[Breast metastases from lung cancers with the EGFR mutation]. / Métastases mammaires des cancers bronchopulmonaires avec mutation de l'EGFR.

INTRODUCTION: The breast is a rare site for metastases from lung cancers. Their occurrence in patients with adenocarcinoma which has the EGFR mutation is exceptional. In this context, it is sometimes difficult to differentiate a second primary breast cancer from a breast metastasis. OBSERVATIONS: We report the cases of two patients who developed breast metastases from lung adenocarcinoma that was TTF1 positive with a deletion of exon 19 of the EGFR gene. A non-smoking woman of Asian origin, presented with a solitary breast metastasis 29 months after being established on first-line chemotherapy. The second case was a Caucasian patient who was an active smoker presented with tumor progression with multiple metastases including involvement of both breasts 10 months after the start of treatment with a tyrosine kinase inhibitor. In both, tumor cells from the breast showed positive immunostaining for TTF1 and genotyping detected the presence of the deletion of exon 19 of the EGFR gene. CONCLUSION: In patients with lung adenocarcinoma and EGFR mutation, immunohistochemical examination, including TTF1 and genotyping of synchronous breast tumour, is needed to confirm its metastatic nature and to guide treatment.

High-throughput somatic mutation profiling in pulmonary sarcomatoid carcinomas using the LungCarta™ Panel: exploring therapeutic targets.

BACKGROUND: Pulmonary sarcomatoid carcinomas (SC) are tumors characterized by poor prognosis and resistance to conventional platinum-based chemotherapy. This study sought to describe the mutational profile of SC using high-throughput genotyping technology. PATIENTS AND METHODS: We used mass spectrometry to test 114 surgical biopsies from 81 patients with SC for 214 mutations affecting 26 oncogenes and tumor suppressor genes. RESULTS: In total, 75 (92.6%) patients were smokers. Within the total 81 tumors, 67 distinct somatic alterations were identified, with 56 tumors (69.1%) harboring at least one mutation. The most frequent mutations were KRAS (27.2%), EGFR (22.2%), TP53 (22.2%), STK11 (7.4%), NOTCH1 (4.9%), NRAS (4.9%), and PI3KCA (4.9%). The EGFR mutations were almost always rare mutations (89%). In 32 tumors (39.5%), two or more mutations co-existed, with up to four mutations in a single case. In six different cases, comparative genetic analysis of different histological areas from the same tumor (giant, spindle, or epithelial component) revealed a 61% concordance rate for all the mutations with a 10% detection threshold, compared with 91.7% with a 20% detection threshold. CONCLUSION: Our results demonstrated a high mutation rate and frequent co-mutations. Despite SC tumors exhibiting a high histological heterogeneity, some intratumoral molecular homogeneity was found. Now with newly developed targeted therapies, SC patients may be eligible for new target mutations, and can now therefore be screened for clinical trials.

Colorectal liver metastases are more often super wild type. Toward treatment based on metastatic site genotyping?

Recent data showed that metastatic colorectal (mCRC) tumors exhibiting extended RAS-BRAF mutations were resistant to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies, making these drugs suitable for the so-called "super" wild-type (WT) patients only. This study aimed to compare the extended RAS-BRAF mutation frequency and characteristics according to location of tumor sampling. All consecutive mCRC specimens (N = 1659) referred to our institution from January 2008 till June 2014 were included in the analysis. Tumor genotyping (first for KRAS exon 2, then for BRAF exon 15, and later for KRAS exons 2, 3, and 4 and NRAS exons 2, 3, and 4) was performed with high-resolution melting analysis or allelic discrimination. The factors predicting for the presence of mutation were explored using multivariate binary logistic regression. Overall, the prevalence of KRAS exon 2 was 36.8%, and it was lower in liver metastases (N = 138/490; 28.2%) in comparison with primary tumors (N = 442/1086; 40.7%), lung metastases (16/32; 50%), or other metastatic sites (15/51; 29.4%; P < 0.0001). Similarly, in the 1428 samples analyzed, BRAF mutations were less often found in liver metastases (N = 9/396; 2.3%) as compared to primary tumors (N = 79/959; 8.2%), lung metastases (N = 2/29; 6.9%), or other metastatic locations (N = 2/44; 4.5%; P < 0.0002). Overall occurrence of extended RAS mutation was 51.7%. Of the 503 samples tested, the prevalence of extended RAS-BRAF mutations was twice as low in liver metastases (N = 53/151; 34.2 %) as compared to primary tumors (N = 191/322; 59.3%, P < 0.0001). Univariate analysis identified age ≤65 years, male gender, and liver localization as predictors of super WT status. At multivariate analysis, only liver metastases were retained (RR 2.85 [95% CI 1.91-4.30]). Colorectal liver metastases are twice as likely to exhibit a super WT genotype as compared to other tumor locations independently from other factors. This molecular feature has the potential to influence therapeutic strategy in mCRC patients.

