Philippe Bertrand

Evidence of involvement of CD44 in endothelial cell proliferation, migration and angiogenesis in vitro

Angiogenesis is essential for tumor growth and metastasis. In the process of angiogenesis, the interaction between adhesive proteins of endothelial cells and extracellular matrix components plays an important role by mediating cell attachment, which is indispensable for their motility, and by transmitting the regulatory signals for cell locomotion and proliferation. In this study, we examined the hypothesis that CD44 expressed on the endothelial cell surface is involved in the angiogenesis process. The experiments using calf pulmonary artery endothelial cells (CPAE) and a human microvascular endothelial cell line (HMEC‐I) show that a monoclonal antibody against CD44 (clone J 173) inhibits endothelial cell proliferation by about 30% and migration by 25–50%, and abolishes the stimulating effect of hyaluronan polysaccharides on endothelial cell migration and proliferation. This antibody also suppresses the capillary formation of CPAE in an in vitro model of angiogenesis using fibrin matrix. These results provide evidence of the involvement of endothelial‐cell‐associated CD44 in angiogenesis.

High-dose chemotherapy with autologous stem cell transplantation as first-line therapy for primary CNS lymphoma in patients younger than 60 years: a multicenter phase II study of the GOELAMS group

The optimum treatment of primary CNS lymphoma (PCNSL) is not yet determined. The objective of this study was to assess the safety and efficacy of initial methotrexate-based chemotherapy followed by high-dose chemotherapy (HDT) with autologous stem cell transplantation (ASCT) in patients with newly diagnosed PCNSL. Twenty-five patients received two courses of initial chemotherapy combining methotrexate, etoposide, carmustine and methylprednisolone, and one course of ifosfamide–cytarabine followed by peripheral stem cell collection. Seventeen responsive patients then received HDT using carmustine, etoposide, cytarabine and melphalan with autologous stem cell rescue. After ASCT for responding patients or after salvage therapy for non-responders, whole brain radiation therapy at a dose of 30 Gy was delivered. The objective response rate to the induction chemotherapy was 84%. Four of the 21 responding patients did not have ASCT because of toxicity or refusal. With a median follow-up time of 34 months, the projected event free survival rate is 46% at 4 years. Projected overall survival is 64% at 4 years. Sixteen patients are actually in continuous complete response. No evidence of late treatment-related toxicity was observed. This treatment approach appears feasible in newly diagnosed PCNSL with encouraging results.

High-dose therapy followed by autologous purged stem cell transplantation and doxorubicin-based chemotherapy in patients with advanced follicular lymphoma: a randomized multicenter study by the GOELAMS with final results after a median follow-up of 9 years

Autologous stem cell transplantation (ASCT) as first-line therapy for follicular lymphoma (FL) remains controversial. The multicenter study randomized 172 patients with untreated FL for either immunochemotherapy or high-dose therapy (HDT) followed by purged ASCT. Conditioning was performed with total body irradiation (TBI) and cyclophosphamide. The 9-year overall survival (OS) was similar in the HDT and conventional chemotherapy groups (76% and 80%, respectively). The 9-year progression-free survival (PFS) was higher in the ASCT than the chemotherapy group (64% vs 39%; P = .004). A PFS plateau was observed in the HDT group after 7 years. On multivariate analysis, OS and PFS were independently affected by the per-formance status score, the number of nodal areas involved, and the treatment group. Secondary malignancies were more frequent in the HDT than in the chemotherapy group (6 secondary myelodysplastic syndrome/acute myeloid leukemia and 6 second solid tumor cancers vs 1 acute myeloid leukemia, P = .01). The occurrence of a PFS plateau suggests that a subgroup of patients might have their FL cured by ASCT. However, the increased rate of secondary malignancies may discourage the use of purged ASCT in combination with TBI as first-line treatment for FL. This trial has been registered with ClinicalTrials.gov under identifier NCT00696735.

