High-depth sequencing of paired primary and metastatic tumours: Implications for personalised medicine.

BACKGROUND: Next-generation sequencing of large panel of genes had been associated with clinical benefit in a significant proportion of patients with advanced cancer. However, the molecular profile of the primary tumour from the initial surgical specimen might significantly differ from the molecular profile in a tumour sample obtained from a biopsy of a metastatic site. PATIENTS AND METHODS: We compare the genetic profile of primary tumours and paired metastases by using a large panel of cancer genes. Training and validation set including a total of 152 primary and metastatic tumour pairs were sequenced (up to 429 genes) focussing on variants described in the Catalogue of Somatic Mutations in Cancer (COSMIC). RESULTS: Training and validation set including a total of 152 primary and metastatic tumour pairs were sequenced focussing on variants described in COSMIC. Agreement rate between the couples of primary and metastasis on COSMIC variants was 65% (24/37) and 43% (49/115) in the training and validation cohort, respectively. That rose to 74% (20/27) and 58% (42/73) when focussing on targetable mutations. In five cases, the discordance was related to appearance of secondary resistance mutation, giving a targetable refined agreement rate of 67% (67/100). CONCLUSION: Up to 40% of paired primary tumour/metastases have discordant molecular profile. Liquid biopsies may overcome, in the near future, the limits of tumour tissue genotyping.

All-trans retinoic acid targets gastric cancer stem cells and inhibits patient-derived gastric carcinoma tumor growth.

Gastric carcinoma is the third leading cause of cancer-related death worldwide. This cancer, most of the time metastatic, is essentially treated by surgery associated with conventional chemotherapy, and has a poor prognosis. The existence of cancer stem cells (CSC) expressing CD44 and a high aldehyde dehydrogenase (ALDH) activity has recently been demonstrated in gastric carcinoma and has opened new perspectives to develop targeted therapy. In this study, we evaluated the effects of all-trans-retinoic acid (ATRA) on CSCs in human gastric carcinoma. ATRA effects were evaluated on the proliferation and tumorigenic properties of gastric carcinoma cells from patient-derived tumors and cell lines in conventional 2D cultures, in 3D culture systems (tumorsphere assay) and in mouse xenograft models. ATRA inhibited both tumorspheres initiation and growth in vitro, which was associated with a cell-cycle arrest through the upregulation of cyclin-dependent kinase (CDK) inhibitors and the downregulation of cell-cycle progression activators. More importantly, ATRA downregulated the expression of the CSC markers CD44 and ALDH as well as stemness genes such as Klf4 and Sox2 and induced differentiation of tumorspheres. Finally, 2 weeks of daily ATRA treatment were sufficient to inhibit gastric tumor progression in vivo, which was associated with a decrease in CD44, ALDH1, Ki67 and PCNA expression in the remaining tumor cells. Administration of ATRA appears to be a potent strategy to efficiently inhibit tumor growth and more importantly to target gastric CSCs in both intestinal and diffuse types of gastric carcinoma.

The MAP3K ZAK, a novel modulator of ERK-dependent migration, is upregulated in colorectal cancer.

Often described as a mediator of cell cycle arrest or as a pro-apoptotic factor in stressful conditions, the MAP3K ZAK (Sterile alpha motif and leucine zipper-containing kinase) has also been proven to positively regulate epidermal growth factor receptor (EGFR) and WNT signaling pathways, cancer cell proliferation and cellular neoplastic transformation. Here, we show that both isoforms of ZAK, ZAK-α and ZAK-ß are key factors in cancer cell migration. While ZAK depletion reduced cell motility of HeLa and HCT116 cells, its overexpression triggered the activation of all three mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK) and p38, as well as an increase in cell motion. On the contrary, the kinase-dead mutants, ZAK-α K45M and ZAK-ß K45M, were not able to provoke such events, and instead exerted a dominant-negative effect on MAPK activation and cell migration. Pharmacological inhibition of ZAK by nilotinib, preventing ZAK-autophosphorylation and thereby auto-activation, led to the same results. Activated by epidermal growth factor (EGF), we further showed that ZAK constitutes an essential element of the EGF/ERK-dependent cell migration pathway. Using public transcriptomic databases and tissue microarrays, we finally established that, as strong factors of the EGFR signaling pathway, ZAK-α and/or ZAK-ß transcripts and protein(s) are frequently upregulated in colorectal adenoma and carcinoma patients. Notably, gene set enrichment analysis disclosed a significant correlation between ZAK+ colorectal premalignant lesions and gene sets belonging to the MAPK/ERK and motility-related signaling pathways of the reactome database, strongly suggesting that ZAK induces such pro-tumoral reaction cascades in human cancers.