Treatment with rituximab, dexamethasone, high-dose cytarabine, and oxaliplatin (R-DHAOx) produces a strong long-term antitumor effect in previously treated patients with follicular non-Hodgkin's lymphoma.

BACKGROUND: We explored the addition of rituximab to high-dose cytarabine (ara-C), oxaliplatin (L-OHP), and dexamethasone [R-DHAOx], in resistant and relapsed patients with CD20-positive follicular non-Hodgkin's lymphoma. METHODS: Twenty-two patients were included; they were treated previously with one to five chemotherapy regimens, including 13 patients who had also received rituximab. R-DHAOx consisted of rituximab, 375mg/m(2), day 1; dexamethasone, 40mg/d, days one to four; L-OHP, 130mg/m(2), day 1; and ara-C, 2000mg/m(2) every 12 h, day 2. Courses were repeated every 21 days for eight courses. RESULTS: Twenty-one patients (95%) achieved a complete response and one had a partial response. Responses were obtained in patients with and without resistance to prior treatment, either alone or combined with rituximab. The median follow-up time was 58.3 months (range, 8.7-92.6 months). Progression-free survival reached a plateau at 84% at 38.2 months. Only two of the 21 complete responders have relapsed. Tumor molecular markers disappeared in all 10 complete responders whose markers were found before treatment. Peripheral neuropathy related to the cumulative dose of L-OHP, and myelosuppression were the most prominent toxic effects. CONCLUSIONS: R-DHAOx is highly active for salvage treatment of patients with follicular non-Hodgkin's lymphoma, and it produces long-term antitumor efficacy.

Co expression of SCF and KIT in gastrointestinal stromal tumours (GISTs) suggests an autocrine/paracrine mechanism.

KIT is a tyrosine kinase receptor expressed by several tumours, which has for specific ligand the stem cell factor (SCF). KIT is the main oncogene in gastrointestinal stromal tumours (GISTs), and gain-of-function KIT mutations are present in 70% of these tumours. The aim of the study was to measure and investigate the mechanisms of KIT activation in 80 KIT-positive GIST patients. KIT activation was quantified by detecting phosphotyrosine residues in Western blotting. SCF production was determined by reverse transcriptase-PCR, ELISA and/or immunohistochemistry. Primary cultures established from three GISTs were also analysed. The results show that KIT activation was detected in all cases, even in absence of KIT mutations. The fraction of activated KIT was not correlated with the mutational status of GISTs. Membrane and soluble isoforms of SCF mRNA were present in all GISTs analysed. Additionally, SCF was also detected in up to 93% of GISTs, and seen to be present within GIST cells. Likewise, the two SCF mRNA isoforms were found to be expressed in GIST-derived primary cultures. Thus, KIT activation in GISTs may in part result from the presence of SCF within the tumours.

Fate and characterization of circulating tumor cells in a NOD/SCID mouse model of human hepatocellular carcinoma.

There is much debate about the way in which epithelial tumors metastasize. It has been proposed that the bone marrow (BM) acts as a tumor cell reservoir. We injected human hepatocellular carcinoma (HCC) cells (Mahlavu cell line) into the livers, circulation or BM of NOD/SCID mice and circulating tumor cells were quantified. When injected under the Glisson capsule, a primary tumor developed and continuously yielded circulating tumor cells. Liver tumor removal led to a very low level of Mahlavu cells both in blood and BM 30 days later. When Mahlavu cells (cultured or from BM of primary mice femurs) were intravenously injected into mice, the number of cells in the bloodstream (BS) steadily decreased, whereas the BM was not significantly colonized. When Mahlavu cells were directly injected into one femur, the controlateral femur was not colonized. Microscopic analysis and a sensitive PCR assay (<1 Mahlavu cell/nuclear cells) both failed to detect human tumor cells in other organs regardless of injection route. In conclusion, our model strongly supports the hypothesis that HCCs continuously release cells into the BS. However, in sharp contrast with the current hypothesis, the BM is not specifically colonized by tumor cells but could store them at a very low level.

[Molecular biology and hepatocellular carcinoma: current status and future prospects]. / Biologie moléculaire et carcinome hépatocellulaire: données actuelles et développements futurs. Prix international de la SFBC 2003.

