ALDH1 and ALCAM as emerging biomarkers in personalized rectal cancer therapy.

50 Background: ALCAM (Activated Leukocyte Cell Adhesion Molecule) is thought to be involved in tumor cell adhesion, migration and metastasis. The ectodomain responsible for adhesion is proteolytically shed. Elevated shedding of ALCAM ectodomain is associated with malignant progression. Considering the proximity of urogentical cancer to both urinary secretions and hematogenous circulation we investigated if ALCAM shed into urine and/or blood could act as a prognostic or diagnostic marker of disease and patient outcome. Methods: Shed ALCAM in serum and urine was measured by ELISA while ALCAM shedding in tissues was detected with a unique immunohistochemical approach. ALCAM shedding was correlated with patient parameters including tumor stage, metastasis, recurrence, overall and disease-specific survival. Results: ALCAM shedding was compared across tissue and fluids from normal age-matched healthy adults, non-urological neoplasia, and non-cancer patients. Elevated ALCAM shedding was readily detected in fluids and tissue from patients with urogenital cancers. However, only the detection of shedding in tissue and urine correlated significantly with disease progression and patient outcome. Specifically, shed ALCAM detected in urine collected from bladder cancer patients undergoing cystectomy (Vanderbilt 2000-2010) correlated strongly with patient outcome. Urine ALCAM correlates positively with invasiveness (80%), and is predictive of recurrence as well as survival (HR=10.2 and 2.9). Urinary ALCAM ROC curves show the optimal sensitivity and specificity of predicting recurrence (90 and 68%) and survival (62 and 83%). Conclusions: We show for the first time that cleaved ALCAM is detectable in the urine and that urinary ALCAM is a novel biomarker in urogenital cancers. ALCAM shedding is marker of cancer progression and patient outcome with high specificity and accuracy. Evaluating levels of shed ALCAM in urine offers a unique and effective method to diagnose, assess, and stratify patients with urogenital cancers. We find this correlation particularly exciting because ALCAM is functionally involved in cancer progression and therefore reports on cellular behavior rather than mere presence of neoplastic disease.

Cancer stem-like cells (CSCs) displaying the properties of normal stem cells have become the main culprit associated with cancer transportation and recurrence. As of now, various CSC functions and marker genes have been identified due to the heterogeneity of cancer, such as aldehyde dehydrogenase (ALDH), the second member of the ABC transporter G-subfamily (ABCG2), activated leukocyte cell adhesion molecule (ALCAM) and CD133. To investigate these markers, most conventional approaches are bulk-based strategies, which may veil the disparity of single cells' gene expression. In this study, one-step digital RT-PCR at the single cell level was developed to co-determine the expression of ALDH1A1, ABCG2, ALCAM and CD133 genes in A549 cancer stem cells that perform high ALDH activities (ALDH+ A549 cells), as well as in ALDH- A549 cells and A549 cells, with 36, 20 and 20 cell samples each. The results demonstrated that, when compared to single ALDH- or A549 cells, the majority of single ALDH+ A549 cells displayed a 1.5- and 2.0-fold increase in the gene expression of ALDH1A1 and ALCAM (P < 0.001), respectively. However, for ABCG2 and CD133, there was no significant difference (P > 0.05), which means that they are not appropriate as co-indicated markers to identify ALDH+ A549 cells. Conclusively, as a single cell level approach, one-step digital RT-PCR has potential in exploring efficient co-detection markers for the classification and identification of CSCs.

Activated leukocyte cell adhesion molecule (ALCAM) has been proposed to function as a cell surface sensor for cell density, controlling the transition between local cell proliferation and tissue invasion in cancer progression. Herein, we determined ALCAM expression in 107 oral squamous cell carcinomas (OSCCs), 78 oral lesions (58 hyperplasias and 20 dysplasias) and 30 histologically normal oral tissues using immunohistochemistry and correlated with clinicopathological parameters. Significant increase in ALCAM immunopositivity was observed from normal oral mucosa, hyperplasia, dysplasia to OSCCs (p(trend) < 0.001). Increased ALCAM expression was observed in cytoplasm of epithelial cells as early as in hyperplasia (p = 0.001, OR = 3.8). Sixty-five of 107 (61%) OSCCs showed significant overexpression of ALCAM protein in cytoplasm/membrane of tumor cells (p = 0.043; OR = 3.3) in comparison with the normal oral tissues. Among OSCCs, cytoplasmic ALCAM was associated with advanced tumor size, tumor stage and tobacco consumption. Importantly, cytoplasmic ALCAM was an independent predictor of poor prognosis of OSCCs in multivariate analysis (p = 0.012, OR = 6.2). In an attempt to understand the molecular basis of cytoplasmic localization of ALCAM, 14-3-3 zeta and 14-3-3 sigma were identified as its novel binding partners in oral cancer cells. In conclusion, increased expression of ALCAM is an early event in oral tumorigenesis; its cytoplasmic accumulation in tumor cells is a predictor of poor prognosis of OSCCs, underscoring its potential as a candidate prognostic marker for oral cancer.