[Mediastinal sarcoidosis in an oncologic context: role of endoscopic ultrasound-guided fine needle aspiration]. / Sarcoïdose médiastinale dans un contexte oncologique : apport de la ponction aspiration guidée par échoendoscopie.

INTRODUCTION: Sarcoidosis and sarcoid reactions have been previously reported in association with cancer. Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) is a minimally invasive test for investigating mediastinal lymph nodes PATIENTS AND METHODS: We conducted a retrospective review of 54 patients undergoing EUS-FNA in a cancer institute for suspected metastatic mediastinal lymph nodes showed by CT-imaging or positron emission tomography (PET). Patients with non-caseating granuloma identified by EUS-FNA were included RESULTS: EUS-FNA identified non-caseating granuloma in seven out of the 54 included patients. Most of them had positive PET. One patient had a prior history of sarcoidosis before the diagnosis of cancer. Another patient developed micrometastasis associated with sarcoid-like reaction. There was no adverse outcome associated with the EUS-FNA procedure CONCLUSIONS: Sarcoidosis must be included in the differential diagnosis of patients with a history of malignancy who develop mediastinal lymphadenopathy. EUS-FNA is a safe and minimally invasive test to obtain tissue diagnosis.

IGHV gene features and MYD88 L265P mutation separate the three marginal zone lymphoma entities and Waldenström macroglobulinemia/lymphoplasmacytic lymphomas.

To clarify the relationships between marginal zone lymphomas (MZLs) and Waldenström macroglobulinemia/lymphoplasmacytic lymphomas (WM/LPLs), immunoglobulin heavy chain variable gene (IGHV) features were analyzed and the occurrence of MYD88 L265P mutations was identified in a series of 123 patients: 53 MZLs from the spleen (SMZLs), 11 from lymph nodes (NMZLs), 28 mucosa-associated lymphatic tissue (MALT) lymphomas and 31 WM/LPLs. SMZLs were characterized by overrepresentation of IGHV1-2 gene rearrangements with a canonical motif, without selection pressure and with long CDR3 segments. NMZLs had increased frequencies of IGHV3 genes. The IGHV gene was unmutated in most cases, often with long CDR3 segments. MALT lymphomas were usually associated with a mutated IGHV gene, but with the absence of selection pressure. WM/LPLs were associated with an IGHV3-23 overrepresentation and high IGHV mutation rate, with features of selection pressure and short CDR3 segments. MYD88 L265P mutations were almost restricted exclusively to WM/LPL patients. Taken all diagnoses together, all patients with MYD88 L265P mutations had an immunoglobulin M peak and almost all patients except one had bone marrow infiltration. These results demonstrate that the history of antigen exposure of the four entities studied was different and MYD88 L265P was specifically associated with WM/LPLs. WM/LPL may thus be functionally associated with constitutive nuclear factor-κB activation.

Therapeutic strategies in elderly and very elderly patients.

Management of the elderly with lymphoma needs specific attention. This means supplementary evaluation as regards to younger patients. The objective is to identify specific weaknesses of the patients and thus to foresee potential unexpected toxicities which may endanger patients' outcome. With this information, the hematologist will be able to propose an optimized treatment strategy i.e. with an adapted efficacy/toxicity ratio. These general principles are consensual but it remains to determine what the practical content of this approach is. Recent results suggest some specific tools for the pre-treatment evaluation. Some specific indexes have been proposed to categorize patients but the ultimate approach remains to be outlined. Finally, while geriatric intervention has demonstrated its capacity to improve survival in the general elderly population, no such demonstration has been made in cancer patients including lymphoma.