Hepatocellular carcinoma (HCC) is among the fifth most common cancers worldwide. Its incidence is still rising in part because of the high level of hepatitis C virus infection. Tumor markers currently used such as serum alpha-foetoprotein are not sufficient for diagnosis of the tumor and satisfying follow-up of the patients. Mechanisms of hepatocarcinogenesis ar not completely understood although several altered genes have been described in HCC. The genetic changes involved can be divided in at least 4 different pathways, each pathway contributing to a limited number of tumors. These are: 1) the p53 pathway involved in response to DNA damage, 2) the retinoblastoma pathway involved in the control of the cell cycle, 3) the transforming growth factor-beta (TGF-beta) pathway involved in growth inhibition, and 4) the Wnt pathway involved in cell-cell adhesion and signal transduction. Alterations of the epigenetic regulation of gene expression have also been described. Evolution of molecular biology methods tends to the development of more global genomic approaches; microsatellite instability analysis, chromosomal instability analysis or gene expression profile analysis have been used to investigate HCC. Finally, attempts to develop molecular biomarkers based on peripheral blood analysis more easily accessible in clinical routine patients have also been developed.

Rapid automated simultaneous screening of (G1691A) Factor V, (G20210A) prothrombin, and (C677T) methylenetetrahydrofolate reductase variants by multiplex PCR using fluorescence scanning technology.

The Factor V Leiden mutation (G1691A), and mutations in the prothrombin (G20210A) and 5,10-methylenetetrahydrofolate reductase (C677T) genes are common hereditary risk factors associated with venous thrombosis. The aim of this study was to develop an automated, PCR-based genotyping assay for rapid simultaneous screening of these three mutations. We adapted multiplex PCR, using primer modifications to introduce cleavage sites for restriction endonucleases into the fragments bearing each of the mutations. The three mutations were analyzed in a single tube by fluorescence scanning. An internal digestion control was introduced to prevent false-negative results due to incomplete digestion or a total lack of digestion. DNA fragment analysis was carried out using an automated capillary electrophoresis instrument (ABI310). This reliable, efficient, easy-to-use assay can be applied to specimens from large clinical trials and epidemiological surveys.

Accumulation of an inactive form of p53 protein in cells treated with TNF alpha.

In MCF-7 cells, TNF alpha induces a G1 arrest with an increased expression of p21/Waf1, an activation of NF-kappa B and an accumulation of p53. NF-kappa B and p53 are two transcriptional factors known to activate p21/Waf1 gene expression. Here we show that p53 inhibition has no effect on p21/Waf1 mRNA accumulation following TNF alpha treatment. In contrast, inactivation of NF-kappa B inhibits p21/Waf1 expression without affecting G1 arrest. The fact that p21/Waf1 gene expression is still stimulated when p53 is inactivated strongly suggests that TNF alpha induces accumulation of an inactive form of p53 protein. This assumption was further supported by the following observations: (i) the p53 DNA-binding activity to its consensus sequence was not stimulated following TNF alpha treatment, (ii) phosphorylation at Ser-15, -20 or -392 was not detected in response to TNF alpha, (iii) the transcription rate of Ddb2, another p53 target gene, was not stimulated by TNF alpha. Finally, the accumulation of p53 in the nuclei of TNF alpha-treated MCF-7 cells was concomitant with an increase in p53 mRNA level, suggesting a regulation at the transcription level.

Detection of gammopathy by serum protein electrophoresis for predicting and managing therapy of lymphoproliferative disorder in 911 recipients of liver transplants.

Monitoring of posttransplantation lymphoproliferative disorder (LPD) is usually based on imaging, which lacks sensitivity. A prospective study in 911 consecutive recipients of liver transplants was conducted to assess the value of gammopathy monitoring by serum protein electrophoresis (SPE) and to compare it with conventional follow-up methods. Patients systematically underwent SPE testing just before transplantation, at least twice during the first year after transplantation, and once a year thereafter. Patients with LPD underwent SPE testing every month. Immunofixation was done if abnormalities were detected by SPE. Gammopathy was observed in 114 patients, 18 of whom had onset of LPD. In 3 other patients, LPD developed, but no gammopathy was detected before onset of LPD or while LPD was present. Multivariate analyses showed gammopathy (relative risk [RR], 65.3), more than one transplantation (RR, 7.5), and viral cirrhosis (RR, 2.8) to be independent prognostic factors associated with occurrence of LPD. LPD was treated by reducing immunosuppression, with or without chemotherapy, administration of anti-CD20 monoclonal antibody, or surgery. The mortality rate was 24% (5 of 21 patients). Remission, which occurred in 13 patients, was associated with disappearance of gammopathy in 10 patients. In 5 patients, normalization of SPE results preceded the diagnosis of remission based on imaging, by a mean of 4 months. For diagnosis of LPD remission, the positive and negative predictive values of disappearance of gammopathy were 91% and 100%, respectively; and gammopathy monitoring was more sensitive than imaging (100% and 38%, respectively). Gammopathy monitoring is an inexpensive, noninvasive, sensitive way to detect LPD and assess the efficacy of treatment. It could be used routinely in follow-up of recipients of transplants.

