Inefficient differentiation response to cell cycle stress leads to genomic instability and malignant progression of squamous carcinoma cells.

Squamous cell carcinoma (SCC) or epidermoid cancer is a frequent and aggressive malignancy. However in apparent paradox it retains the squamous differentiation phenotype except for very dysplastic lesions. We have shown that cell cycle stress in normal epidermal keratinocytes triggers a squamous differentiation response involving irreversible mitosis block and polyploidisation. Here we show that cutaneous SCC cells conserve a partial squamous DNA damage-induced differentiation response that allows them to overcome the cell division block. The capacity to divide in spite of drug-induced mitotic stress and DNA damage made well-differentiated SCC cells more genomically instable and more malignant in vivo. Consistently, in a series of human biopsies, non-metastatic SCCs displayed a higher degree of chromosomal alterations and higher expression of the S phase regulator Cyclin E and the DNA damage signal γH2AX than the less aggressive, non-squamous, basal cell carcinomas. However, metastatic SCCs lost the γH2AX signal and Cyclin E, or accumulated cytoplasmic Cyclin E. Conversely, inhibition of endogenous Cyclin E in well-differentiated SCC cells interfered with the squamous phenotype. The results suggest a dual role of cell cycle stress-induced differentiation in squamous cancer: the resulting mitotic blocks would impose, when irreversible, a proliferative barrier, when reversible, a source of genomic instability, thus contributing to malignancy.

Childhood florid follicular hyperplasia with immunoglobulin light-chain restriction in the gastrointestinal tract.

AIMS: Immunoglobulin light-chain expression is used routinely as an indirect marker of clonality for recognizing B cell lymphoproliferative disorders. METHODS AND RESULTS: Here we describe four floral follicular hyperplasia cases in the gastrointestinal tract (appendix and rectum) of children (4 to 6 years). Immunohistochemical studies revealed lambda light-chain restriction that was associated with polyclonal IgH pattern. Clinical features and follow-up of the patients did not reveal any other systemic symptoms, laboratory abnormalities or organ alterations. CONCLUSIONS: Recognition of this phenomenon is useful in the diagnosis of nodular lymphoid hyperplasia of the gastrointestinal tract, for avoiding overdiagnosis of lymphoid malignancies, and raises concerns that the identification of light-chain restriction is not necessarily a marker of monoclonality.

NFκB expression is a feature of both activated B-cell-like and germinal center B-cell-like subtypes of diffuse large B-cell lymphoma.

The activation of nuclear factor kappa B (NFκB) transcription factor family is considered to have a key role in diffuse large B-cell lymphoma (DLBCL) pathogenesis and is associated with a specific molecular subtype, the activated B-cell-like (ABC) subtype. We evaluated the expression of NFκB by immunohistochemistry in a large series of DLBCL cases. The five different NFκB family members (NFκB1, NFκB2, RELA, RELB, and REL) showed a heterogeneous expression pattern with the vast majority of cases being positive for at least one factor. Two independent series of tumor samples were classified into germinal center B-cell-like (GCB) or ABC subtypes using different approaches, immunohistochemistry, or gene expression profiling, and the expression of NFκB family members was assessed. Notably, no significant differences regarding the expression of the different NFκB members were detected between the two subtypes, suggesting that NFκB signaling is a prominent feature not only in the ABC subtype, but also in the GCB tumors. Of the five transcription factors, only REL expression had a significant clinical impact on R-CHOP-treated diffuse large B-cell lymphoma, identifying a subgroup of patients with superior clinical outcome.

Persistent polyclonal B-cell lymphocytosis with splenomegaly: histologic description of 2 cases.

Persistent polyclonal B-cell lymphocytosis is a rare, benign lymphoproliferative disorder characterized by a stable, polyclonal CD19-positive CD5-negative lymphocytosis, the presence of binucleated lymphocytes in peripheral blood, and a polyclonal increase in serum immunoglobulin-M that may occasionally be accompanied by splenomegaly. Histopathologic diagnosis of these splenectomy specimens is difficult because of the massive spleen infiltration and the rarity of the descriptions of this condition. We describe the histopathologic findings from 2 splenectomy specimens. These included a partially preserved architecture with infiltration of the red pulp by small lymphocytes and partial replacement of the white pulp. Suggestions for identifying the disorder are made.

EBV-positive diffuse large B-cell lymphoma of the elderly is an aggressive post-germinal center B-cell neoplasm characterized by prominent nuclear factor-kB activation.

