Ley Expression Research Articles

Preclinical Evidence of the Efficacy of Lewis Y Car T Cells in Patient-Derived Models of Prostate Cancer

Chimeric antigen receptor T (CAR T) cell therapy is an adoptive immunotherapy that has led to new treatments for lymphoma, leukemia, and other blood cancers; however, its efficacy for prostate cancer remains unproven. Here we report pre-clinical evidence of the efficacy of CAR T cell therapy against the Lewis Y antigen (LeY) using patient-derived models of prostate cancer. To assess the expression of LeY on prostate tumours, we performed immunohistochemistry on a cohort of 41 patient-derived xenografts (PDXs). Cytoplasmic and membrane expression were separately assessed and quantified, for each patient. Overall, 61% (25/41) of PDXs were positive for membrane LeY expression, of which 18 PDXs had greater than 50% membrane-positive cells, and considered most suitable to detection and stable binding by anti-LeY CAR T’s. To determine the in vitro sensitivity to CAR T cytotoxicity, we selected 4 PDXs with high and 2 PDXs with low LeY expression using 3 androgen receptor (AR)-positive adenocarcinomas and 3 AR-negative tumors expressing neuroendocrine markers. Next we established organoids for in vitro co-culture assays where organoids were co-incubated with an equal number of anti-LeY+ CAR T cells or Empty vector control CAR T cells (Ev CAR T). Using time-lapse microscopy we reported destruction of organoids by LeY+ CAR T cells as indicated by their morphological collapse and uptake of propidium iodide from the culture medium; control Ev CAR T cells produced no cytotoxicity. Over the 48h assay, the level of target cell death of the LeY+ organoids was correlated to the intensity LeY surface expression. Target cell death mediated by the CAR T cells required perforin and granzyme B, as potent and highly specific small molecule inhibitors of perforin (SN34960) and granzyme B (C20) applied alone or in combination greatly decreased PI uptake, indicating organoid survival. Neither inhibitor adversely affected CAR T cell viability as measured by PI and Annexin V staining. This demonstrated canonical activation of granule exocytosis pathway by the CAR T cells, leading to organoid cell death. To assess CAR T cell efficacy in vivo, we selected one PDX with high LeY expression. Monotherapy with CAR T cells failed to decrease tumour volume compared to vehicle control. However, CAR T cells given after a single dose of the chemotherapeutic agent carboplatin greatly and durably reduced tumour burden, with residual tumour mass being less than 1% of their original size (0.56 ± 0.23% of tumour volume at the start of treatment). Overall, these data provide preclinical evidence that: i) high membrane expression of LeY correlates with in vitro and in vivo CAR T cell-induced tumour cell death via the canonical perforin/granzyme B mechanism; and, ii) membrane LeY can be used as a biomarker for patient selection.

Blood group-related antigen Ley on human platelets and its involvement in platelet aggregation via a possible interaction with CD61.

The Ley antigen is a carbohydrate chain belonging to the ABH-Lewis blood group family. Ley has been reported to be present on red blood cells (RBCs) and granulocytes, but its distribution and function in platelets remain unknown. There are a variety of glycoproteins on platelets, which may carry the Ley antigen. This study aims to investigate the expression pattern and the function of Ley in human platelets. Flow cytometry, Western blot, and immunofluorescence assays were performed to determine Ley expression on human platelets. ADP (1.25-10 μM) and thrombin (0.05-1 IU/mL) were used to activate platelets in the presence or absence of prostaglandin E1 (PGE1) and the Ley expression was evaluated again by flow cytometry. Blockade was performed with an anti-Ley monoclonal antibody to verify the role of this epitope in platelet function. Finally, coimmunoprecipitation was performed to identify glycoproteins associated with Ley . Ley was expressed on human platelets independent of ABO blood type. Ley expression was decreased in a dose-dependent manner after activation with either ADP or thrombin, and this effect could be partially reversed by PGE1. Anti- Ley mAb treatment increased alpha-granule release and neutralized the inhibitory effect of the anti-CD61 antibody on platelet aggregation. In addition, Ley was proven to interact and colocalize with CD61. These results demonstrate nondifferential expression of Ley in platelets of different ABO blood types and suggest the involvement of Ley in platelet function, possibly via interaction with CD61.

