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Sequencing of genes encoding mitogen-activated protein kinase (MAPK) pathway proteins in colorectal cancer (CRC) has established as dogma that of the genes in a pathway only a single one is ever mutated. We searched for cases with a mutation in more than one MAPK pathway gene (co-mutations). Tumor tissue samples of all patients presenting with CRC, and referred between 01/01/2008 and 01/06/2015 to three French cancer centers for determination of mutation status of RAS/RAF+/-PIK3CA, were retrospectively screened for co-mutations using Sanger sequencing or next-generation sequencing. We found that of 1791 colorectal patients with mutations in the MAPK pathway, 20 had a co-mutation, 8 of KRAS/NRAS, and some even with a third mutation. More than half of the mutations were in codons 12 and 13. We also found 3 cases with a co-mutation of NRAS/BRAF and 9 with a co-mutation of KRAS/BRAF. In 2 patients with a co-mutation of KRAS/NRAS, the co-mutation existed in the primary as well as in a metastasis, which suggests that co-mutations occur early during carcinogenesis and are maintained when a tumor disseminates. We conclude that co-mutations exist in the MAPK genes but with low frequency and as yet with unknown outcome implications.

Cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of diseases primarily involving the skin that could have an aggressive course with circulating blood cells, especially in Sézary syndrome and transformed mycosis fungoides. So far, few CTCL cell lines have been adapted for in vivo experiments and their tumorigenicity has not been adequately assessed, hampering the use of a reproducible model for CTCL biological evaluation. In fact, both patient-derived xenografts and cell line xenografts at subcutaneous sites failed to provide a robust tool, because engraftment was dependent on mice strain and cell line subtype. Herein, we describe an original method of intrahepatic injection into adult NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ mice liver of both aggressive (My-La, HUT78, HH, MAC2A, and MAC2B) and indolent (FE-PD and MAC1) CTCL cell lines. Six of the seven CTCL cell lines were grafted with a high rate of success (80%). Moreover, this model provided a quick (15 days) and robust assay for in vivo evaluation of CTCL cell lines tumorigenicity and therapeutic response in preclinical studies. Such a reproducible model can be therefore used for further functional studies and in vivo drug testing., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: It can be useful to assess the NRAS mutation status in patients with metastatic melanoma because NRAS-activating mutations confer resistance to RAF inhibitors, and NRAS-mutated patients appear to be sensitive to mitogen-activated protein kinase (MEK) inhibitors., Objective: We aimed to assess the diagnostic accuracy of an immunohistochemistry (IHC) approach using a novel anti-NRAS (Q61R) monoclonal antibody on formalin-fixed paraffin-embedded tissue samples from patients with metastatic melanoma., Methods: We conducted a retrospective multicenter cohort study on 170 patients with metastatic melanoma. The automated IHC assay was performed using the SP174 clone, and compared with results of the molecular testing., Results: Evaluation of a test cohort with knowledge of the mutation status established a specific IHC pattern for the mutation. In the independent blinded analysis of the remaining cases, the anti-NRAS (Q61R) antibody accurately identified all NRAS Q61R-mutated tumors, and demonstrated 100% sensitivity and specificity., Limitations: Limitations include retrospective design and lack of multicenter interobserver reproducibility., Conclusion: The NRAS (Q61R) IHC assay is reliable and specific for the evaluation of the Q61R mutation status in metastatic melanoma and may be an alternative to molecular biology in evaluation of metastatic melanoma in routine practice., (Copyright © 2015 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.)