Real-time quantitation of bcr-abl transcripts in haematological malignancies.

We have applied an automated real-time quantitative PCR assay using a double-labeled fluorogenic probe to detect t(9;22)-positive cells in haematological malignancies. The results are expressed as the ratio of chimeric bcr-abl transcripts on abl transcripts. Highly reproducible results were obtained for t(9;22)-positive K562 RNA. Ten copies of bcr-abl DNA from a recombinant KW-3 plasmid and one positive cell in 10(4) can be detected. Thirty-two patients with chronic myeloid leukaemia (CML), 25 with acute leukaemia, 12 with myelodysplastic syndromes and 7 with other myeloproliferative syndromes were tested. Follow-up data were obtained in bcr-abl positive cases. Results were compared with those of conventional nested RT-PCR and cytogenetics. Real-time quantitative RT-PCR values correlated well with both these methods. However, in some cases the only means of detecting early relapse or blastic transformation was to examine the kinetics of real-time quantitative RT-PCR. Thus, real-time quantitative RT-PCR appears suitable for the diagnosis and follow-up of patients with the t(9;22) translocation.

Microsatellite instability in European hepatocellular carcinoma.

Genetic instability has been detected in many types of cancers but poorly investigated in hepatocellular carcinoma (HCC). We have studied the incidence of microsatellite instability (MI) at eight highly polymorphic microsatellite markers and the poly A tract BAT26 and tested for mutations at two sites of repetitive sequence (poly-A nucleotides 709-718 and GT repeat-nucleotides 1931-1936) in the Transforming Growth Factor beta (TGFbeta) type II receptor (RII) gene, in a group of 46 European HCCs and the surrounding nontumour tissue. This analysis showed that 63% of HCCs exhibit MI in at least one chromosome locus and 41% in two or more loci. No mutations of the TGFbetaRII gene were found in the MI positive tumours. No correlation was found with clinicopathological characteristics of the tumours such as cirrhosis, etiology, number of nodules, Edmondson's grade and vascular invasion. However, in patients who had a rearranged D16S402 microsatellite in their tumour, the recurrent disease and the number of nodules were significantly higher than in the others (P<0.005 and P<0.02, respectively). We propose to consider D16S402 rearrangement in HCC as a prognostic factor to identify patients presenting a higher risk of recurrence.

Clinical significance of circulating anti-p53 antibodies in European patients with hepatocellular carcinoma.

p53 alterations are considered to be predictive of poor prognosis in hepatocellular carcinoma (HCC) and may induce a humoral response. Anti-p53 serum antibodies were assessed by enzyme-linked immunosorbent assay (ELISA) using purified recombinant human p53 on 130 European HCC patients before treatment and during the clinical course of the disease. p53 immunohistochemistry was performed on tumours from the 52 patients who underwent surgery, and DNA sequencing analysis was initiated when circulating anti-p53 antibodies were detected. Nine (7%) HCC patients had anti-p53 serum antibodies before treatment. During a mean period of 30 months of follow-up, all the negative patients remained negative, even when recurrence was observed. Of the nine positive patients, eight were still positive 12-30 months after surgery. The presence of anti-p53 serum antibodies was correlated neither with mutation of the p53 gene nor the serum alpha-fetoprotein levels and clinicopathological characteristics of the tumours. However, a greater incidence of vascular invasion and accumulation of p53 protein were observed in the tumours of these patients (P<0.03 and P<0.01 respectively) as well as a better survival rate without recurrence (P = 0.05). In conclusion, as was recently shown in pancreatic cancer, anti-p53 serum antibodies may constitute a marker of relative 'good prognosis' in a subgroup of patients exhibiting one or several markers traditionally thought to be of bad prognosis.

Importance of the FcgammaRIIa-Arg/His-131 polymorphism in heparin-induced thrombocytopenia diagnosis.