Role of difucosylated Lewis Y antigen in outcome of locally advanced cervical squamous cell carcinoma treated with cisplatin regimen.

Several mechanisms are involved in the development of resistance to therapy in locally advanced cervical squamous cell carcinoma (LACSCC). Studies have shown that CD44 and Lewis Y antigen (LeY) form a complex that is associated with chemoresistance, tumor invasion and metastasis. We assessed the role of CD44 and LeY in the outcome of LACSCC patients treated with different chemotherapy regimens. 126 LACSCC patients at FIGO stages IIB-IVA were selected from the GOCS database: 74 patients included in 3 different prospective phase II trials in the neoadjuvant setting (vinorelbine, docetaxel, ifosfamide-vinorelbine-cisplatin) and 52 patients treated with standard radiochemotherapy based on cisplatin (RCBC). Clinical data at baseline, disease-free survival (DFS) and overall survival (OS) were recorded. Univariate and multivariate Cox models were employed. Median age was 45.6 years (range: 24.9-80.5). Sixty-three and 47 tumors were CD44+ and LeY+, respectively. Tumors with expansive growth showed higher grade (p = 0.0024), mitotic index (p = 0.0505), tumor necrosis (p = 0.0191), LeY+ (p = 0.0034) and CD44+/LeY+ coexpression (p = 0.0334). CD44+ cells were present in 91.3% of patients with local recurrence (p = 0.0317). Advanced stage was associated with LeY+ tumors. Patients treated with RCBC had worse DFS and OS when their tumors expressed LeY (p = 0.0083 and p = 0.0137, respectively). Pre-treatment hemoglobin level, FIGO stage and tumor response remained the most significant prognostic factors in Cox regression. In our cohort of LACSCC patients, the coexpression of CD44 and LeY was not associated with worse outcome. However, in the subgroup of patients receiving RCBC, LeY expression was correlated with shorter DFS and OS.

Lewisy promotes migration of oral cancer cells by glycosylation of epidermal growth factor receptor.

Aberrant glycosylation changes normal cellular functions and represents a specific hallmark of cancer. Lewisy (Ley) carbohydrate upregulation has been reported in a variety of cancers, including oral squamous cell carcinoma (OSCC). A high level of Ley expression is related to poor prognosis of patients with oral cancer. However, it is unclear how Ley mediates oral cancer progression. In this study, the role of Ley in OSCC was explored. Our data showed that Ley was upregulated in HSC-3 and OC-2 OSCC cell lines. Particularly, glycosylation of epidermal growth factor receptor (EGFR) with Ley was found in OC-2 cells, and this modification was absent upon inhibition of Ley synthesis. The absence of Ley glycosylation of EGFR weakened phosphorylation of AKT and ERK in response to epidermal growth factor (EGF). Additionally, EGF-triggered cell migration was reduced, but cell proliferation was not affected. Ley modification stabilized EGFR upon ligand activation. Conversely, absence of Ley glycosylation accelerated EGFR degradation. In summary, these results indicate that increased expression of Ley in OSCC cells is able to promote cell migration by modifying EGFR which in turn stabilizes EGFR expression and downstream signaling. Targeting Ley on EGFR could have a potential therapeutic effect on oral cancer.

Relevant Role of Di-Fucosylated Ley Antigen in the Outcome of Locally Advanced Cervical Squamous Cell Carcinoma Patients Treated with Cisplatin Regimen