Introduction: Taking as a base our retrospective study of 2760 cases of cutaneous lymphoproliferations from the LYMPHOPATH and GFELC networks, we analyzed the doubtful and discordant cases between non-expert and expert pathologists, and the interest of clinicopathological confrontation., Material and Methods: We defined the main diagnostic difficulties presented by cutaneous lymphoproliferations. We then designed and tested the algorithms on 20 random cases with 20 pathologists, in order to be used by any pathologist (not necessarily specialised in dermatopathology)., Results: The problematic differential diagnoses most frequently encountered are the following: MF or reactive dermatose; lymphoma without any other precision or reactive infiltrate; small B cell lymphoproliferation: lymphoma or reactive infiltrate; phenotyping of large B cell lymphoproliferation. We also analyzed less common problematic differential diagnoses, on the grounds that they are over- or under- diagnosed. Our test had a 72% success rate among the 20 randomly tested cases. The use of several algorithms for the same case is possible., Discussion: Our study shows that an expert second-opinion is of interest in the area of cutaneous lymphoproliferations. A second opinion is useful for distinguishing a small B cell lymphoma from a HLR, and for defining a final diagnosis when the first pathologist doubts between lymphoma and reactive infiltrate. However, we demonstrate that for the problem MF or reactive dermatose, an initial clinicopathological confrontation produces more results than a second-opinion pathology review., Conclusion: This is the first study of cutaneous lymphoproliferations that, without excluding reactionary infiltrates, concentrates on doubtful and discordant diagnoses between non expert and expert pathologists, and which has produced tested diagnostic algorithms., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Importance: The activating mutation of MYD88 L265P is a frequent feature of primary cutaneous diffuse large B-cell lymphoma, leg-type (PCLBCL-LT), reported in up to 69% of the cases. Whether patients with MYD88 mutation display specific clinical and evolutive features has not been evaluated., Objective: To identify the clinical characteristics associated with MYD88 mutation, confirm its high prevalence, and evaluate its effect on prognosis in patients with PCLBCL-LT., Design, Setting, and Participants: A retrospective multicenter study was conducted using the medical records of patients from dermatology departments belonging to the French Study Group for Cutaneous Lymphomas. Sixty-one patients with a diagnosis of PCLBCL-LT made between 1988 and 2010 who were available for molecular study were included. Of these, 58 patients displaying interpretable results constituted the study group. Median follow-up was 33 months, and 39 patients (67%) were monitored until death., Main Outcomes and Measures: Clinical features (age, sex, number of skin lesions, tumor stage, and location as leg vs elsewhere), MYD88 mutation (allele-specific TaqMan polymerase chain reaction assay), treatment regimen, and outcome were recorded. Baseline characteristics and outcome were compared according to the status of MYD88., Results: The median age of the patients was 79 years, and 59% were female. Skin lesions were located on the leg in 76% of the cases. Thirty-four of 58 patients (59%) harbored the MYD88 L265P mutation. Patients had similar clinical characteristics at presentation regardless of their MYD88 status, except that those harboring the MYD88 mutation were older (P = .006) and had more frequent involvement of the leg (P = .008). Patients harboring the MYD88 mutation had 3- and 5-year-specific survival rates of 65.7% and 60.2% vs 85.4% and 71.7% in patients with the wild-type allele. The MYD88 mutation was significantly associated with shorter disease-specific survival in univariate (P = .03) and multivariate (odds ratio, 3.01; 95% CI, 1.03-8.78; P = .04) analysis. There was no significant difference between the groups in their treatment regimens. Considering overall survival, in univariate (P = .002) and multivariate (odds ratio, 2.94; 95% CI, 1.18-7.30; P = .02) analysis, MYD88 L265P mutation was an independent adverse prognostic factor., Conclusions and Relevance: This study confirms the high prevalence of MYD88 L265P mutation in PCLBCL-LT and shows its association with shorter survival. The clinical effect of MYD88 mutation activating the nuclear factor-κB pathway supports the use of targeted therapies at the time of relapse after conventional therapies.

Primary cutaneous follicle center lymphoma (PCFCL) is the most frequent cutaneous B-cell lymphoma. A 62-year-old man presented with a solitary indolent subcutaneous nodule for 3 years duration, without other abnormalities. Histological examination showed lymphoproliferation with a nodular growth pattern characterized by fibrous collagen bands surrounding nodules. The nodules were composed of medium-sized centrocytes admixed with many large multilobulated and lacunar cells without eosinophils or granulomatous aspect. Hodgkin-like cells were CD30+, CD15+, PAX5+, OCT2+, BOB1+, MUM1+, Ki67+, Bcl6+ and focally CD20+ and EMA-, CD79a-, Bcl2- and CD10-. The medium-sized cells were CD20+, CD79a+, Bcl2+, Bcl6+ and CD10+, enmeshed in a network of CD21-positive follicular dendritic cells. Epstein-Barr virus detection was negative. Interphase fluorescence in situ hybridization showed the absence of BCL2 or BCL6 rearrangement. In such a case, the presence of Hodgkin-like cells intermixed with the tumor population may result in a pitfall diagnosis of classical Hodgkin lymphoma (CHL). Differential diagnoses to be ruled out are secondary or primary skin localization of rather CHL, or systemic follicular lymphoma. Several clinical, radiological, histological, immunohistochemical and molecular arguments indicated the diagnosis of PCFCL. To our knowledge, this is the first report of PCFCL with Hodgkin-like cells., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The BRAF(V600E) mutation confers worse prognosis to metastatic colorectal cancer (mCRC) patients. In addition, this mutation has a negative predictive value for response to treatment with monoclonal antibodies against EGFR in patients with KRAS wild-type (wt) mCRC. The utility of immunohistochemistry (IHC) as an alternative approach for detection of BRAF(V600E) in the thoracic metastases of sporadic mCRC patients has not been evaluated until now. The purpose of this study was to compare BRAF(V600E) IHC staining with molecular biology methods and to define the diagnostic value of the VE1 antibody for the detection of BRAF(V600E) in this population. BRAF mutations were analysed by two DNA sequencing methods (pyrosequencing and Sanger sequencing) in a Caucasian population of 310 sporadic mCRC with thoracic metastases patients expressing KRAS wt. Detection of the BRAF(V600E) mutation was performed in the corresponding tumours by IHC using the VE1 antibody and compared to results of the DNA-based assays. Thirty-nine out of 310 (13%) of tumours harboured a BRAF mutation, which corresponded to either a BRAF(V600E) in 34 of 310 (11%) cases or a non-BRAF(V600E) mutation in 5 of 310 (2%) cases. IHC with VE1 was strongly positive in 32 of 34 (88%) BRAF(V600E) mutated tumours and negative in non-BRAF(V600E) mutated tumours. IHC using the VE1 clone is a specific and sensitive method for the detection of BRAF(V600E) and may be either a complementary or an alternative method to molecular testing in mCRC patients.

