Martina Prochazkova-Carlotti

Fluorescence in situ hybridization, a diagnostic aid in ambiguous melanocytic tumors: European study of 113 cases.

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.

Human Bone Marrow-Derived Stem Cells Acquire Epithelial Characteristics through Fusion with Gastrointestinal Epithelial Cells

Bone marrow-derived mesenchymal stem cells (MSC) have the ability to differentiate into a variety of cell types and are a potential source for epithelial tissue repair. Several studies have demonstrated their ability to repopulate the gastrointestinal tract (GIT) in bone marrow transplanted patients or in animal models of gastrointestinal carcinogenesis where they were the source of epithelial cancers. However, mechanism of MSC epithelial differentiation still remains unclear and controversial with trans-differentiation or fusion events being evoked. This study aimed to investigate the ability of MSC to acquire epithelial characteristics in the particular context of the gastrointestinal epithelium and to evaluate the role of cell fusion in this process. In vitro coculture experiments were performed with three gastrointestinal epithelial cell lines and MSC originating from two patients. After an 8 day coculture, MSC expressed epithelial markers. Use of a semi-permeable insert did not reproduce this effect, suggesting importance of cell contacts. Tagged cells coculture or FISH on gender-mismatched cells revealed clearly that epithelial differentiation resulted from cellular fusion events, while expression of mesenchymal markers on fused cells decreased over time. In vivo cell xenograft in immunodeficient mice confirmed fusion of MSC with gastrointestinal epithelial cells and self-renewal abilities of these fused cells. In conclusion, our results indicate that fusion could be the predominant mechanism by which human MSC may acquire epithelial characteristics when in close contact with epithelial cells from gastrointestinal origin . These results could contribute to a better understanding of the cellular and molecular mechanisms allowing MSC engraftment into the GIT epithelium.

IRF4 Gene Rearrangements Define a Subgroup of CD30-Positive Cutaneous T-Cell Lymphoma: A Study of 54 Cases

The identification of IFN regulatory factor 4 gene (IRF4) translocation in 8 out of 14 cases of cutaneous anaplastic large cell lymphomas (C-ALCLs) (Leukemia, 2009; 23(3):574-80) prompted us to study IRF4 locus status in different cutaneous T-cell lymphoma (CTCL) subtypes. Fluorescence in situ hybridization (FISH) was used with break-apart dual-color probes. Sixty samples from 54 patients were analyzed with 23 C-ALCL, 11 transformed mycosis fungoides (T-MF), 7 lymphomatoid papulosis (LyP), and 13 Sézary syndrome (SS) cases. IRF4 immunostaining was performed in 32 cases. We observed a split FISH signal pattern indicating a translocation at the IRF4 locus in 6 out of 23 C-ALCL (26%) and 2 out of 11 T-MF (18.2%) cases. Extra copies of the IRF4 locus were found in 4 out of 13 SS, 1 out of 11 T-MF, and 1 out of 23 C-ALCL cases, corresponding to either aneuploidy, chromosome 6 trisomy, or 6p partial gain. The IRF4 expression was not correlated with the presence of IRF4 alteration in C-ALCL or T-MF. Interestingly, IRF4 rearrangements define a subgroup of CD30-positive C-ALCL and T-MF cases. Conversely, T-MF cases with IRF4 rearrangements may correspond to the development of C-ALCL in MF patients and not to large cell transformation. Investigation of the biological pathways triggered by IRF4 rearrangements and/or expression in CTCL will provide a biological basis for IRF4-directed therapy.

Multiple genetic alterations in primary cutaneous large B-cell lymphoma, leg type support a common lymphomagenesis with activated B-cell-like diffuse large B-cell lymphoma

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.

Assessment of diagnostic criteria between primary cutaneous anaplastic large‐cell lymphoma and CD30‐rich transformed mycosis fungoides; a study of 66 cases

Transformed mycosis fungoides (TMF) large cells may express CD30 antigen, and because of this, the differential diagnosis between CD30‐rich TMF and primary cutaneous anaplastic large‐cell lymphoma (cALCL) may be difficult, and especially in distinguishing cALCL associated with MF vs. CD30‐rich TMF.