Here, we report a retrospective series of 47 EBV-positive diffuse large B-cell lymphoma associated with advanced age. Histopathology allowed to the identification of different histological patterns: cases with polymorphic diffuse large B-cell lymphoma (29 cases), Hodgkin-like (8 cases) and polymorphic lymphoproliferative disorder-like (9 cases) patterns. One case was purely monomorphic diffuse large B-cell lymphoma. We show that this lymphoma type is a neoplasm with prominent classical and alternative nuclear factor-kB pathway activation in neoplastic cells (79% of the cases showed nuclear staining for p105/p50, 74% for p100/p52 and 63% for both proteins), with higher frequency than that observed in a control series of EBV-negative diffuse large B-cell lymphoma (χ(2) <0.001). Most cases showed an activated phenotype (95% non-germinal center (Hans algorithm); 78% activated B cell (Choi algorithm)). Clonality testing demonstrated IgH and/or K/Kde/L monoclonal rearrangements in 64% of cases and clonal T-cell populations in 24% of cases. C-MYC (1 case), BCL6 (2 cases) or IgH (3 cases) translocations were detected by FISH in 18% cases. These tumors had a poor overall survival and progression-free survival (the estimated 2-year overall survival was 40 ± 10% and the estimated 2-year progression-free survival was 36 ± 9%). Thus, alternative therapies, based on the tumor biology, need to be tested in patients with EBV-positive diffuse large B-cell lymphoma of the elderly.

B-cell overexpression of Bcl-2 cooperates with p21 deficiency for the induction of autoimmunity and lymphomas.

Genetic abnormalities predisposing to autoimmunity generally act in a cooperative manner affecting one or several mechanisms regulating immunological tolerance. In addition, many of these genetic abnormalities are also involved in the development of lymphoproliferative diseases. In the present study, we have determined the possible cooperation between deficiencies in members of the Cip/Kip family of cell cycle regulators (p21(WAF1/Cip1) or p27(kip1)) and the overexpression of human Bcl-2 in B lymphocytes in the induction of autoimmune and lymphoproliferative diseases in non-autoimmune C57BL/6 (B6) mice. Unlike single mutant mice, B6.p21(-/-) mice transgenic for human Bcl-2 in B cells developed a lethal autoimmune syndrome characterized by the production of autoantibodies, the prominent expansion of memory B and CD4(+) T cells and the development of severe glomerular lesions resembling IgA nephropathy. Furthermore, these mice presented a high incidence of B-cell lymphoproliferative disorders. Such genetic cooperation in the induction of autoimmunity was not observed in B6.p27(-/-) mice transgenic for human Bcl-2 in B cells. Altogether, what we have demonstrated here is the existence of preferential interactions among particular regulators of the G(1)/S transition of the cell cycle and B-cell survival in the induction of systemic autoimmune and lymphoproliferative diseases.

Simultaneous cytomorphologic and multiparametric flow cytometric analysis on lymph node samples is faster than and as valid as histopathologic study to diagnose most non-Hodgkin lymphomas.

We evaluated the validity and accuracy of cytomorphology and multiparametric flow cytometry (C-FCM) in diagnosing oncohematologic disease in 223 consecutive lymph node biopsy specimens from patients with lymphadenopathy, from 2004 to 2007. C-FCM and histopathologic studies were interpreted independently by hematologists and pathologists, respectively. C-FCM detected neoplastic disorders in 133 samples (59.6%): 92 non-Hodgkin lymphomas (NHLs; 41.3%), 21 Hodgkin lymphomas (HLs; 9.4%), 19 malignant nonhematologic neoplasms (8.5%), and 1 multiple myeloma (0.4%). Sensitivity and specificity were 87.25% and 95.95%, respectively. Positive predictive value and negative predictive value (NPV) were 97.74% and 78.89%, respectively. Sensitivity and NPV were 94.79% and 96.81% upon excluding HL and malignant nonhematologic neoplasms from the analysis. Of the 92 NHLs, 89 (97%) were categorized according to the 2001 World Health Organization classification of hematolymphoid neoplasms with a concordance of 87%. The C-FCM study was significantly faster than the histopathologic study. C-FCM has high sensitivity and specificity, allowing for a valid and reliable diagnosis, especially in NHLs and enabling their subclassification. C-FCM is faster than the histopathologic examination, allowing for therapeutic decisions to be made quickly. However, in the samples in which C-FCM cannot establish a diagnosis, histopathologic results are needed.

A TaqMan low-density array to predict outcome in advanced Hodgkin's lymphoma using paraffin-embedded samples.