Introduction: Anaplastic lymphoma kinase (ALK) rearrangements occur in 1% to 7% of non-small-cell lung cancers (NSCLCs). Crizotinib, an ALK inhibitor, has been demonstrated to provide dramatic clinical benefits in ALK-positive advanced-stage NSCLC. Fluorescent in situ hybridization (FISH) has been established in clinical trials as the standard procedure method for detecting ALK rearrangements. Although the detection of ALK by immunohistochemistry (IHC) has been proposed for the screening of patients, large-scale studies are warranted to validate such a hierarchical approach., Methods: In this article, we report the largest series thus far of parallel FISH and IHC ALK testing in 3244 consecutive NSCLC cases analyzed at two independent French centers., Results: FISH-positive and/or IHC-positive results were demonstrated in 150 of 3244 cases (4.6%). An imbalanced sex ratio was detected, with women exhibiting a 2.2-fold relative risk for an alteration. Strikingly, only 80 of 150 specimens were classified as ALK positive by both techniques. The specimens with discordant FISH/IHC analyses were FISH-positive/IHC-negative (36), FISH-negative/IHC-positive (19), or FISH-noncontributive/IHC-positive (15). Thus, a single FISH or IHC analysis performed alone would have failed to detect approximately one-fourth of the ALK-positive cases with similar findings in our two centers., Conclusions: This study highlights the feasibility of systematic NSCLC testing by both FISH and IHC in routine practice. Many preanalytical factors may account for the apparent discrepancies between both methods, suggesting that hierarchical screening may underscore ALK-positive cases. This significant level of discrepancy supports the need of combined testing to optimize the detection of ALK-inhibitor-eligible patients given that some patients with discordant testing were found to respond to crizotinib.

Primary cutaneous large B-cell lymphoma, leg type has been individualized from nodal diffuse large B-cell lymphoma. The objective of this study was to screen primary cutaneous large B-cell lymphoma, leg type for genetic alterations recently described in nodal diffuse large B-cell lymphoma. Skin biopsies from 23 patients were analyzed for IRF4, BCL2, BCL6, and MYC expression. FISH testing was performed for BCL2, BCL6, MYC with separation probes and for CDKN2A and PRDM1/BLIMP1 deletion. Multiple sequential FISH analyses with up to six probes were performed to define samples with multiple cytogenetic alterations. MYD88 mutations were studied by Sanger sequencing. All cases but one displayed at least one genetic alteration (96%). Nine patients exhibited a single genetic mutation and 12 combined several alterations (52%). We observed a split for BCL2, BCL6, or MYC in 1/23, 6/23, and 3/23 of cases, respectively. No double-hit lymphoma was observed. CDKN2A deletion was detected by FISH in only 5/23 cases. BLIMP1 and/or 6q deletion was observed at a higher rate in 10/20 of cases. No correlation between rearrangement and immunohistochemical expression was found for BCL2 or MYC. FISH tracking of sequential hybridizations showed that several alterations were carried by the same nuclei. The p.L265P MYD88 mutation was found in 11/18 (61%) of cases. Contrary to most cutaneous lymphomas that rarely harbor primary genetic alteration of their nodal histological equivalent, primary cutaneous large B-cell lymphoma, leg type seems to be a 'cutaneous counterpart' of activated B-cell-like diffuse large B-cell lymphoma with a similar cytogenetic profile and a high rate of MYD88 oncogenic L265P mutation. This also suggests a common lymphomagenesis with NF-κB activation, strong IRF4 expression and terminal B-cell differentiation blockage. Our data support the use of therapies targeting NF-κB, as most patients displayed disease progression and resistance to conventional therapies.