[Natural killer cell nasal lymphoma mimicking localized Wegener's disease]. / Lymphome nasal natural killer mimant une maladie de Wegener localisée.

INTRODUCTION: Primary non-Hodgkin's lymphoma of the nasal cavity is particular. Pathological characteristics mainly associate a prevalent NK lymphocyte phenotype, a frequent exposure to the Epstein-Barr virus and a poor sensitivity to radiotherapy compared to other lymph node localizations. EXEGESIS: The authors report the case of a 38-year-old man. The patient had previously presented a chronic maxillary sinusitis. After a diagnosis of Wegener's disease, the poor course under therapy resulted in a nasal lymphoma. Natural killer cell nasal lymphoma was confirmed with a leading biopsy at the same time as a serious clinical outcome. The patient died of septic shock with multivisceral failure. CONCLUSION: The two differential diagnoses of ulcerative lymphoma of the midface are ulcerative infectious diseases and Wegener's disease. We must not miss this severe disease, with its poor prognosis and variable, though sometimes rapid speed of evolution.

A 1-kb Bcl-2-PCR fragment detection in a patient with follicular lymphoma and development of a new diagnostic PCR method.

The t(14;18) translocation is a useful marker to characterize follicular lymphoma and to monitor residual disease. The polymerase chain reaction (PCR) is a powerful technique to detect this translocation. Located on chromosome 18, within the Bcl-2 gene, breakpoints occur mainly in the 3; untranslated region, in the third exon of Bcl-2 (MBR region). In this study, the authors amplified MBR breakpoints by PCR and found an unexpectedly large fragment of 1 Kb that corresponds to a recently described new breakpoint in the Bcl-2 gene. With a new primer set, a further previously considered t(14;18)-unrelated tumor was in fact positive for this new breakpoint.

Small B-cell lymphoproliferative disorders: an overview of diagnostic approach.

Despite their common origin from the B-cell mature lymphoid system, small B-cell lymphomas/leukaemias represent in fact an heterogeneous group of diseases. Recent advances in immunohistochemistry and molecular techniques have improved our knowledge of the immune system and lymphoid neoplasms. An international consensus has been recently reached among pathologists and clinicians, that recognises clinico-pathological entities which are defined by a combination of morphological, immunophenotypical, genetic and clinical features. In each entity, a range of histological grade and clinical aggressiveness can be encountered. Recognition of these entities, combined with clinical prognostic factors has clinical implications in terms of response to treatment and prognosis. The purpose of this paper is to focus on a practical approach, either clinical or pathological, of the diagnosis of small B-cell lymphoma/leukaemia.

Current treatment of Hodgkin's disease.

In spite of the fact that Hodgkin's disease (HD) remains still an enigma its management and treatment yield a cure rate of about 80% of all patients. However, this management has two limits: on one side favourable cases which should not be overtreated because of unacceptable side-effects, and on the other side very unfavourable cases which should be treated differently because of a very high rate of failure and/or relapse. Then it becomes necessary to precise as thoroughly as possible these two limits in order to choose the adequate treatment for the patient. Prognostic factors based on patient and disease characteristics allow a relatively exact classification of favourable and unfavourable cases. This distinction in two prognostic groups has therapeutic implications in terms of chemotherapy (regimen, duration) and radiotherapy (extension, doses). Other specific situations have to be considered, e.g. pediatric cases, pregnancy, old age and HIV-infected patients who need an adapted management according to very different situations.

Thyroid pathologic findings in patients with Cowden disease.