Diagnostic and Prognostic Value of BCL2 Rearrangement in 53 Patients With Follicular Lymphoma Presenting as Primary Skin Lesions

OBJECTIVES To study the diagnostic value of BCL2 rearrangement in follicle center lymphoma (FCL) presenting as primary skin lesions, evaluate its prevalence and the prognostic value in primary cutaneous FCL (PCFCL), and assess prognostic factors in PCFCL. METHODS Fifty-three patients with a cutaneous presentation of FCL without a history of nodal lymphoma were selected retrospectively. Clinical and histologic data were collected together with staging and follow-up data. A fluorescence in situ hybridization (FISH) test for BCL2 split probes was performed on skin biopsy specimens. RESULTS Initial staging procedures identified 47 PCFCLs and six cases of secondary skin involvement of FCL (SSIFCL). FISH detected seven cases carrying a BCL2 rearrangement: four (8.5%) of 47 PCFCLs and three (50%) of six SSIFCLs. These seven cases coexpressed BCL2 and CD10. In PCFCL, cutaneous relapse rate was 42.6%. A small/medium centrocytic cell population was associated with a higher probability of skin relapse in univariate (P = .008) and multivariate (P = .028) analysis, and BCL2 rearrangement detection was associated with secondary extracutaneous spreading (P = .05). CONCLUSIONS We observed that BCL2 rearrangement in PCFCL is rare, associated with initial positivity of staging (diagnostic value) or with secondary extracutaneous spreading (prognostic value). In selected cases with BCL2-CD10 coexpression, FISH testing could detect patients with poor outcome and require closer monitoring.

Molecular alterations and tumor suppressive function of the DUSP22 (Dual Specificity Phosphatase 22) gene in peripheral T-cell lymphoma subtypes

Monoallelic 6p25.3 rearrangements associated with DUSP22 (Dual Specificity Phosphatase 22) gene silencing have been reported in CD30+ peripheral T-cell lymphomas (PTCL), mostly with anaplastic morphology and of cutaneous origin. However, the mechanism of second allele silencing and the putative tumor suppressor function of DUSP22 have not been investigated so far. Here, we show that the presence, in most individuals, of an inactive paralog hampers genetic and epigenetic evaluation of the DUSP22 gene. Identification of DUSP22-specific single-nucleotide polymorphisms haplotypes and fluorescence in situ hybridization and epigenetic characterization of the paralog status led us to develop a comprehensive strategy enabling reliable identification of DUSP22 alterations. We showed that one cutaneous anaplastic large T-cell lymphomas (cALCL) case with monoallelic 6p25.3 rearrangement and DUSP22 silencing harbored exon 1 somatic mutations associated with second allele inactivation. Another cALCL case carried an intron 1 somatic splice site mutation with predicted deleterious exon skipping effect. Other tested PTCL cases with 6p25.3 rearrangement exhibited neither mutation nor deletion nor methylation accounting for silencing of the non-rearranged DUSP22 allele, thus inactivated by a so far unknown mechanism. We also characterized the expression status of four DUSP22 splice variants and found that they are all silenced in cALCL cases with 6p25.3 breakpoints. We finally showed that restoring expression of the physiologically predominant isoform in DUSP22-deficient malignant T cells inhibits cellular expansion by stimulating apoptosis and impairs soft agar clonogenicity and tumorigenicity. This study therefore shows that DUSP22 behaves as a tumor suppressor gene in PTCL.

The Cytolethal Distending Toxin Subunit CdtB of Helicobacter hepaticus Promotes Senescence and Endoreplication in Xenograft Mouse Models of Hepatic and Intestinal Cell Lines

Cytolethal distending toxins (CDTs) are common among pathogenic bacteria of the human and animal microbiota. CDTs exert cytopathic effets, via their active CdtB subunit. No clear description of those cytopathic effects has been reported at the cellular level in the target organs in vivo. In the present study, xenograft mouse models of colon and liver cell lines were set up to study the effects of the CdtB subunit of Helicobacter hepaticus. Conditional transgenic cell lines were established, validated in vitro and then engrafted into immunodeficient mice. After successful engraftment, mice were treated with doxycyclin to induce the expression of transgenes (red fluorescent protein, CdtB, and mutated CdtB). For both engrafted cell lines, results revealed a delayed tumor growth and a reduced tumor weight in CdtB-expressing tumors compared to controls. CdtB-derived tumors showed γ-H2AX foci formation, an increase in apoptosis, senescence, p21 and Ki-67 nuclear antigen expression. No difference in proliferating cells undergoing mitosis (phospho-histone H3) was observed. CdtB intoxication was also associated with an overexpression of cytokeratins in cells at the invasive front of the tumor as well as an increase in ploidy. All these features are hallmarks of endoreplication, as well as aggressiveness in cancer. These effects were dependent on the histidine residue at position 265 of the CdtB, underlying the importance of this residue in CdtB catalytic activity. Taken together, these data indicate that the CdtB triggers senescence and cell endoreplication leading to giant polyploid cells in these xenograft mouse models.