STAT3 protein phosphorylation is a frequent event in various hematologic malignancies and solid tumors. Acquired STAT3 mutations have been recently identified in 40% of patients with T-cell large granular lymphocytic leukemia, a rare T-cell disorder. In this study, we investigated the mutational status of STAT3 in a large series of patients with lymphoid and myeloid diseases. STAT3 mutations were identified in 1.6% (4 of 258) of patients with T-cell neoplasms, in 2.5% (2 of 79) of patients with diffuse large B-cell lymphoma but in no other B-cell lymphoma patients (0 of 104) or patients with myeloid malignancies (0 of 96). Functional in vitro assays indicated that the STAT3Y640F mutation leads to a constitutive phosphorylation of the protein. STA21, a STAT3 small molecule inhibitor, inhibited the proliferation of two distinct STAT3 mutated cell lines. Using a mouse bone marrow transplantation assay, we observed that STAT3Y640F expression leads to the development of myeloproliferative neoplasms with expansion of either myeloid cells or megakaryocytes. Together, these data indicate that the STAT3Y640F mutation leads to constitutive activation of STAT3, induces malignant hematopoiesis in vivo, and may represent a novel therapeutic target in some lymphoid malignancies.

Purpose: Rapid advances in the understanding of cancer biology have transformed drug development thus leading to the approval of targeted therapies and to the development of molecular tests to select patients that will respond to treatments. KRAS status has emerged as a negative predictor of clinical benefit from anti-EGFR antibodies in colorectal cancer, and anti-EGFR antibodies use was limited to KRAS wild type tumors. In order to ensure wide access to tumor molecular profiling, the French National Cancer Institute (INCa) has set up a national network of 28 regional molecular genetics centers. Concurrently, a nationwide external quality assessment for KRAS testing (MOKAECM) was granted to analyze reproducibility and costs., Methods: 96 cell-line DNAs and 24 DNA samples from paraffin embedded tumor tissues were sent to 40 French laboratories. A total of 5448 KRAS results were collected and analyzed and a micro-costing study was performed on sites for 5 common methods by an independent team of health economists., Results: This work provided a baseline picture of the accuracy and reliability of KRAS analysis in routine testing conditions at a nationwide level. Inter-laboratory Kappa values were >0.8 for KRAS results despite differences detection methods and the use of in-house technologies. Specificity was excellent with only one false positive in 1128 FFPE data, and sensitivity was higher for targeted techniques as compared to Sanger sequencing based methods that were dependent upon local expertise. Estimated reagent costs per patient ranged from €5.5 to €19.0., Conclusion: The INCa has set-up a network of public laboratories dedicated to molecular oncology tests. Our results showed almost perfect agreements in KRAS testing at a nationwide level despite different testing methods ensuring a cost-effective equal access to personalized colorectal cancer treatment.