Low activated leukocyte cell adhesion molecule expression is associated with advanced tumor stage and early prostate-specific antigen relapse in prostate cancer

Proteins involved in tumor cell migration can potentially serve as markers of invasive disease. Activated Leukocyte Cell Adhesion Molecule (ALCAM) promotes adhesion, while shedding of its extracellular domain is associated with migration. We hypothesized that shed ALCAM in biofluids could be predictive of progressive disease. ALCAM expression in tumor (n = 198) and shedding in biofluids (n = 120) were measured in two separate VUMC bladder cancer cystectomy cohorts by immunofluorescence and enzyme-linked immunosorbent assay, respectively. The primary outcome measure was accuracy of predicting 3-year overall survival (OS) with shed ALCAM compared to standard clinical indicators alone, assessed by multivariable Cox regression and concordance-indices. Validation was performed by internal bootstrap, a cohort from a second institution (n = 64), and treatment of missing data with multiple-imputation. While ALCAM mRNA expression was unchanged, histological detection of ALCAM decreased with increasing stage (P = 0.004). Importantly, urine ALCAM was elevated 17.0-fold (P < 0.0001) above non-cancer controls, correlated positively with tumor stage (P = 0.018), was an independent predictor of OS after adjusting for age, tumor stage, lymph-node status, and hematuria (HR, 1.46; 95% CI, 1.03–2.06; P = 0.002), and improved prediction of OS by 3.3% (concordance-index, 78.5% vs. 75.2%). Urine ALCAM remained an independent predictor of OS after accounting for treatment with Bacillus Calmette-Guerin, carcinoma in situ, lymph-node dissection, lymphovascular invasion, urine creatinine, and adjuvant chemotherapy (HR, 1.10; 95% CI, 1.02–1.19; P = 0.011). In conclusion, shed ALCAM may be a novel prognostic biomarker in bladder cancer, although prospective validation studies are warranted. These findings demonstrate that markers reporting on cell motility can act as prognostic indicators.

Activated leukocyte cell adhesion molecule (ALCAM) has been identified as a novel potential molecular marker of human tumors. The present study aimed to assess ALCAM as a prognostic marker for gastric cancer (GC), and to explore the mRNA deregulation underlying the abnormal expression of ALCAM. The mRNA and protein expression of ALCAM in GC and adjacent non‑tumor tissues from 66 patients with GC were analyzed. The association between miR‑9 and ALCAM mRNA expression was determined by quantitative polymerase chain reaction. Serum soluble ALCAM (sALCAM) was analyzed by ELISA in 72 patients with GC, 82 patients with gastric precancerous lesions and 73 controls. ALCAM and sALCAM levels were associated with certain clinicopathological variables, including overall survival. Compared with the non‑tumor tissues, the expression of ALCAM mRNA in the GC tissues was significantly upregulated (P=0.013). The expression of miR‑9 was reduced and inversely correlated with ALCAM mRNA levels in GC tissues and cell lines. The ALCAM mRNA level was reduced following ectopic overexpression of miR‑9 in SGC‑7901 human gastric cancer cells. The rates of membranous and cytoplasmic expression of ALCAM in GC tissues were 59.1 and 48.48%, respectively, and the serum sALCAM levels were significantly elevated in patients with GC. Elevated ALCAM mRNA, membranous ALCAM expression in GC tissues and high sALCAM levels are associated with advanced tumor stage, lymphatic invasion and shorter overall survival duration. The results of the current study indicated that membranous ALCAM expression and high serum sALCAM levels are independent prognostic markers of poor survival for patients with GC, and that the overexpression of ALCAM may be due to the downregulation of miR‑9.

