Antonello Domenico Cabras

B-cell lymphomas with features intermediate between distinct pathologic entities. From pathogenesis to pathology

Published in September 2008, the updated World Health Organization Classification of Tumors of Hematopoietic and Lymphoid Tissues introduces provisional borderline categories for lymphoma cases that demonstrate overlapping clinical, morphological, and/or immunophenotypic features between well-established entities. These overlapping features pose real diagnostic challenges especially in identifying atypical cases of diffuse large B-cell lymphoma, Hodgkin lymphoma, and Burkitt lymphoma. Lymphoma cases showing borderline features between T-cell/histiocyte-rich large B-cell lymphoma and nodular lymphocyte predominant Hodgkin lymphoma are not included within the borderline categories provisionally recognized by the updated classification. Within the borderline categories, there are cases combining features of primary mediastinal large B-cell lymphoma and classical Hodgkin lymphoma. Many of these cases resemble classical Hodgkin lymphoma but have a large number of tumor cells expressing CD20, CD45, and B-cell transcription factors. Alternatively, these cases may resemble primary mediastinal large B-cell lymphoma but contain tumor cells resembling Reed-Sternberg cells and displaying an aberrant phenotype such as CD20(-), CD15(-/+) CD45(+), CD30(+), Pax5(+), OCT2(+/-), and BOB1(+/-). Another new borderline category defining B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, represents a biologically heterogeneous group. Cases with morphologic features intermediate and with CD10/BCL6 coexpression should be placed in diffuse large B-cell lymphoma/Burkitt lymphoma category if tumor cells also show strong BCL2 staining and/or a Ki67 proliferation index of less than 90%. When MYC rearrangements are present in these cases, the lymphomas often have atypical features, including concurrent rearrangements of BCL2 and/or BCL6 genes (so-called double/triple-hit lymphomas) and more aggressive behavior. For the provisional borderline categories, unresolved issues include understanding molecular pathogenesis and defining an effective treatment.

Species identification of mycobacteria in paraffin-embedded tissues: frequent detection of nontuberculous mycobacteria

Diagnosis of infections caused by mycobacteria, especially nontuberculous mycobacteria still represents a difficult task both in microbiology and pathology. The aim of this study was to determine the frequency of mycobacterial DNA detectable by PCR in formalin-fixed paraffin-embedded tissues showing suspicious granulomatous lesions. A total of 190 archival specimens were analyzed, using a nested PCR protocol, which amplifies a fragment of the mycobacterial 65-kDa heat-shock protein gene. Restriction fragment-length polymorphisms and sequencing were utilized to further analyze the obtained PCR products. Corresponding microbiological culture results were available for 41 cases. We detected mycobacterial DNA in 119 cases (63%), of which 71 (60%) were positive for Mycobacterium tuberculosis complex DNA and 41 (34%) for DNA of nontuberculous mycobacteria. Seven cases (6%) could not be subtyped for technical reasons. The largest group of nontuberculous mycobacteria comprised 29 cases (25% of the 119 positive cases), which were assigned to Mycobacterium fortuitum complex. Mycobacterium avium–intracellulare complex was detected in eight (7%) cases, Mycobacterium gordonae in three (2.5%) and Mycobacterium rhodesiae in a single case (0.8%). All cases of Mycobacterium tuberculosis were unequivocally identified by restriction fragment-length polymorphism analysis. In contrast, sequencing provided a gain of information over restriction fragment-length polymorphism analysis in 37% of the nontuberculous mycobacteria cases (15 of 41). Alignment studies on DNA of nontuberculous mycobacteria showed frequent sequence variations, supporting the existence of sequevars. Comparison of molecular data to available results of microbiological culture assays showed a good concordance of 83%. In conclusion, amplification and sequencing of the mycobacterial 65-kDa heat-shock protein gene is an excellent tool for species identification of mycobacteria, especially nontuberculous mycobacteria, in formalin-fixed paraffin-embedded tissues.