Introduction: The prescription of some anti-cancer therapies is now based on the detection of specific genetic alterations that should be determined as early as possible not to put patients at a disadvantage. In 2009, the 'Aquitaine platform of molecular tumour genetics' (PGMC) developed a programme to evaluate and to improve the organisation of molecular cancer analyses, particularly the analysis of the KRAS gene. The objective was to describe the analysis process, the organization of pathology laboratories and the delays between the different phases of the process., Methods: We established a working group to describe the different steps between the prescription of molecular biology analyses and the analysis report. A retrospective study based on the first quarter of 2009 allowed us to measure management delays. In addition, a pathology laboratory organisational questionnaire allowed us to identify organisational features hindering rapid delivery., Results: The median delay between the analysis prescription and the results was 15 days (range: 7-78 days). Practices explaining longer delays were highlighted not only in the pathology laboratories (for example, pending of the prescription before sending the analysis, waiting for several cases before sending the material, sample slicing, sending by standard mail), but also within the PGMC (for example, sample testing by another technique or new extraction for non-contributory samples)., Conclusion: The results of this study emphasise the necessity of speeding up pre-analytical phases, and of creating an electronic procedure and regional facilities in order to provide results more rapidly to clinicians., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Objectives: In Africa, lymphomas are widely represented by pediatric Burkitt lymphomas. In Abidjan, cytology performed after needle biopsy may be an examination of choice for pediatric lymphomas because of its low cost. We evaluated the value of this cytological examination in comparison with MYC rearrangement assessment., Patients and Methods: A cytological examination was performed after needle biopsy of masses suspected for lymphoma. The reliability of this cytological examination was assessed versus a cytogenetic technique of fluorescence in situ hybridization with a probe targeting locus 8q24 (MYC) which is recurrently rearranged in Burkitt lymphomas., Results: Thirty-four patients were enrolled in this study. The median age was 8 years old. The main locations of the suspicious masses were maxillofacial and abdominal. Thirty cytological examinations identified cytological aspects of Burkitt lymphoma. Among these cases, the cytogenetic technique identified 24 cytogenetic rearrangements compatible with a translocation involving MYC as described in Burkitt lymphoma. Six cytological examinations identified cytological aspects of Burkitt lymphoma without MYC translocation. Two cytological examinations were not compatible with Burkitt lymphoma and a normal MYC status was observed. Two cytological examinations were technically not contributive., Conclusions: The cytological examination showed good performance, notably with excellent sensitivity. The cytological examinations compatible with a Burkitt lymphoma without MYC translocation (6/30=20.0%) could be explained by the absence of translocation involving locus 8q24 (MYC) in some endemic Burkitt lymphomas., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Some melanocytic tumors are ambiguous, so the reproducible histopathological diagnosis of benign or malignant lesion is difficult. This study evaluated the contribution of fluorescence in situ hybridization (FISH) first in 43 non-equivocal melanomas and nevi, and then in 113 ambiguous melanocytic tumors selected by expert pathologists from six different European institutions. We included two groups of ambiguous tumors: patients without recurrence (5-year minimal follow-up) and with metastases. An independent triple-blind histopathological review was performed to classify tumors as 'favor benign' (A-) or 'favor malignant' (A+). A four-color probe set targeting 6p25, 6q23, 11q13 and CEP6 was used for FISH. In the 43 non-equivocal melanomas and nevi, sensitivity was 85% and specificity 90%. Ninety out of 95 ambiguous melanocytic tumors included were FISH interpretable (67 FISH negative and 23 FISH positive). Of the 90 patients, 69 presented no recurrence and 21/90 exhibited metastases. These ambiguous tumors were mostly spitzoid tumors (45/90). Histopathological reviewers classified these tumors as favor malignant (49/90) and favor benign (32/90), whereas nine cases had a discordant diagnosis. By comparison with outcome, the sensitivity and specificity of histopathological review were 95 and 52%, and the sensitivity and specificity of FISH were 43 and 80%. Compared with histopathological review, the sensitivity and specificity of FISH were 34.5 and 91%. Interestingly, by combining the histopathological diagnosis with FISH results, the diagnosis was optimized, especially by increasing specificity (76% instead of 52% for expert diagnosis alone) and by improving sensitivity compared with FISH alone (90 vs 43% for FISH result alone). The value of this FISH test is to add a reproducible demonstration of malignancy to the histopathological diagnosis, especially in doubtful/ambiguous melanocytic tumors. A positive FISH test reinforces the diagnosis of melanoma, allowing such tumors (particularly thick tumors) to be managed as melanomas.

Inactivation of the CDKN2A-CDKN2B locus has been reported in the most frequent subtypes of cutaneous T-cell lymphomas (CTCLs), mycosis fungoides, Sézary syndrome (SS) and CD30+ cutaneous anaplastic large cell lymphoma. To investigate whether genetic or epigenetic inactivation of CDKN2A-CDKN2B is more specifically observed in certain CTCL subtypes with clinical impact, we used array-comparative genomic hybridization, quantitative PCR, interphase fluorescent in situ hybridization and methylation analyses of p14(ARF) p16(INK4A) and p15(INK4B) promoters. We studied 67 samples from 58 patients with either transformed mycosis fungoides (n=24), SS (n=16) or CD30+ cutaneous anaplastic large cell lymphoma (n=18). We observed combined CDKN2A-CDKN2B deletion in both transformed mycosis fungoides (n=17, 71%) and SS patients (n=7, 44%), but, surprisingly, in only one CD30+ cutaneous anaplastic large cell lymphoma case. Interphase fluorescent in situ hybridization showed 9p21 loss in 17 out of 19 cases, with 9p21 deletion indicating either hemizygous (n=4) or homozygous (n=2) deletion, with mixed patterns in most patients (n=11). The limited size of 9p21 deletion was found to account for false-negative detection by either BAC arrays (n=9) or fluorescent in situ hybridization (n=2), especially in patients with Sézary syndrome (n=6). Methylation was found to be restricted to the p15(INK4B) gene promoter in patients with or without 9p21 deletion and did not correlate with prognosis. In contrast, CDKN2A-CDKN2B genetic loss was strongly associated with a shorter survival in CTCL patients (P=0.002) and more specifically at 24 months in transformed mycosis fungoides and SS patients (P=0.02). As immunohistochemistry for p16(INK4A) protein was not found to be informative, the genetic status of the CDKN2A-CDKN2B locus would be relevant in assessing patients with epidermotropic CTCLs in order to identify those cases where the disease was more aggressive.