BackgroundChronic rhinosinusitis with polyps (CRSwNP) is characterized by high heterogeneity and postoperative recurrence rate. This study aimed to explore the clinical significance of activated leukocyte cell adhesion molecule (ALCAM) in endotyping CRSwNP and predicting its recurrence.MethodsWe recruited 120 CRSwNP patients including 70 non-eosinophilic CRSwNP (neCRSwNP) and 50 eosinophilic CRSwNP (eCRSwNP) patients, and 40 healthy controls (HCs). Serum and tissue samples were collected. Serum ALCAM levels were detected by enzyme-linked immunosorbent assay (ELISA), and tissue ALCAM expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR), Western blotting (WB) and immunohistochemistry (IHC). The predictive values of ALCAM expression for CRSwNP endotypes and postoperative recurrence were assessed.ResultsThe serum levels of ALCAM were significantly increased in CRSwNP patients in comparison with HCs and were correlated with the peripheral eosinophil count, tissue eosinophil counts, and percentage. Multivariate analysis and receiver operating characteristic (ROC) curve highlighted that serum ALCAM levels were associated with CRSwNP endotypes. Tissue ALCAM expression was significantly enhanced in CRSwNP patients, especially in eCRSwNP patients. At the end of the study, 110 patients completed the follow-up schedule, 78 patients were categorized into the non-recurrent group, and the other 32 patients were included in the recurrent group. The serum ALCAM levels were elevated in the recurrent group compared with the non-recurrent group, and ALCAM expression in the tissue was significantly elevated. The ROC curve exhibited a high predictive ability of serum ALCAM in predicting postoperative recurrence. Logistic regression and Kaplan–Meier curves demonstrated that serum ALCAM was an independent risk factor for postoperative recurrence.ConclusionThis is the first report suggesting that ALCAM expression was upregulated and associated with mucosal eosinophil infiltration and CRSwNP recurrence. Serum ALCAM could be a promising biomarker for distinguishing endotypes and predicting postoperative recurrence in CRwNP patients.

Activated leukocyte cell adhesion molecule (ALCAM) is a membranous cell adhesion protein that is often expressed in breast cancer. Data on the prognostic impact of ALCAM expression is highly controversial in this cancer. To evaluate the clinical impact of ALCAM expression in a sufficiently large patient cohort, we utilized a tissue microarray (TMA) containing more than 2,100 primary breast cancers with clinical follow-up data by immunohistochemistry. TMA spots containing normal breast epithelium showed moderate to strong membranous ALCAM staining. ALCAM staining was strong in 66.2%, moderate in 10.9%, weak in 11.1% and absent in 11.8% of 1,778 (80.9%) interpretable breast cancer tissue spots. Decreased ALCAM expression was significantly associated with advanced tumor size (p=0.0017), unfavorable tumor grade (p<0.0001), negative ER and PR status (p<0.0001 each) as well as high Ki67 labeling index (p<0.0001). Cancers with ACLAM expression loss had a significantly poorer overall (p<0.0001) and disease-specific survival (p=0.0088). This association also held true in the subset of nodal positive cancers (p<0.0001). In conclusion, these data demonstrate that ALCAM is generally expressed in normal and cancerous breast epithelium and that a marked reduction of ALCAM expression characterizes a subset of breast cancer patients with adverse tumor characteristics and unfavorable clinical outcome.

Long-term survival in cases of head and neck squamous cell carcinoma, particularly oral squamous cell carcinoma (OSCC), remains a rare achievement in the field of clinical oncology. In recent years, the theory of cancer stem cells (CSCs) has emerged and been used to offer explanations for tumour recurrence and metastasis. The present aim was to investigate the role of aldehyde dehydrogenase 1 (ALDH1) as a CSC-marker for OSCC and to determine the role of p16ink4a, which is also a surrogate marker of human papilloma virus (HPV), in the expression of ALDH1. The study cohort comprised of 186 surgically-treated cases of OSCC. The primaries were located in the oral cavity. The expression of the CSC marker (CSCM) ALDH1 was evaluated via immunohistochemistry (IHC) of a tissue microarray. HPV detection was performed by polymerase chain reaction and an HPV Array kit. Furthermore, the IHC expression of p16ink4a was also analysed. Risk regression models as the Kruskal Wallis test was used to assess the association of CSCM and p16ink4a expression with tumour size and lymph node metastasis, and cox proportional hazards were analysed. Additionally, coexpression of the markers ALDH1 and p16ink4a was analysed with regard to associations with tumour classification. Overall, high expression of ALDH1 in lymph nodes was significantly associated with Union for International Cancer Control (UICC) stage IV (P=0.044) and T4 stage cancer (P=0.03). p16ink4a positivity, in cases of HPV negativity, was associated with worse survival rate compared with that of the total cohort (P=0.048). Collectively the data indicate that ALDH1 expression may be suitable for detection of unfavourable prognosis in OSCC patients, based in part on its apparent role as a marker of metastasis. HPV status was not statistically predictive of patient outcome or CSCM expression; however, p16ink4a remains a potential marker in HNSCC Further in vitro studies with ALDH1 and p16ink4a should be performed to evaluate the expression of ALDH1 and HPV in cell culture and to clarify the role of ALDH1 as a marker for increased invasiveness of OSCC cells.