Is Tumour Angiogenesis a Prognostic Factor in Patients with Colorectal Cancer and No Involved Nodes

OBJECTIVEnTo examine a possible association between tumour angiogenesis and conventional prognostic variables and to assess the prognostic value of the variables examined in patients with colorectal cancer, with no involved nodes.nnnDESIGNnRetrospective study.nnnSETTINGnUniversity hospital, Italy.nnnSUBJECTSn119 patients who had had colorectal cancers resected for cure with no involved nodes between 1985-1990.nnnINTERVENTIONSnThe three microscopic fields with the most microvessels were identified by immunohistochemical techniques. 10 high-power fields in each area were used for the microvessel count and the mean values indicated the microvessel density.nnnMAIN OUTCOME MEASURESnCorrelation of microvessel density with conventional prognostic factors, recurrence rates, and survival.nnnRESULTSnThere was a significant correlation between microvessel density and sex, women having a higher density than men (p < 0.05), but no significant correlations between density and recurrence rates or survival. Multivariate analysis did not indicate that microvessel density had a prognostic role.nnnCONCLUSIONnMicrovessel density in colorectal cancer without involved nodes does not correlate with conventional prognostic factors and provides no prognostic information.

Serological identification of HSP105 as a novel non-Hodgkin lymphoma therapeutic target

We reported that the clinical efficacy of dendritic cell-based vaccination is strongly associated with immunologic responses in relapsed B-cell non-Hodgkin lymphoma (B-NHL) patients. We have now investigated whether postvaccination antibodies from responders recognize novel shared NHL-restricted antigens. Immunohistochemistry and flow cytometry showed that they cross-react with allogeneic B-NHLs at significantly higher levels than their matched prevaccination samples or nonresponders antibodies. Western blot analysis of DOHH-2 lymphoma proteome revealed a sharp band migrating at approximately 100 to 110 kDa only with postvaccine repertoires from responders. Mass spectrometry identified heat shock protein-105 (HSP105) in that molecular weight interval. Flow cytometry and immunohistochemistry disclosed HSP105 on the cell membrane and in the cytoplasm of B-NHL cell lines and 97 diagnostic specimens. A direct correlation between HSP105 expression and lymphoma aggressiveness was also apparent. Treatment of aggressive human B-NHL cell lines with an anti-HSP105 antibody had no direct effects on cell cycle or apoptosis but significantly reduced the tumor burden in xenotransplanted immunodeficient mice. In vivo antilymphoma activity of HSP105 engagement was associated with a significant local increase of Granzyme B(+) killer cells that very likely contributed to the tumor-restricted necrosis. Our study adds HSP105 to the list of nononcogenes that can be exploited as antilymphoma targets.

Herpesvirus 8-associated Penile Kaposi's Sarcoma in an Hiv-negative Patient: First Report Of A Solitary Lesion

&NA; Kaposis sarcoma is a neoplastic vascular lesion. Its form of onset is frequently disseminated, especially in HIV‐positive patients. Its association with the infection caused by a virus of the Epstein‐Barr family, human herpesvirus 8 (HHV‐8), has been recently demonstrated. In this article we discuss the unusual presentation of a solitary manifestation of Kaposis sarcoma on the penis of a 53‐year‐old HIV‐negative patient. Polymerase chain reaction analysis of the tumor tissue was positive for HHV‐8 in the tumor cells but not in the reactive stroma cells surrounding the tumor. The case is interesting for its unusual site of presentation, the young age of onset, the association with HHV‐8 infection, the HIV‐negative serology, and the benign course of the disease.

Oligoclonality of a "composite" gastric diffuse large B-cell lymphoma with areas of marginal zone B-cell lymphoma of the mucosa-associated lymphoid tissue type.

Abstract.We analyzed a case of oligoclonal Helicobacter pylori-associated, primary gastric diffuse large B-cell lymphoma (DLBCL) with areas of extranodal marginal zone B-cell lymphoma (MZBL) of mucosa-associated lymphoid tissue (MALT) type in a 61-year-old male patient. The patient had two separate tumors, in the corpus and in the antrum, each showing two morphologically distinct components (DLBCL and MZBL of MALT type). To determine the clonal relationship between the large- and small-cell components, we microdissected four samples from morphologically distinct tumor components at both tumor sites. PCR analysis revealed rearranged immunoglobulin heavy chain (IgH) genes of different sizes in three of the four microdissected samples. Cloning and sequencing of the PCR products displayed different IgH gene rearrangements in the two small cell components of the antrum and the corpus. A third genuinely differently rearranged IgH gene was found in the large-cell components of both antrum and corpus. Our results suggest that in primary gastric DLBCL with areas of MZBL of MALT type, the small-cell component may comprise more than one tumor clone. Furthermore, the large-cell component may evolve independently from coexisting MZBL.