The tyrosine kinase JAK3 plays a well-established role during normal lymphocyte development and is constitutively phosphorylated in several lymphoid malignancies. However, its contribution to lymphomagenesis remains elusive. In this study, we used the newly identified activating JAK3A572V mutation to elucidate the effect of constitutive JAK3 signaling on murine lymphopoiesis. In a bone marrow transplantation model, JAK3A572V induces an aggressive, fatal, and transplantable lymphoproliferative disorder characterized by the expansion of CD8(+)TCRalphabeta(+)CD44(+)CD122(+)Ly-6C(+) T cells that closely resemble an effector/memory T-cell subtype. Compared with wild-type counterparts, these cells show increased proliferative capacities in response to polyclonal stimulation, enhanced survival rates with elevated expression of Bcl-2, and increased production of interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha), correlating with enhanced cytotoxic abilities against allogeneic target cells. Of interest, the JAK3A572V disease is epidermotropic and produces intraepidermal microabscesses. Taken together, these clinical features are reminiscent of those observed in an uncommon but aggressive subset of CD8(+) human cutaneous T-cell lymphomas (CTCLs). However, we also observed a CD4(+) CTCL-like phenotype when cells are transplanted in an MHC-I-deficient background. These data demonstrate that constitutive JAK3 activation disrupts T-cell homeostasis and induces lymphoproliferative diseases in mice.

Aims: The goal of this work was to evaluate the impact of expert pathological second opinion on the diagnosis and management of patients with cancer, in a French region (Aquitaine) and with an economic point of view., Material and Methods: The study was first quantitative, performed retrospectively on all cases of cancer, voluntary sent for a second opinion to an expert pathologist of two centers. Secondly, we restricted the study to lymphoid, melanocytic and soft tissue tumors sent for second opinion. We considered that the expert review had an important diagnostic impact either when the initial pathologist sent the specimen to identify or classify malignant tumor or hesitated between benign and malignant tumor or had no hypothesis, or if there were discordant diagnoses (malignant/benign) between the two pathologists. We considered that the expert review had a high therapeutic impact if the disagreement between initial and expert diagnoses induced a complete modification in therapy. We evaluated the cost of second opinion for the expert centers and the cost of care management., Results: Over the year 2006, the expert centers received 5077 lesions for consultation: 3769 specimens were sent by a pathologist for a second review, 1324 by pathologists of Aquitania and of these, 751 samples were submitted for lymphoid (55%), soft tissues (30%) or melanocytic tumors (15%). There was an important diagnostic impact for 75% of the samples; the impact of the expert review on patient management was considered high for 46% of specimens and the expert pathological diagnosis modified the clinical prognosis for 40% of the specimens. We estimated that for 53 discordant diagnoses (malignant/benign), second opinion allowed an economy of 500,000 euro., Conclusion: Expert second opinion is very important not only for diagnosis and management for patient with cancer but also for economic reasons.

The mapping of a balanced t(12;18)(q21;q21.2) translocation in a Sézary syndrome (SS) case led Karenko et al. to identify NAV3 gene (12q21-22) deletion by interphase fluorescence in situ hybridization (FISH) in 15/21 patients with mycosis fungoides (MF) or SS. To determine whether the NAV3 deletion is the result of a specific gene breakpoint, we used FISH with dual-color split or break-apart bacterial artificial chromosome (BAC) probes covering the NAV3 locus. A total of 31 samples (18 skin, 11 blood, 1 lymph node, and 1 spleen) from 24 patients with advanced MF/SS (18 with large-cell transformation) were studied. Chromosome 12 imbalances were analyzed by comparative genomic hybridization (CGH) array with a 3K BAC probes in 24 samples from 22 patients. Both normal FISH and CGH array patterns were observed in 22 samples from 18 patients. In 6 patients, abnormal patterns were observed with an abnormal number of chromosome 12 set in 5 of them. Chromosome 12 structural abnormalities were seen in four of these six patients. An imbalanced FISH pattern between NAV3 and pericentromeric control probes was seen in three patients in accordance with CGH array data (one with a pericentromeric deletion and two with a large 12q deletion including NAV3). No NAV3 specific breakpoint or partial deletion was detected.