We describe the histologic findings in thyroid glands from six female and five male patients with Cowden disease. The patients were aged 9 to 43 years (mean age, 26 years). The salient thyroid lesions in this syndrome were multicentric follicular adenomas and adenomatous (parenchymatous, hyperplastic) nodules showing a wide range of nonspecific cytoarchitectural patterns. Multiple tiny cellular foci, so-called microadenomas, were also a feature. Specific lesions composed of oxyphil or clear cells, a tumor with features of hyalinizing trabecular adenoma, and an adenolipoma also occurred. Two cases showed a follicular carcinoma in addition to multiple benign follicular cell proliferations. The follicular carcinomas occurred at an older age and were larger in size than the clinically significant benign nodular lesions, suggesting tumor progression. All tumors showed thyroglobulin immunoreactivity and were negative for calcitonin. The histologic findings of a multiple adenomatous goiter or multiple follicular adenomas, particularly in children and young adults, should alert the pathologist and physician to the possibility of an inherited trait, such as Cowden disease, with its implications for family screening. The tumors are usually benign and well demarcated, but, because of multicentricity and increased risk of recurrence or progression to carcinoma, total thyroidectomy should be advocated.

Competitive polymerase chain reaction to quantify tumor cells in peripheral blood of patients with T(14;18)-bearing follicular non-Hodgkin's lymphoma: an exploratory study in 8 patients.

Detection of residual disease in follicular lymphoma is hampered by the observation of t(14;18)-bearing cells in the blood of healthy adult humans. To overcome this problem, we decided to validate a quantification method of t(14;18)-bearing cells and test it in t(14;18)-bearing follicular lymphomas (FL). We designed a competitive PCR method to quantify t(14;18)-bearing cells in peripheral blood. First, we controlled overall reliability (specificity, sensitivity, reproducibility, precision and accuracy); then we used our method to study 16 peripheral blood samples collected in 8 patients with t(14;18)-bearing FL. There were considerable variations in the number of circulating tumor cell (CTC) in FL patients, ranging from zero to 17,813 cells/ml. In 2 patients who were sampled before and after treatment and who attained complete remission (CR), a significant decrease in the number of CTC was observed. In 3 patients with detectable CTC during CR, relapse occurred 4 to 11 months later. Of 3 patients with no detectable CTC, 2 remain in CR 35 and 95 months later, but one relapsed 11 months after sample collection. These preliminary results suggest that quantification of CTC may be worthwhile in follicular lymphoma. It may improve our ability to predict relapse occurrence, but also may help in understanding this peculiar disease. Int. J. Cancer (Pred. Oncol.) 84:558-561, 1999.

Evolution of BCL-2/IgH hybrid gene RNA expression during treatment of T(14;18)-bearing follicular lymphomas.

Bcl-2, the gene over-expressed in follicular lymphomas (FL), is able to block chemotherapy-induced apoptosis. Consequently, we wondered whether bcl-2/IgH expression variations during treatment of FL could predict the outcome of patients with t(14;18)-bearing FL. For this purpose, we used a reverse transcription polymerase chain reaction (RT-PCR) assay to analyse 180 serial peripheral blood samples (PBS) during 34 treatment phases in 25 patients with t(14;18)-bearing FL. In all patients but two, bcl-2/IgH gene expression was demonstrated in pre-treatment samples. During 16 out of the 34 treatment phases (47%), bcl-2/IgH expression became negative: all but one were responders to chemotherapy. This conversion was transient in six cases. In 18 treatment phases, bcl2/IgH expression remained detectable: eight were clinically considered as treatment failures, while eight others achieved PR and two achieved CR. We observed a significant correlation between treatment response and RNA PCR results (P = 0.002). Three-year overall survival of patients with stable bcl2/IgH-negative conversion was 100% compared to 54% for the remaining patients (P = 0.069); 3-year freedom from progression was respectively 87.5% and 13% (P = 0.005). These results indicate a correlation between bcl-2/IgH expression variations and both clinical response and outcome. Whether this might predict disease outcome early remains to be confirmed.

[Adult's anaplastic CD30+ large cell lymphomas]. / Lymphomes anaplasiques T ou nuls à grandes cellules CD30+ (Ki-1) de l'adulte.