Heparin-induced thrombocytopenia (HIT) involves heparin-dependent antibodies which induce platelet activation. In the present study, we searched for a relationship between the polymorphism of the Fc receptor (FcgammaRIIa) and the development of HIT. In this purpose, all the donors were genotyped for their FcgammaRIIA and HIT patients were selected on the basis of at least one positive answer by 14C-serotonin release assay (SRA). The frequency distribution of the FcgammaRIIa polymorphism in the HIT patient group was similar to that observed in the healthy control group. Moreover, a statistical analysis taking into account our results and those of 3 previously published studies, suggested at most only a weak association between HIT and the FcgammaRIIa-131 polymorphism. Laboratory tests used to diagnose HIT rely on the activation of normal donor platelets but fail to detect every HIT positive patient. We determined the role of FcgammaRIIa-131 polymorphism on the reactivity of control platelets to HIT plasmas. When control platelet FcgammaRIIa-131 was of Arg/Arg form, only 47% of the HIT plasmas were positive by SRA, compared to 81% and 74% for His/His or His/Arg forms, respectively. We also compared the level of anti PF4/heparin antibodies in the HIT plasmas with the response obtained by SRA. The mean anti PF4/heparin antibodies level in HIT plasma was significantly lower in negative SRA than in positive tests when using control platelets from FcgammaRIIa-Arg/Arg 131 and heterozygous donors. Thus, the variability of control platelets to respond to HIT plasmas in the SRA test is related to both the FcgammaRIIa-131 polymorphism, and to the amount of anti PF4/heparin antibodies.

Role of Fc gamma RIIA gene polymorphism in human platelet activation by monoclonal antibodies.

The aim of this study was to determine if there is a correlation between the activity of a MoAb as an agonist and its ability to bind to the Fc platelet receptor, Fc gamma RIIa. A polymorphism at amino acid 131 [arginine (Arg) or histidine (His)] of Fc gamma RIIa was first shown to be determinant for MoAb-IgG1 binding on monocytes. To clarify the role of this polymorphism in platelet activation by MoAb-IgG1 we (i) established the Fc gamma RIIa polymorphism at the gene level by adapting the denaturating gradient gel electrophoresis method, (ii) analyzed the binding affinity of the MoAbs to Fc gamma RIIa on platelets from homozygous Arg, homozygous His, and heterozygous Arg/His donors, and (iii) characterized the different reactivities of platelets according to the Fc gamma RIIA polymorphism. Among 167 caucasian donors we found 46% heterozygous Arg/His, 36% homozygous His and 18% homozygous Arg. ALB6, and anti CD9, P256 an anti GPIIb-IIIa, and AP3 an anti-GPIIIa were chosen according to their ability (ALB6, P256) or not (AP3) to activate platelets. These 3 MoAbs-IgG1 bind to Fc gamma RIIa with a stronger affinity for the Arg-form of Fc gamma RIIa, a result which was confirmed with the use of diverse MoAbs directed against various antigens. The different abilities of MoAbs to bind to the two Fc gamma RIIa forms were well correlated to the different platelet responses induced by ALB6 and P256. However, low concentrations of ALB6, which allow full activation of platelets from homozygous Arg donors, as did P256, did not induce any activation of platelets from homozygous His donors, whereas P256 is able to induce a low aggregation. The results further define the respective roles of the antigen and the Fc receptor, depending on the MoAb, and the role of the Fc gamma RIIa polymorphism in platelet activation induced by MoAbs. In addition, the results obtained with MoAbs unable to induce platelet activation provided evidence that the binding of a MoAb on Fc gamma RIIa does not predict its ability to activate platelets.

[Antiplatelet glycoprotein monoclonal antibodies: properties and practical value]. / Anticorps monoclonaux antiglycoprotéines plaquettaires: propriétés et intérêts pratiques.

The aim of this review is (i) to classify the different monoclonal antibodies against platelet glycoproteins according to their properties and (ii) to take stock of their many diagnostic and therapeutic uses. Most of these antibodies recognize antigens on resting platelets without inducing activation, sometimes however they inhibit platelet function. Some of these antibodies, especially those against specific antigens (GPIIb/IIIa, CD9, CD36), have the capacity to activate platelets in vitro, either by direct binding of antibodies on the antigen or through the Fc domain binding to its platelet receptor Fc gamma RII. Other antibodies are directed against activation-dependent antigens that are expressed as a result of (i) a modification of a glycoprotein structure during platelet activation (as for GPIIb/IIIa), (ii) platelet release of granular antigens (GMP140, CD63, granulophysin...) or (iii) binding of soluble antigens on the activated platelet surface. Monoclonal antibodies find practical applications for both in vitro and in vivo diagnosis of bleeding or thrombotic pathology with some of them, notably anti-GPIIb/IIIa, having a promising future for antithrombotic therapy.