Background: Several mechanisms are involved in the development of resistance to therapy in LACSCC. Studies have shown that CD44 and Lewis Y antigen (LeY) form a complex that is associated with chemoresistance, tumor invasion and metastasis. We assessed the role of CD44 and LeY in the outcome of LACSCC patients (pts) treated with different chemotherapy regimens. Methods: A total of 126 LACSCC pts FIGO stages IIB-IVA were selected from GOCS databases: 74 pts included in three different prospective phase II trials in the neoadjuvant setting (vinorelbine, docetaxel, ifosfamide-vinorelbinecisplatin) and 52 pts treated with standard radio-chemotherapy based in cisplatin (RCBC). Clinical data at baseline, disease free survival (DFS) and overall survival (OS) were recorded. Univariate and multivariate Cox models were employed. Results: Median age was 45.6 years (range: 24.9 - 80.5). Sixty-three and 47 tumors were CD44+ and LeY+, respectively. Expansive growth tumors showed a higher grade (p=.0024), mitotic index (p=.0505), tumoral necrosis (p=.0191), LeY+ (p=.0034) and CD44+/LeY+ co-expression (p=.0334). CD44+ cells were present in 91.3% of those with local recurrence (p=.0317). Advanced stage was associated with LeY+ tumors (p=.0057). Pts treated with RCBC had worse DFS and OS when their tumors expressed LeY antigen (p=.0083 and p=.0137, respectively). Pre-treatment hemoglobin level, FIGO stage and tumor response remained the most significant prognostic factors in Cox regression. Conclusions: In our cohort of LACSCC pts, the co-expression of CD44+/LeY+ was not associated with worse outcome. However, in the subgroup of pts receiving RCBC, LeY expression was correlated with shorter DFS and OS.

Safety and Therapeutic Efficacy of the Lewis Y Carbohydrate Specific Humanized Antibody MB311 in Patients with Malignant Effusion

Purpose: Investigation of safety and tolerability as well as therapeutic efficacy of the LeY specific humanized mAb MB311 in cancer pts with malignant effusions in a Phase II clinical trial. Experimental Design: An openlabel, single treatment arm, uncontrolled study with MB311 (100 mg per dose, intravenous infusion on day 1 and 7) in pts with malignant effusion (ascites or pleural effusion) was conducted with the primary objective to examine safety and tolerability as well as pharmacokinetics. Secondary objectives were assessment of pharmacodynamics, volumetric measurement of the malignant effusion and obtaining data for several immunological parameters. Results: Five pts (2 pts with gastric cancer and malignant ascites, 3 pts with breast cancer and malignant pleural effusion/ascites) have completed the study. MB311 was well tolerated with only two pts showing the easily manageable side effects nausea, vomiting (up to grade 2) and one episode of skin rash (grade 2) after the first application. Data of 4 pts were available for evaluating immunologic results and efficacy. In all pts significant levels of MB311 could be detected in the systemic blood circulation and the effusion leading to increased infiltration of CD45 positive immune cells (4/5 pts) and resulting in a reduction of tumor cell counts as detected by immunocytochemistry of effusion samples in 3/5 pts). Most interestingly, the pt with the highest LeY positive tumor showed a significant reduction of effusion volume after treatment—this decrease was also evident for Her2/neu positive tumor cells which were dramatically reduced after MB311 treatment in this breast cancer pt. Conclusion: MB311 was well tolerated in patients with malignant effusions, permeated into malignant effusion and attracted immune cells leading to decreased tumor cell counts in the effusion. In the case of strong LeY expression of malignant cells in the effusion a pronounced decrease in LeY, EpCAM and Her2/neu positive tumor cells and a significant reduction of the effusion volume could be demonstrated.

619 - Differential Antigenic Expression in Colorectal Cancer, Mucosa Adjacent to Colorectal Cancer, and Normal Colorectal Mucosa