The role of activated leukocyte cell adhesion molecule (ALCAM) in cancer progression, invasion, metastasis and recurrence: A novel cancer stem cell marker and tumor-specific prognostic marker

Tropomyosin 4 (TPM4) regulates contractility and migration in many cell types and is important in the migration of colon epithelial cells. Several different alternatively-spliced TPM4 isoforms are predicted to exist in mammalian cells. However, TPM4.1 and TPM4.2, differing only in the inclusion of 1 (TPM4.2) or 2 (TPM4.2) unique exon(s) at the 5’ end, are the only two isoforms experimentally validated. We previously showed that TPM4 expression may be an early, premalignant molecular event that contributes to adoption of a tumorigenic state and, specifically, that overexpressed TPM4.2 blocks certain aspects of induced differentiation in colon epithelial cells and promotes a highly proliferative state. Many studies have shown that in vivo, tumors are heterogeneous and contain a very small subpopulation of cancer stem cells that not only drive growth of the tumor via robust proliferation but also are highly invasive and resistant to conventional anti-cancer treatments. Identifying molecular characteristics that promote cancer stem cell formation is therefore critical to developing effective strategies to treat and prevent colon cancer. Adherent LS123 cells (LS123 2D), representing minimally-invasive colon cancer cells at an early stage of tumor development, express both TPM4.1 and TPM4.2 transcripts, while nonadherent LS123 tumorspheres (LS123 3D), having a highly invasive, robustly proliferating phenotype similar to cancer stem cells, exclusively express TPM4.2. Therefore, we hypothesize that TPM4.2 overexpression, either by itself or in combination with TPM4.1 suppression, plays a role in transforming colon epithelial cells to a cancer stem cell phenotype. To test this hypothesis, we determined whether knockdown of TPM4.1 and/or overexpression of TPM4.2 will induce a gene expression pattern characteristic of cancer stem cells in the LS123 2D cell line. Specifically, we measured the expression of Sox2, Lgr5, epithelial cell adhesion molecule (EpCAM), activated leukocyte cell adhesion molecule (ALCAM)/CD166, aldehyde dehydrogenase I (ALDH1), and CD44 in LS123 2D cells treated with siRNA targeting TPM4.1 or TPM4.2 or in these cells in which TPM4.1 or TPM4.2 is overexpressed. Our results indicate that favoring TPM4.2 over TPM4.1 expression in LS123 cells increases expression of certain stem cell markers, particularly CD44 and to a lesser extent EPCAM and ALCAM, whereas favoring TPM4.1 over TPM4.2 expression shows the reciprocal effect on cancer stem cell marker expression. These results may identify distinct roles for expression of individual TPM4 isoforms in colon epithelial cells and may specifically elucidate whether TPM4.2 overexpression can contribute to a more aggressive tumorigenic phenotype. Ultimately, our studies may identify therapeutic strategies that target perturbations in cell contractility and migration at early stages of colon tumorigenesis. Citation Format: Nia Lowe, Giulio Ferrero, Barbara Pardini, Alessio Naccarati, Michael Papetti. TPM4 isoform expression modulates a cancer stem cell phenotype in human colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1451.

Rectal cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up.

Aldehyde dehydrogenase isozymes: markers of cancer stem cells in human melanoma.

