, younger ages, and shorter duration of disease. Those patients who had undergone total colectomy showed levels of circulating CEA and frequency of CEA positivity similar to those of an age-matched normal control group. Levels of CEA did not correspond with known cancer risk factors in patients with inflammatory bowel disease. Although rising or persisting plasma CEA values unrelated to severity and extent of disease may indicate an unfavourable prognosis in cancer, this study shows that a single CEA value in patients with chronic inflammatory bowel disease is not a reliable indicator of cancer risk.