PURPOSE: Despite major advances in the treatment of classic Hodgkin's lymphoma (cHL), approximately 30% of patients in advanced stages may eventually die as result of the disease, and current methods to predict prognosis are rather unreliable. Thus, the application of robust techniques for the identification of biomarkers associated with treatment response is essential if new predictive tools are to be developed. EXPERIMENTAL DESIGN: We used gene expression data from advanced cHL patients to identify transcriptional patterns from the tumoral cells and their nonneoplastic microenvironment, associated with lack of maintained treatment response. Gene-Set Enrichment Analysis was used to identify functional pathways associated with unfavorable outcome that were significantly enriched in either the Hodgkin's and Reed-Sternberg cells (regulation of the G2-M checkpoint, chaperones, histone modification, and signaling pathways) or the reactive cell microenvironment (mainly represented by specific T-cell populations and macrophage activation markers). RESULTS: To explore the pathways identified previously, we used a series of 52 formalin-fixed paraffin-embedded advanced cHL samples and designed a real-time PCR-based low-density array that included the most relevant genes. A large majority of the samples (82.7%) and all selected genes were analyzed successfully with this approach. CONCLUSIONS: The results of this assay can be combined in a single risk score integrating these biological pathways associated with treatment response and eventually used in a larger series to develop a new molecular outcome predictor for advanced cHL.

Myeloid sarcoma of the extrahepatic bile ducts presenting as obstructive jaundice.

We report a rare case of myeloid sarcoma (MS) of the extrahepatic bile ducts presenting as obstructive jaundice in a patient without leukemia at time of diagnosis. A 75-year-old female presented with a one-month history of abdominal pain and jaundice. Computerized tomography scan of the abdomen showed stenosis of the extrahepatic bile ducts. Endoscopic retrograde cholangiography disclosed an irregular narrowing of the common biliary duct, suggestive of a cholangiocarcinoma, and resection was performed. Histologic examination showed diffuse transmural infiltration of malignant cells. These cells exhibited medium-sized round nuclei with central nucleoli and eosinophilic cytoplasm, and were strongly positive for myeloperoxidase, CD68, lysozyme, CD45, CD117 (c-kit protein) and CD43. Eight months following surgery the patient presented with multiple cutaneous nodules and bone marrow trephine biopsy showed acute myelomonocytic leukemia. A literature search identified two previously reported cases of MS of the extrahepatic biliary duct. MS should be taken into consideration in the differential diagnosis of a patient with obstructive jaundice. Immunohistochemistry is essential for a correct diagnosis.

Hodgkin and Reed-Sternberg cells harbor alterations in the major tumor suppressor pathways and cell-cycle checkpoints: analyses using tissue microarrays.

Tumoral cells in Hodgkin lymphoma (HL) display an increased growth fraction and diminished apoptosis, implying a profound disturbance of the cell cycle and apoptosis regulation. However, limitations of molecular techniques have prevented the analysis of the tumor suppressor pathways and cell-cycle checkpoints. Tissue microarray (TMA) is a powerful tool for analyzing a large number of molecular variables in a large series of tumors, although the feasibility of this technique has not yet been demonstrated in heterogeneous tumors. The expression of 29 genes regulating the cell cycle and apoptosis were analyzed by immunohistochemistry and in situ hybridization in 288 HL biopsies using TMA. The sensitivity of the technique was validated by comparing the results with those obtained in standard tissue sections. The results revealed multiple alterations in different pathways and checkpoints, including G1/S and G2/M transition and apoptosis. Striking findings were the overexpression of cyclin E, CDK2, CDK6, STAT3, Hdm2, Bcl2, Bcl-X(L), survivin, and NF-kappaB proteins. A multiparametric analysis identified proteins associated with increased growth fraction (Hdm2, p53, p21, Rb, cyclins A, B1, D3, and E, CDK2, CDK6, SKP2, Bcl-X(L), survivin, STAT1, and STAT3), and proteins associated with apoptosis (NF-kappaB, STAT1, and RB). The analysis also demonstrated that Epstein-Barr virus (EBV)-positive cases displayed a characteristic profile, confirming the pathogenic role of EBV in HL. Survival probability depends on multiple biologic factors, including overexpression of Bcl2, p53, Bax, Bcl-X(L), MIB1, and apoptotic index. In conclusion, Hodgkin and Reed-Sternberg cells harbor concurrent and overlapping alterations in the major tumor suppressor pathways and cell-cycle checkpoints. This appears to determine the viability of the tumoral cells and the clinical outcome.