To evaluate urinary activated leucocyte cell adhesion molecule (ALCAM) and CD6 as predictors of LN progression or disease resolution across a 1-year study. Serum and urine samples from biopsy proven LN subjects (n = 122) were prospectively collected over the course of a year at 3- or 6-month intervals (weeks 0, 12, 26 and 52) across multiple study sites and assessed for soluble ALCAM and CD6 levels. Urine creatinine from the same urine sample was used to normalize the levels of urinary ALCAM and urinary CD6. Measured levels of serum and urine ALCAM and CD6 were then analysed against disease metrics cross-sectionally and longitudinally. Cross-sectional analysis at baseline revealed that urinary ALCAM significantly correlated with urine protein creatinine ratio, renal SLEDAI, and the Physician Global Assessment (PGA), and negatively correlated with serum C3 and C4. Receiver operating characteristic curve analysis demonstrated that urinary ALCAM is a predictor of LN with an area under the curve (AUC) of 0.97, compared with urinary CD6 with an AUC of 0.71. Importantly, the change in urinary ALCAM over a 3-month period distinguished between non-responders and responders at week 52. Urinary ALCAM is reflective of changes in LN and may be predictive of response status.

Simple SummaryThis study reveals that circulating Hsp70 levels could serve as a tumor biomarker for patients with NSCLC in advanced UICC stages. All patients in advanced tumor stages had significantly elevated Hsp70 levels in the circulation compared to a healthy control cohort and an early-stage tumor cohort, and Hsp70 levels progressively increased with higher UICC tumor stages. These findings demonstrate the potential of Hsp70 measurements to predict an advanced tumor stage in NSCLC patients. We have also demonstrated that the prevalence of CD3−/CD94+ NK cells and CD8+ cytotoxic T cells were greater in advanced tumor stages, whereas that of CD4+ T helper cells was decreased. We hypothesize that raised levels of circulating Hsp70 in higher tumor stages might support NK cell proliferation, but that a lowered prevalence of CD4+ T helper cells could temper the capacity of cytolytic CD8+ T cells and NK cells to control tumor growth.Non-small cell lung cancer (NSCLC) is the second most frequently diagnosed tumor worldwide. Despite the clinical progress which has been achieved by multimodal therapies, including radiochemotherapy, and immune checkpoint inhibitor blockade, the overall survival of patients with advanced-stage NSCLC remains poor, with less than 16 months. It is well established that many aggressive tumor entities, including NSCLC, overexpress the major stress-inducible heat shock protein 70 (Hsp70) in the cytosol, present it on the plasma membrane in a tumor-specific manner, and release Hsp70 into circulation. Although high Hsp70 levels are associated with tumor aggressiveness and therapy resistance, membrane-bound Hsp70 can serve as a tumor-specific antigen for Hsp70-primed natural killer (NK) cells, expressing the C-type lectin receptor CD94, which is part of the activator receptor complex CD94/NKG2C. Therefore, we investigated circulating Hsp70 levels and changes in the composition of peripheral blood lymphocyte subsets as potential biomarkers for the advanced Union for International Cancer Control (UICC) stages in NSCLC. As expected, circulating Hsp70 levels were significantly higher in NSCLC patients compared to the healthy controls, as well as in patients with advanced UICC stages compared to those in UICC stage I. Smoking status did not influence the circulating Hsp70 levels significantly. Concomitantly, the proportions of CD4+ T helper cells were lower compared to the healthy controls and stage I tumor patients, whereas that of CD8+ cytotoxic T cells was progressively higher. The prevalence of CD3−/CD56+, CD3−/NKp30, CD3−/NKp46+, and CD3−/NKG2D+ NK cells was higher in stage IV/IIIB of the disease than in stage IIIA but were not statistically different from that in healthy individuals. However, the proportion of NK cells expressing CD94 and the activation/exhaustion marker CD69 significantly increased in higher tumor stages compared with stage I and the healthy controls. We speculate that although elevated circulating Hsp70 levels might promote the prevalence of CD94+ NK cells in patients with advanced-stage NSCLC, the cytolytic activity of these NK cells also failed to control tumor growth due to insufficient support by pro-inflammatory cytokines from CD4+ T helper cells. This hypothesis is supported by a comparative multiplex cytokine analysis of the blood in lung cancer patients with a low proportion of CD4+ T cells, a high proportion of NK cells, and high Hsp70 levels versus patients with a high proportion of CD4+ T cells exhibiting lower IL-2, IL-4, IL-6, IFN-γ, granzyme B levels.