Background: Cytogenetic analysis of classical Hodgkin's lymphoma is limited by the low content of the neoplastic Hodgkin-Reed-Sternberg cells in the affected tissues. However, available cytogenetic data point to an extreme karyotype complexity. To obtain insights into chromosomal imbalances in classical Hodgkin's lymphoma, we applied array-based comparative genomic hybridization (array comparative genomic hybridization) using DNA from microdissected Hodgkin-Reed-Sternberg cells., Design and Methods: To avoid biases introduced by DNA amplification for array comparative genomic hybridization, cHL cases rich in Hodgkin-Reed-Sternberg cells were selected. DNA obtained from approximately 100,000 microdissected Hodgkin-Reed-Sternberg cells of each of ten classical Hodgkin's lymphoma cases was hybridized onto commercial 105 K oligonucleotide comparative genomic hybridization microarrays. Selected imbalances were confirmed by interphase cytogenetics and quantitative polymerase chain reaction analysis and further studied in an independent series of classical Hodgkin's lymphoma., Results: Gains identified in at least five cHL affected 2p12-16, 5q15-23, 6p22, 8q13, 8q24, 9p21-24, 9q34, 12q13-14, 17q12, 19p13, 19q13 and 20q11 whereas losses recurrent in at least five cases involved Xp21, 6q23-24 and 13q22. Copy number changes of selected genes and a small deletion (156 kb) of the CDKN2B (p15) gene were confirmed by interphase cytogenetics and polymerase chain reaction analysis, respectively. Several gained regions included genes constitutively expressed in cHL. Among these, gains of STAT6 (12q13), NOTCH1 (9q34) and JUNB (19p13) were present in additional cHL with the usual low Hodgkin-Reed-Sternberg cell content., Conclusions: The present study demonstrates that array comparative genomic hybridization of microdissected Hodgkin-Reed-Sternberg cells is suitable for identifying and characterizing chromosomal imbalances. Regions affected by genomic changes in Hodgkin-Reed-Sternberg cells recurrently include genes constitutively expressed in cHL.

The World Health Organization-European Organization for Research and Treatment of Cancer has individualized three main categories among the primary cutaneous B cell lymphoma (PCBCL): leg-type primary cutaneous large B cell lymphoma (PCLBCL leg type), primary cutaneous follicle center lymphoma (PCFCL), and primary cutaneous marginal zone lymphoma (PCMZL). The genetic features of 21 PCBCL cases (six PCLBCL leg type four PCFCL large cells, seven PCFCL small cells, and four PCMZL) were investigated by comparative genomic hybridization (CGH array). Fluorescent in situ hybridization (FISH) analysis was performed to confirm CGH array data and to detect lymphoma-associated gene rearrangements. p14(ARF)/p16(INK4a) CDKN2A gene quantification, methylation analysis, and immunohistochemical detection were also performed. CGH array showed a higher number of recurrent genetic imbalances in PCLBCL leg type (mean 62) than in PCFCL large cells (mean 34). PCFCL small cells and PCMZL exhibited fewer chromosomal alterations (mean 24 and 9). FISH analysis provided concordant results with CGH array data in 97% (98 of 101) assays and demonstrated a t(8;14)(q24;q32) in two of six PCLBCL leg type. Recurrent deletions in 9p21 (p14(ARF)/p16(INK4a)CDKN2A) were a constant finding in PCLBCL leg type (six of six). Conversely, PCFCL large cells exhibited recurrent 1p36 deletions (four of four) without deletion in 9p21 (zero of four). The diagnostic and prognostic impact of the p16(INK4a)CDKN2A gene status in PCBCL should therefore be confirmed on a larger series.

We report the case of a 71-year-old woman presenting with composite lymphoma (CL) composed of a follicular lymphoma and a B-cell chronic lymphocytic leukemia. CL is a rare lymphoproliferative disorder, characterized by two distinct morphological and immunophenotypical patterns in the same anatomical site, most frequently of biclonal origin. This entity must be distinguished from transformation of low-grade lymphoma into high-grade lymphoma and from lymphoma with differentiation such as follicular lymphoma with marginal differentiation. In this context, molecular analysis including immunoglobulin rearrangement, sequencing and FISH analyses is determinant and can be improved by tissue microdissection. Routinely, CL must not be misdiagnosed because of its prognosis and treatment implication.