Proliferative Nodules vs Melanoma Arising in Giant Congenital Melanocytic Nevi During Childhood

Importance The differential diagnosis between proliferative nodules (PNs) and melanoma arising in congenital melanocytic nevi (CMN) is crucial, as patients with PNs most often experience no increased risk of melanoma with metastases and death. Objective To analyze the utility of immunohistochemistry and fluorescence in situ hybridization (FISH) in distinguishing PNs from childhood and adult-onset melanoma arising in CMN. Design, Setting, and Participants A case series was conducted from June 29, 1989, to November 12, 2009, of 13 children with PNs arising in CMN in childhood and 5 children with melanomas arising in CMN in childhood. Five patients with giant CMN with no nodules were included as negative controls, and 6 patients with melanomas arising in CMN in adulthood were included as positive controls. Follow-up ranged from 3 to 21 years in all children (mean, 9.9 years) and from 3 months to 7 years in adults. Specimens were selected for immunohistochemistry and FISH. All histopathologic sections were reviewed by 2 dermatopathologists who examined all nodules arising at different ages in the same patient and, in the case of melanoma, all locations. Data analysis was performed from January 1, 2013, to January 31, 2015. Main Outcomes and Measures The ability to distinguish melanoma from PN using immunohistochemistry and/or FISH. Results Of the 13 patients (5 boys and 8 girls) with PNs present at birth, all PNs were stable (mean follow-up, 9 years). Eight patients with PNs and 4 of 5 patients with childhood-onset melanoma showed homogeneous staining for HMB45, while heterogeneous staining for HMB45 was seen in 3 of 6 patients with adult-onset melanoma. Expression of p16 was strongly positive in most patients with childhood-onset PNs (10 of 11 patients) and melanoma (all patients) but negative in 4 patients with adult-onset melanoma. Patients with PNs and the 5 patients with childhood-onset melanoma had numerical chromosomal aberrations never observed in the adjacent CMN. The 2 children with FISH-positive PNs are melanoma free after 7 and 4 years. Only 1 patient with childhood-onset melanoma had a FISH aberration compared with 4 patients with adult-onset melanoma. Conclusions and Relevance Immunohistochemistry and the 4-probe FISH melanoma analysis are not useful for distinguishing PN from childhood-onset melanoma as opposed to adult-onset melanoma. Numerical anomalies seen in PNs but not in the adjacent CMN could be the result of a chromosomal segregation malfunction resulting in the development of nodules.

Evaluation of Quantitative Fluorescence in situ Hybridization for Relative Measurement of Telomere Length in Placental Mesenchymal Core Cells

Background: Reduced telomere length in placental mesenchymal core cells has been reported during pregnancies complicated by intrauterine growth restriction. To estimate telomere length, a precise, accurate and reproducible technique must be used. Objective: We evaluated the characteristics of a quantitative fluorescence in situ hybridization (Q-FISH) technique for measuring relative telomere length in placental mesenchymal core cells. Methods: From late chorionic villus samplings, telomere length in placental mesenchymal core cells was estimated by a Q-FISH technique using peptide nucleic acid telomere probes. The main characteristics of the Q-FISH technique, such as precision and reproducibility, were evaluated. Results: The telomere length of the cultured placental mesenchymal cells did not follow a normal distribution. When the Q-FISH technique was performed on interphase nuclei of uncultured mesenchymal core cells, normal telomere length distribution was observed. The precision of the technique when applied to cultured placental mesenchymal core cells was estimated to be <6%, and its reproducibility ranged from to 92.9 to 104.7%. Conclusion: Our results showed that cell culture of placental villi produced a non-normal telomere length distribution, probably related to telomere DNA replication during the cell cycle. Despite the influence of cell culture, the Q-FISH technique reported herein showed good precision and reproducibility.