ABSTRACT Introduction Despite of an excellent oncologic surgery with broad normal tissue margins, the mucosa next to the bed anastomosed is a frequent place of tumoral recurrence on colorectal cancer. Objective: This study was performed to evaluate the expression of mucins and carbohydrates associated antigens in normal mucosa colorectal specimens (NMC), mucosa adjacent to colorectal cancer (MACCR) and malignant colorectal tumour samples (CCR). Methods Ninety colorectal cancer tumour samples and their MACCR, and also 68 NMC were included. Monoclonal antibodies (MAbs) against the following antigens were employed: anti-MUC1 (HMFG1), MUC2 (H-300) and MUC5AC (45M1), anti-Tn (HB-Tn1), Lex (KM380), sLex (KM93), Ley (C70) and sLea (1116-N5-19-9). An immunohistochemical approach following standard procedures was performed. Statistical analysis: an univariate statistical analysis with Chi2 was applied. Results Malignant samples expressed MUC1 in 94% of cases, MUC2 in 52.4%, MUC5AC in 14.3%, Tn in 41%, Lex in 74.4%, sLex in 66.7%, Ley in 91.8% and sLea in 90.7%. Taking into account MUC2 and Ley expression, a positive correlation was found between MACCR and CCR: MUC2, P = .0006 and Ley, P = .0008. In malignant samples, MUC1 expression was increased compared to NMC (P = .04), also this mucin was 22.2% higher in MACCR than NMC. The expression of sLex and Ley was higher in CCR than NMC (179% and 33.6%, respectively). Expression of most antigens showed an increased tendency from NMC to MACCR and CCR: MUC1 (27.9%, 34.1% and 94%, respectively), Lex (60.9%, 30.5% and 74.4%, respectively), sLex (23.9%, 16.9% and 66.7%, respectively), Ley (68.7%, 78.3% and 91.8%, respectively) and sLea (62.1%, 45.6% and 90.7%, respectively). On the other hand, MUC5AC expression decreased in the mentioned sequence (51.5%, 20.7% and 14.3, respectively). Conclusion Our results may support the hypothesis that the MACCR could be an intermediate point or “transitional zone” between NMC and CCR. Further studies are needed in order to correlate these findings with clinical outcomes. Disclosure All authors have declared no conflicts of interest.

LeY oligosaccharide upregulates DAG/PKC signaling pathway in the human endometrial cells

LeY oligosaccharide is stage specifically expressed by the embryo and uterine endometrium, and it plays important roles in embryo implantation. In addition to participating in the recognition and adhesion on fetal-maternal interface, LeY potentially regulates the expression of some implantation-related factors. However, it remains elusive whether it can mediate the involved signaling pathway. In this study, agarose-LeY beads were used to mimic the embryos, and the effects of LeY oligosaccharide on DAG/PKC signaling pathway was studied in human endometrial epithelial cells. Results showed that LeY could significantly trigger the activation of cPKCalpha and cPKCbeta2, and their translocation from the cytosol to the plasma membrane. The cellular DAG content was also upregulated, and the activation of PLCgamma1 was promoted. On the contrary, DAG/PKC signaling pathway was significantly inhibited when anti-LeY antibody was used after confirmation of LeY expression in human endometrial epithelial cells by immunohistochemistry and flow cytometry. These results suggest that LeY oligosaccharide acts as a signal molecule to modulate DAG/PKC signaling pathway.

Clinical Evaluation of BR96 sFv-PE40 Immunotoxin Therapy in Canine Models of Spontaneously Occurring Invasive Carcinoma

The immunotoxin BR96 sFv-PE40 is an effective antitumor agent against human breast and lung carcinoma xenografts in rodents. This study was designed to (a) determine the frequency with which canine carcinoma cells express Lewis(y) (Le(y)) antigen, thereby identifying canine carcinoma types suitable for the clinical evaluation of BR96 sFv-PE40, and (b) determine the safety and efficacy of BR96 sFv-PE40 in a canine model of spontaneously occurring cancers for investigation of targeted therapy. Carcinoma tissue samples were obtained from client-owned dogs presented for medical care. The tissues were assessed for Le(y) antigen expression using immunohistochemical methods. Dogs with tumors expressing Le(y) antigen were offered enrollment in a clinical trial to receive twice-weekly infusions of 4 to 12 mg/m(2) BR96 sFv-PE40. Clinical toxicity and response data were assessed at each treatment. Twenty-two of 61 carcinomas evaluated were positive for Le(y) expression, including mammary, prostate, lung, and rectal carcinomas, and 12 dogs were enrolled in the clinical trial. The primary side effect was transient emesis. Partial responses or disease stabilization were noted in dogs with inflammatory mammary, bronchogenic, rectal, and tonsillar carcinoma. At least nine of the dogs developed antibodies to the immunotoxin after two to five infusions. Although development of anti-BR96 sFv-PE40 antibodies limited the long-term effectiveness of this immunotoxin in dogs, rapid clinical responses in several aggressive canine carcinomas suggest the immunotoxin has utility for treatment of certain naturally occurring tumors and that its clinical evaluation for treatment of similar human carcinomas is warranted.