We report a case of lymphomatoid granulomatosis (LG), arising in a 60-year-old man in the setting of an acute myeloid leukemia. LG is a rare Epstein Barr virus (EBV) lymphoproliferative disorder, generally occurring in a context of immunodeficiency. Patients usually present with respiratory symptoms and bilateral pulmonary nodules. Histologically LG is characterized by an angiocientric and angiodestructive lymphoproliferation of B/EBV+ cells admixed with numerous reactive T cells. The differential diagnosis mainly includes pulmonary vasculitis and Hodgkin's lymphoma. The outcome of this lymphoproliferation is highly variable, ranging from an indolent process to an aggressive large cell lymphoma.

The persistence of gastric lymphoma after Helicobacter pylori eradication may be difficult to evidence on endoscopic and histological examination. The aims of the study were to evaluate the detection of monoclonal immunoglobulin H (IgH) gene rearrangement in endoscopically infiltrated and normal mucosa at diagnosis and during follow-up in order to determine its clinical and prognostic impact. We studied 60 gastric marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT), and IgH monoclonality was detected at diagnosis in 52 patients (87%). The endoscopically normal mucosa contained clonal lymphomatous cells in 69% of cases before remission. A complete histological remission (HR) was observed in 28 patients (47%). Among them, 23 were followed for molecular remission (MR). The median delay was 10 months to achieve HR and 18 months to achieve MR. Interestingly, patients with HR but not MR had a longer delay to achieve HR (21 months) (P=0.0006) and a more frequent clonal normal mucosa at diagnosis (88%) than patients with both HR and MR (10 months and 39%, respectively). The presence of monoclonal B cells at both infiltrated and normal sites may therefore identify patients with a longer delay to achieve complete response, suggesting that molecular dissemination may require therapeutic intensification.

Congenital mesoblastic nephroma (CMN) is a rare renal tumor of early infancy with a favorable outcome after complete surgical removal. CMN consists of a heterogeneous group of spindle cell tumors subdivided into "classical", "cellular or atypical" and "mixed" forms based on histologic features. We describe a new case of cellular CMN diagnosed by antenatal ultrasonography with complete remission five years after nephrectomy. Cytogenetic study evidenced a trisomy 11, and real time RT-PCR, but not conventional karyotype, allowed for the detection of the Tel-ETV6/TrkC-NTRK3 fusion transcript as a consequence of a cryptic t(12-15)(p13;q25). As in congenital fibrosarcoma (CFS), two Tel-ETV6/ TrkC-NTRK3 fusion transcripts different by a 42 bp insert in the TrkC kinase domain were expressed. Our observations outline the close links between cellular CMN and CFS. Both tumors have the clinical presentation and histologic features as well as identical cytogenetic and molecular markers in common. Therefore, they are likely to represent the same neoplasm, but occurring at different locations.

Most of the primary thyroid malignant lymphomas have been considered of mucosa-associated lymphoid tissue (MALT) type and arise from lymphocytic thyroiditis. We report an uncommon case of a 67-yr-old man who underwent total thyroidectomy for multinodular goiter with tracheal compression. At histopathologic examination, we discovered a minute (3-mm diameter) lesion of low-grade thyroid lymphoma of MALT type without any lymphocytic thyroiditis lesion on 33 section levels of the entire thyroid gland. No general inflammatory, autoimmune, or lymphomatous disorder has been evidenced both at staging and after 30 mo of follow-up. MALT-type low-grade lymphoma may, in some instances, develop de novo within the thyroid without an antecedent MALT-type lymphoma.

We describe an interphase FISH analysis for formol-fixed, paraffin-embedded tissue sections with commercial probes detecting the t(11;14)(q13;q32) in mantle cell lymphoma and the t(8;14)(q24;q32) in Burkitt's lymphoma. Staining of an adjacent section allowed identification of tumoral areas. The cut-off value was evaluated in reactive lymph nodes at 5% for both probes. An incomplete hybridization pattern was found in 18 to 26% of the nuclei but was always lower than the percentages of translocated cells in tumoral regions. A t(11;14) was detected in 4/4 mantle cell lymphomas and a t(8;14) in 2/3 Burkitt's lymphomas. Interphase FISH analysis of fixed-tissue sections is a reliable technique for the direct detection of lymphoma-associated translocations on routine histological material.