Increased hyaluronidase levels in breast tumor metastases

Hyaluronidase, a matrix‐degrading enzyme, was assayed in extracts from breast primary tumors and regional metastases using a pool of human sera as a standard. Optimal activities of tumor extracts and serum were found for concentrations of 0.15–0.20 M NaCl in pH 3.8–4.0 buffer. In evaluating contamination by serum due to vascular proliferation, we expressed our results as the ratio of the entire activity (mU/l extract) on serum albumin content of tumors (g/l). Median (interquartile range) activities were 9.02 (6.04–14.34) for primary tumors and 37.36 (24.06–99.63) mU/g albumin for metastases. The difference was significant. Zymographic analysis showed that 3 bands of activity were detected which corresponded to 68, 53 and 49 kDa for tumoral hyaluronidase. The same pattern was observed for cellular extracts of breast cancer cell line CAL51, demonstrating that hyaluronidase detected in tumor extracts had mainly a cellular origin. Our results suggest that hyaluronidase may be involved in the metastatic process. Int. J. Cancer 73:327–331, 1997.

Mapping of MYC breakpoints in 8q24 rearrangements involving non-immunoglobulin partners in B-cell lymphomas.

Chromosomal translocations joining the immunoglobulin (IG) and MYC genes have been extensively reported in Burkitts and non-Burkitts lymphomas but data concerning MYC rearrangements with non-IG partners are scarce. In this study, 8q24 breakpoints from 17 B-cell lymphomas involving non-IG loci were mapped by fluorescence in situ hybridization (FISH). In seven cases the breakpoint was inside a small region encompassing MYC: in one t(7;8)(p12;q24) and two t(3;8)(q27;q24), it was telomeric to MYC whereas in four cases, one t(2;8)(p15;q24) and three t(8;9)(q24;p13) it was located in a 85 kb region encompassing MYC. In these seven cases, partner regions identified by FISH contained genes known to be involved in lymphomagenesis, namely BCL6, BCL11A, PAX5 and IKAROS. Breakpoints were cloned in two t(8;9)(q24;p13), 2.5 and 7 kb downstream from MYC and several hundred kb 5′ to PAX5 on chromosome 9, joining MYC to ZCCHC7 and to ZBTB5 exon 2, two genes encoding zinc-finger proteins. In these seven cases, MYC expression measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR) was significantly higher when compared to that of patients without 8q24 rearrangement (P=0.006). These results suggest that these rearrangements are the consequence of a non-random process targeting MYC together with non-IG genes involved in lymphocyte differentiation and lymphoma progression.

Immunoenzymoassay of the hyaluronic acid-hyaluronectin interaction: application to the detection of hyaluronic acid in serum of normal subjects and cancer patients.

The binding of a hyaluronic acid-binding glycoprotein, hyaluronectin (HN), isolated from human brain, to hyaluronic acid (HA) was investigated with the enzyme-linked immunosorbent assay technique using plastic microtest plates coated with a 50 mg/liter solution of HA in 0.1 M bicarbonate. Optimum conditions for HN binding to HA were in 0.2 M NaCl buffered with 0.1 M sodium phosphate at pH 7. An assay for HA in solution was set up exploiting the fact that HN binding could be inhibited by soluble HA. HA was preincubated for 1 h in a test tube with a 30-ng/ml HN solution (v/v) in the buffer containing 0.1% bovine serum albumin. Incubation on HA-coated microtest plate lasted 4 h and maximum sensitivity was achieved when incubation was carried out at 4 degrees C. HN bound to the plate was revealed by means of alkaline phosphatase-conjugated anti-HN antibodies. The test was used to measure HA inhibitory activity after depolymerization by ferrous ions. No difference was found between inhibitory activity or smaller fragments and that of high-molecular-weight HA. The assay was applied to determination of HA in sera. Specificity was demonstrated by Streptomyces hyaluronidase digestion of reactive material in sera. Other glycosaminoglycans did not interfere with the assay. Recovery of HA was good and intra- and interassay variation coefficients were 6 +/- 2.2 and 12%. In 103 blood donor sera, HA was found at 22.4 +/- 16.7 micrograms/liter. HA was elevated in most of the cancer patient sera tested.

Serum hyaluronate in malignant pleural mesothelioma.