Phase I trial of hu3S193 in patients with advanced epithelial cancers which express the Lewis-y antigen

2567 Background: The Lewis-y (Ley) antigen is a blood group related antigen that is expressed in a high proportion of epithelial cancers. We have generated a humanised antibody (hu3S193) against Ley, which has potent immune effector function, and efficacy in murine tumour models (Scott et al, Cancer Res 60: 3254–3261, 2000). Methods: An open label dose escalation Phase I trial of hu3S193 in patients (pts) with advanced Ley positive epithelial cancers has been conducted. Inclusion criteria included +ve Ley expression in tumour assessed prior to study entry. Pts received 4 infusions of hu3S193 at weekly intervals, with four dose levels (5, 10, 20 and 40 mg/m2). The first infusion of hu3S193 was trace labelled with 111In to evaluate targeting. Biodistribution, pharmacokinetics, and immune response were evaluated in all patients. Results: A total of 12 pts (6M:6F; age range 42–76 yrs; 5 breast, 7 colorectal cancer) have been accrued into the study, completing the 5 (3 pts), 10 (6 pts) and 20 (3 pts) mg/m2 dose levels. No infusion related AEs were observed. There was one episode of dose limiting toxicity, at the 10mg/m2 dose level, with Grade 3 liver enzyme rise observed in a pt with extensive liver metastases, which resolved following cessation of hu3S193 infusions. The biodistribution of 111In-hu3S193 showed no normal tissue uptake, and 111In-hu3S193 uptake was observed in cutaneous, lymph node and hepatic metastases. Pharmacokinetic analysis showed no dose dependence of 111In-hu3S193 serum clearance, with a (mean ± SD) T½α 6.2 ± 5.5 hrs, T½β 183.1 ± 66.4 hrs, CL 0.025 ± 0.014 L/hr, V1 3.43 ± 0.63 L. No immune responses (HAHA) were observed in any pt. Two pts had stable disease and 10 PD at restaging. Conclusions: Hu3S193 has promise for immunotherapy of Ley +ve tumours, and further Phase I/II clinical trials are planned. No significant financial relationships to disclose.

Lewis Antigen Expression in Gastric Mucosa of Children: Relationship With Helicobacter pylori Infection

ABSTRACTObjectiveLewis epithelial antigen expression has a role in Helicobacter pylori adherence, presumably mainly in cag A‐positive strains. The authors investigated whether Lewis antigen expression in children's gastric mucosa was associated with H. pylori infection, cag A status, patient age, or presence of duodenal ulcer (DU).MethodsThe expression of Lewis A (Lea), B (Leb), X (Lex), and Y (Ley) was detected by immunohistochemistry in the antral and oxyntic mucosae of 70 children. Children were divided in four age groups (<4 years; 4–8 years; 9–12 years; and 13–18 years).ResultsForty‐seven of the 70 children had H. pylori and 17 had DU. The cag A status was determined by polymerase chain reaction in 34 patients. Lea and Leb were expressed in 64% and 44% of the patients, respectively; Lex and Ley were expressed in the glands in all of the patients and in the superficial epithelium. Leb expression was more common among patients without H. pylori (15/23, 65%) than in those with H. pylori (16/47, 34%) (P = 0.03). In noninfected patients, Leb and superficial Ley expression were associated with increased age. Leb expression was more common in patients with chronic gastritis than in those with DU. Lex superficial expression was significantly associated with DU in patients with H. pylori.ConclusionIn children, the expression of Leb and Ley in the superficial gastric epithelium depends on age. Other receptors, such as Lex, may have a role in H. pylori colonization, especially in patients with DU. Studies assessing the expression of Lewis antigens in children may contribute to an understanding of the mechanisms of acquisition of H. pylori infection.

Ley glycolipid-recognizing monoclonal antibody inhibits procoagulant activity and metastasis of human adenocarcinoma.