Anaplastic large-cell lymphomas were recognized by Stein in 1985. Less than fifteen years were necessary to confirm this entity, as well as her phenotype and to characterize the t(2;5) (p23;q35) chromosomal abnormality. This rare subgroup of non-Hodgkin's lymphomas (15% of peripheral T cell lymphomas and 8% of all diffuse aggressive lymphomas) is individualized in the Real classification. This disease, which had a bimodal age distribution, is clinically characterized by a diffuse nodal involvement and the frequency of extranodal involvement, especially skin and lungs. Primitive cutaneous anaplastic large cell lymphomas belong to the cutaneous CD30+ lymphoproliferative diseases spectrum. Among peripheral T cell and diffuse aggressive lymphomas, they have the better prognosis. We present in this paper a review of the recent advances in the knowledge, treatment and prognosis of this peculiar entity.

Polymerase chain reaction diagnosis of t(14;18) from paraffin-embedded tissues fixed with Holland Bouin fluid.

The t(14;18) translocation and its molecular counterpart, the bcl-2/IgH gene rearrangement, are highly characteristic of follicular non-Hodgkin lymphomas. The identification of the tumor-specific t(14;18) clone is mandatory for any molecular studies on residual disease because of the existence of circulating t(14;18)-bearing benign cells. In this study, the ability to specifically polymerase chain reaction (PCR) amplify t(14;18) with DNA purified from tissues fixed with Holland Bouin fluid is demonstrated. The specificity of the PCR product was confirmed by internal probe hybridization and with comparison of the nucleotidic sequences of this PCR product with those obtained from the corresponding frozen material. Although the sensitivity of the technique is 50% to 60%, paraffin-embedded tissues fixed with bouin fluid may be a good alternative to frozen tissues to detect t(14;18) in tumors.

[Large core of nonpalpable breast lesions]. / Microbiopsies des lésions mammaires non palpables.

The development of breast cancer screening has radically changed the diagnostic approach to breast lesions. Stereotactic large-core breast biopsy was developed to reduce the number of unnecessary surgical excisional biopsies in the increasing number of patients with doubtful or suspicious mammogram findings. The methods used to evaluate this new technique are discussed, as well as results in terms of efficacy, factors that may influence efficacy, and difficulties in interpreting large-core biopsies. Recommendations regarding the harvesting and interpretation of stereotactic large-core biopsies are made.

Obvious peritumoral emboli: an elusive prognostic factor reappraised. Multivariate analysis of 1320 node-negative breast cancers.

This study was conducted to determine the prognostic influence of obvious peritumoral vascular emboli as prospectively determined by a simple routine slide examination in patients with operable node-negative breast cancer. Obvious peritumoral emboli (OPE) were defined by the presence of neoplastic emboli within unequivocal vascular lumina (including both lymphatic spaces and blood capillaries) in areas adjacent to but outside the margins of the carcinoma. OPE were assessed routinely on 5 microns thick haematoxylin and eosin-stained sections for each of 1320 primary operable node-negative breast cancers from 1975 to 1992 at our institution. OPE and other prognostic variables (tumour size, SBR grade, oestrogen and progesterone receptor status) were correlated to overall survival (OS) and metastasis-free interval (MFI) by means of univariate and multivariate analysis with a median follow-up of 103 months. OPE were found in 19.5% of tumours. In univariate analysis, OPE were related to tumour size (P = 6.3 x 10(-5)) and histologic grade (P = 4.9 x 10(-7)). Statistically significant correlations were found with OS (P = 4.6 x 10(-5)) and MFI (P = 6.4 x 10(-9)). Furthermore, in multivariate analysis, OPE was an independent prognostic variable, the most predictive factor for MFI (P = 7.7 x 10(-7)) before tumour size and grade, and was second after tumour grade for OS (P = 0.002). This study on a large unicentric series and with a long follow-up confirms the prognostic significance of vascular emboli in patients with operable node-negative breast carcinoma. Importantly, vascular emboli were found to be accurately detectable by a simple routine and non-time-consuming method. Therefore, such obvious vascular emboli should be considered as an important cost-effective, prognostic variable in patients with node-negative breast carcinoma.