The diagnostic value of hyaluronate concentration in effusions of malignant mesothelioma has been extensively reported but no information is available about serum hyaluronate in patients with this cancer. Using a new enzymoimmunologic assay based on hyaluronate‐hyaluronectin interaction, serum levels of hyaluronate were measured in 16 patients with malignant pleural mesothelioma, 50 patients with other pleural effusions, and 94 healthy blood donors. The mean serum hyaluronate level in patients with mesothelioma (mean, 750 μg/l; range, 29 to 5833 μg/l) was significantly higher than in patients with other pleural effusions (mean, 56 μg/l; range, 4 to 137 μg/l) and than in blood donors (mean, 24 μg/l; range, 0 to 94 μg/l). Comparison of serum hyaluronate values observed in mesotheliomas with the clinical course of the disease suggests that serum hyaluronate might increase only at an advanced stage of the cancer. Therefore, serum hyaluronate determination has probably no clinical value for early detection of malignant mesothelioma, but might be useful to evaluate the clinical course of this malignancy.

High rate of TNFRSF14 gene alterations related to 1p36 region in de novo follicular lymphoma and impact on prognosis

High rate of TNFRSF14 gene alterations related to 1p36 region in de novo follicular lymphoma and impact on prognosis

Distribution of BCL2 breakpoints in follicular lymphoma and correlation with clinical features: specific subtypes or same disease?

The t(14;18)(q32;q21) translocation is closely associated with follicular lymphoma (FL), and is routinely assessed with molecular methods exploring BCL2 breakpoints for both diagnosis and minimal residual disease (MRD) monitoring. We and others have previously reported new recurrent breakpoints (3′BCL2 and 5′mcr) which could be easily analyzed. In this study, we characterized the BCL2 breakpoints in 113 untreated patients with t(14;18)-positive FL and correlated their location with the location of JH break and with the clinical features. Breakpoints were respectively located at the major breakpoint region (MBR) in 73 cases (65%), at the minor cluster region (mcr) in 10 cases (9%), at 3′BCL2 in 14 cases (12%) and at 5′mcr in seven cases (6%). Finally, the breakpoint could not be located in nine patients (8%). 5′mcr cases were associated with bulky and high-stage disease, with frequent extranodal involvement and bone marrow infiltration. Survival studies did not show any correlation between breakpoint location and clinical outcome. The joining JH6 segment was the most frequently involved whatever the breakpoint location. In conclusion, unusual BCL2 breakpoints are found in about 20% of newly diagnosed follicular lymphomas and their study should be considered in the investigation of BCL2-JH rearrangement. It was not possible, in this series, to demonstrate any correlation between breakpoint location and either initial characteristics of the disease or survival of the patients.

Targetable Activating Mutations are Very Frequent in GCB and ABC Diffuse Large B-Cell Lymphoma

Diffuse large B cell lymphoma (DLBCL) is an aggressive and heterogeneous malignancy that can be divided in two major subgroups, germinal center B‐cell‐like (GCB) and activated B‐cell‐like (ABC). Activating mutations of genes involved in the BCR and NF‐κB pathways (CD79A, CD79B, MYD88, and CARD11) or in epigenetic regulation (EZH2) have been recently reported, preferentially in one of the two DLBCL subtypes. We analyzed the mutational status of these five recurrently mutated genes in a cohort of 161 untreated de novo DLBCL. Overall, 93 mutations were detected, in 61 (38%) of the patients. The L265P MYD88 mutation was the most frequent MYD88 variant (n = 18), observed exclusively in the ABC subtype. CD79A/CD79B ITAM domains were targeted in ABC DLBCL (12/77; 16%), whereas CARD11 mutations were equally distributed in the two subtypes. The EZH2 Y641 substitution was found almost exclusively in the GCB subgroup (15/62; 24%). Twenty cases (12%) displayed two activating mutations, including the most frequent CD79/MYD88 variants combination (n = 8) which is observed exclusively in the ABC subtype. When considering only ABC DLBCL patients treated by rituximab plus chemotherapy, the presence of an activating NF‐κB mutation was associated with an unfavorable outcome (3‐years OS 26% for mutated cases versus 67% for the cases without mutations, P = 0.0337). Our study demonstrates that activating and targetable mutations are observed at a very high frequency in DLBCL at the time of diagnosis, indicating that sequencing of a limited number of genes could help tailor an optimal treatment strategy in DLBCL.