Tumor procoagulant is associated with cancer at advanced stages of malignancy such as infiltration and metastasis. In the present study, we investigated the role of Ley glycolipid in the mechanism of cancer metastasis. Ley glycolipid acts as an important cofactor in the expression of the blood-coagulating activity of cancer cell-derived coagulating activity 1 (CCA-1), which is one of the known tumor procoagulants. Monoclonal antibody (MoAb) FS01, which serves as the Ley-recognizing epitope, inhibits the procoagulant activity of CCA-1 was found to dose-dependently inhibit the procoagulant activity of normal plasma induced by the human lung adenocarcinoma cell line, HAL8, which shows a high level of Ley expression. It did not, however, inhibit the procoagulant activity of the human colon cancer cell line, RPMI4788, which does not express Ley. Administration of FS01 MoAb inhibited lung metastasis of HAL8 cells, but not that of RPMI4788. The absence of antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity of FS01 MoAb against the HAL8 cell line suggests that the inhibition of HAL8 metastasis by FS01 MoAb derives from the inhibition of blood-coagulating activity of the latter. These findings indicate that Ley glycolipid plays an important role in the mechanism of cancer metastasis via the procoagulant activity of CCA-1.

Stage-specific expression of alpha1,2-fucosyltransferase and alpha1, 3-fucosyltransferase (FT) during mouse embryogenesis.

Lex [Galbeta1-4(Fucalpha1-3)GlcNAc] and Ley [Fucalpha1-2Galbeta1-4(Fucalpha1-3)GlcNAc] are both stage-specific embryonic antigens. Lex is first detected on the blastomeres of the 8-cell stage embryo, which correlates with the onset of blastomere compaction. Ley is highly expressed on the surface of the blastocyst, which has been shown to be involved in blastocyst attachment in the mouse. In the present study, mouse alpha1,2-FT (also known as FUT1) and alpha1,3-FT (also known as Fuc-TIV), which were responsible for Lex and Ley formation, were examined in preimplantation stage embryos by reverse transcription-PCR and in situ hybridization. alpha1,3-FT mRNA was detected in all embryos of preimplantation stage, while alpha1,2-FT mRNA emerged in the later stage embryos from 8-cell to 16-cell to the blastocyst. These results indicated the expression of Ley was regulated by alpha1,2-FT. In situ hybridization showed that these two enzyme mRNAs were detected only in morula and blastocyst stage embryos. The alpha1,2-FT and alpha1, 3-FT mRNAs were located in both the inner cell mass and the trophoblast cells. 2-Cell and 4-cell embryos were isolated from the oviduct and cultured in vitro to the 8-cell, morula and blastocyst stage. The expression of alpha1,2-FT and alpha1,3-FT were observed in these embryos developed in vitro; immunohistochemical analysis also showed that Ley expression was positive. These results suggested the stage-specific expression of Ley on the embryos was synthesized by endogenous alpha1,2-FT and alpha1,3-FT rather than transfer from other sources. In addition, the expression of alpha1, 2-FT was differentially regulated and the uterine factor was not prerequisite of the expression of Ley.

Correlation of prognosis of breast cancer patients and expression of Ley which acts as a cofactor of tumor procoagulant.

Association between Ley expression and prognosis of breast cancer was investigated using monoclonal antibody (MoAb) FS01, which recognizes Ley as an epitope, inhibits the procoagulant activity of cancer cell-derived coagulating activity 1 (CCA-1). Expression intensity and procoagulant activity of CCA-1, tissue factor and HLA-DR on breast cancer cell lines were also examined. Immunohistochemical staining of Ley was performed on primary lesions of 223 breast cancer patients who received absolute curative operation. Flow cytometric analysis and clot timer was used to detect expression and activity of each procoagulant on cancer cell lines. The Ley expression was 73.5%, and no significant relation was observed between clinicopathological factors and intensity of Ley expression. The group showing strong Ley positivity had a significantly poorer prognosis than the Ley-negative group in 5-year disease-free survival (p=0.019). Multivariate analysis using the Cox's proportional hazards' regression model showed that Ley expression is an independent prognostic factor (p=0.018), following tumor size and lymph node metastasis. Ley expression on cancer cell surface is higher than tissue factor and HLA-DR. FS01 and anti-tissue factor MoAb inhibited the coagulating activity of tissue factor-expressing lines, but no cells were inhibited by staphylococcal enterotoxin A, which is known to inhibit the coagulating activity of HLA-DR. CCA-1 and tissue factor plays a important role in the blood coagulating activity of breast cancer cell lines. Breast cancer patients are thought to have a poor prognosis because Ley expression on the surface of the cancer cell induces blood coagulation via CCA-1.

Expression of proliferating cell nuclear antigen (PCNA) and apoptosis related antigen (LeY) in epithelial skin tumors.

We semiquantitatively analyzed expression of PCNA and LeY in seborrheic keratosis (SK), actinic keratosis (AK), Bowen's disease (BD), and squamous cell carcinoma (SCC), using immunocytochemically stained tissue sections. PCNA expression increased in a stepwise fashion from low levels in normal skin to higher expressions within SK, AK, BD, and SCC. The levels of LeY protein also increased in this order. The PCNA expression pattern shifted from expression limited to the basal and suprabasal cell layers (in normal skin and SK) to expression extending to the upper squamous and granular layers (in AK, BD, and SCC). On the other hand, the pattern of LeY expression shifted from the granular (in normal skin) to the upper squamous (in SK and AK) and suprabasal layers (in BD and SCC). These findings suggest that PCNA expression is related to the degree of cell proliferation and that LeY expression is related to the degree of differentiation or keratinization of tumor cells. In addition, PCNA and LeY show a reciprocal relationship in their expression.

Therapy for oral squamous cell carcinoma by tegafur and streptococcal agent OK-432 in combination with radiotherapy: association of the therapeutic effect with differentiation and apoptosis in the cancer cells.

Twenty patients with oral squamous cell carcinoma having mainly stage II or III lesions without distant metastasis, were treated with tegafur and streptococcal agent, OK-432, in combination with radiotherapy. As a consequence, 16 cases among the treated 20 cases showed complete remission by this therapy alone. Especially, we have found that the squamous cell carcinoma arising in non-keratinizing oral epithelium rather than in keratinizing oral epithelium has better response to this therapy. Among the 16 cases with complete remission (CR) by the current therapy, 10 cases were histopathologically diagnosed as well-differentiated squamous cell carcinoma and six cases as moderately differentiated squamous cell carcinoma. When we examined immunohistochemically the expression of various antigens such as proliferating cell nuclear antigen (PCNA), p53 and LeY or the presence of DNA fragmentation by nick-end labelling in the biopsy materials taken at the first visit to our clinic from 20 patients treated with the current therapy, the CR group showed a significantly increased LeY expres-sion level ( p< 0.05) and DNA fragmentation rate (p< 0.05) as compared with the partial response (PR, n= 3) + no change (NC, n= 1) group. On the other hand, the CR group with respect to PCNA expression level was significantly decreased as compared with the PR + NC group ( p< 0.05). From these findings, it can be considered that the therapy for oral squamous cell carcinoma by UFT and OK-432 in combination with radiotherapy is very effective, which may be associated with differentiation or apoptosis in oral squamous carcinoma cells. In addition, we present the clinical findings and results of immunohistochemical staining for the biopsy materials obtained from four CR cases treated with the current therapeutic method.

The treatment with differentiation- and apoptosis-inducing agent, vesnarinone, of a patient with oral squamous cell carcinoma.

A patient with histopathological recurrent oral cancer with well-differentiated squamous cell carcinoma, was treated with differentiation- and apoptosis-inducing agent, vesnarinone, per os at a dose of 180 mg/day for 56 days and then at a dose of 60 mg/day for 93 days. The vesnarinone administration caused complete remission of the tumour. It has been found by immunohistochemical staining and PCR-SSCP analysis that the recurrent tumour has wild type p53 gene and relative high level of LeY expression as well as DNA fragmentation in the cancer cells, as assessed by nick-end labelling. These findings suggest that the cure of oral squamous cell carcinoma observed in this case might be associated with induction of differentiation and apoptosis of cancer cells by vesnarinone.

Apoptosis of human embryonal carcinoma cells with in vitro differentiation.

An in vitro model of apoptosis and differentiation in human embryonal carcinoma (EC) cells was developed to study the mode of cell death and mechanisms of cell death in early development. Death of these cells was induced by treatment with retinoic acid (RA) under the same conditions as those for induction of differentiation. The manner of this cell death was apoptosis, not necrosis, with the morphological criterion for apoptosis. serum deprivation likewise caused apoptosis in both undifferentiated and differentiated EC cells. In differentiated EC cells, DNA fragmentation was observed in a smear pattern lacking the ladder pattern typically associated with apoptosis. However, in differentiated EC cells, DNA fragmentation occurred in various sizes. The expression of a carbohydrate antigen, LeY, a reported marker of apoptotic cancer cells, was increased by the treatment with RA. However, two-color analysis by flow cytometry with nick end labelling method revealed that LeY expression was closely correlated with cellular differentiation but not apoptosis after RA treatment in the human EC cell system. Collection of LeY positive cells by the magnetic bead method demonstrated that this expression was not due to apoptosis but rather to differentiation. On the other hand, LeY expression associated with apoptosis was induced by serum deprivation in both undifferentiated and differentiated EC cells. These results show that a subpopulation of undifferentiated EC cells takes the apoptotic pathway by induction of differentiation. The results also suggest that the population of cells taking an apoptotic pathway differs from a population of cells taking a differentiation pathway. This in vitro system for apoptosis in human EC cells will be useful for studies concerning apoptosis or programmed cell death in human developmental biology.

Proliferative and apoptotic status in endometrial adenocarcinoma.

The contribution of cell proliferation and apoptosis to growth patterns in endometrial adenocarcinoma were investigated. Immunohistochemical staining was carried out by an antibody for Ki-67 proliferative antigen, Ley apoptotic antigen, and oncogene products bcl-2 and p53. Forty cases of endometrial adenocarcinoma were classified as exophytic, endophytic, and mixed exo- and endophytic in light of their vertical growth pattern, and, in each case, the carcinomatous area was divided into three layers by its vertical axis. In all but one case, no zonal distribution of the antigen expression was observed. In one case, an exophytic tumor, Ki-67 expression was intense in the surface layer and Ley expression in the deep layer was also intense, suggesting a correlation between macroscopic growth pattern and cellular growth and apoptotic potential. However, in general, zonal distribution of cell proliferation and apoptosis could not explain the growth morphology of endometrial adenocarcinoma of the uterus and it was suggested that factor(s) other than cell proliferation and apoptosis determine macroscopic growth patterns.

In vitro and in vivo characterization of BR96 sFv-PE40. A single-chain immunotoxin fusion protein that cures human breast carcinoma xenografts in athymic mice and rats.

Abstract BR96 sFv-PE40 is a single-chain immunotoxin fusion protein targeted to the Ley Ag, which is expressed in many different human carcinomas as well as in normal gastrointestinal epithelium of humans and certain animals, including athymic rats but not mice. In vitro binding analysis determined that BR96 sFv-PE40 was similar in affinity to BR96 Fab. BR96 sFv-PE40 internalizes rapidly, similar to BR96 IgG. H3396 cells, derived from metastatic human breast carcinoma, have been established as tumor xenografts in estradiol-supplemented athymic mice and rats. H3396 tumor xenografts established in athymic mice (up to 350 mm3) and rats (up to 100 mm3) completely regressed after i.v. administration of BR96 sFv-PE40, given as 0.625 mg/kg (1.975 mg/m2) every 4th day for a total of five doses (mice) or 0.25 mg/kg (1.475 mg/m2) every 4th day for a total of four doses (rats). The tumors remained regressed for the duration of the study (&amp;gt; 85 days post-implant), which represents &amp;gt; 10 doubling times, indicating that the animals were cured. There was no toxicity in rats receiving a curative dose of 0.25 mg/kg, although liver and lung toxicity could be detected at a 16 times higher dose, 4 mg/kg or 23.6 mg/m2. We conclude, therefore, that BR96 sFv-PE40 can cure tumor xenografts at well tolerated doses and also in the presence of Ley expression in normal tissues.