Epstein-Barr virus-negative Hodgkin's lymphoma after mycosis fungoides: molecular evidence for distinct clonal origin.

The association of mycosis fungoides (MF) and Hodgkins lymphoma is a relatively frequent occurrence, but the potential clonal relationship of the two neoplasms is still controversial. We report a case of a patient with a history of MF in Clinical Stage 1A who developed retroperitoneal lymphadenopathy 9 years after the initial diagnosis of MF. A bone marrow biopsy obtained at this time showed nodular involvement by a mixed cellular infiltrate with large, atypical cells consistent with Hodgkin and Reed-Sternberg (RS) cells. These atypical cells were positive for CD30 and CD15 and did not express B- or T-cell markers. In addition, they lacked evidence of infection by Epstein-Barr virus, both by immunohistochemical staining for latent membrane protein 1 and by in situ hybridization for EBER1/2. The background population consisted mainly of small T cells without morphological or phenotypical signs of malignancy. Review of the skin biopsy obtained 9 years before showed the typical features of MF. Polymerase chain reaction analysis of the T-cell receptor γ-gene confirmed the presence of a clonal T-cell rearrangement in the skin specimen. The bone marrow biopsy, however, showed a polyclonal pattern both for the T-cell receptor γ-gene, as well as for immunoglobulin heavy chain genes. Isolation of RS cells stained for CD30 was performed by laser capture microdissection. Polymerase chain reaction analysis of several groups of RS cells showed a reproducible biallelic rearrangement of IgH genes, which was confirmed by cloning and sequencing of polymerase chain reaction products. To our knowledge, this is the first case in which a distinct clonal origin of MF and Hodgkins lymphoma arising in the same patient is clearly demonstrated, based on molecular analysis of microdissected RS cells.

Biclonality of Gastric Lymphomas

The pathogenesis and clonal evolution of gastric diffuse large B-cell lymphoma (DLBCL) and its relationship to extranodal marginal zone B-cell lymphoma (MZBL), mucosa-associated lymphoid tissue (MALT) type, are still controversial. The aim of this study was to establish the clonality of morphologically distinct areas of gastric lymphomas as well as their genetic relationship to each other. Six gastric lymphomas, consisting of two MZBL, MALT type, two DLBCL, and two “composite” lymphomas were subjected to laser capture microdissection and subsequent PCR-based amplification of the immunoglobulin heavy chain gene. One DLBCL showed a biclonal pattern of rearranged immunoglobulin heavy chain (IgH) genes of two different areas without evidence of a common origin. Two composite DLBCL with areas of extranodal MZBL, MALT type, were also biclonal and displayed different IgH gene rearrangements in the small-cell and in the large-cell components, respectively. Sequencing of the CDR3 region revealed unique VH-N-D and D-N-JH junctions, thus corroborating the presence of two genuinely distinct tumor clones in each of these three cases. In contrast, the remaining three gastric lymphomas (one DLBCL and two MZBL, MALT type) showed IgH gene rearrangements in which CDR3 regions were identical in the different tumor areas. Our results suggest that gastric DLBCL may be composed of more than one tumor cell clone. Further, DLBCL may not necessarily evolve by transformation of a low-grade lymphoma, but may also originate de novo. An ongoing emergence of new tumor clones may considerably hamper molecular diagnosis and follow-up of gastric DLBCL.

Using Q-RT-PCR to measure cyclin D1, TS, TP, DPD, and Her-2/neu as predictors for response, survival, and recurrence in patients with esophageal squamous cell carcinoma following radiochemotherapy

PurposeThe purpose of this study was to evaluate the use of thymidilate synthetase (TS), thymidilate phosphorylase (TP), dihydropyrimidin dehydrogenase (DPD), Her-2/neu, and cyclin D1 as predictors of therapy response, survival, and recurrence in patients with esophageal squamous cell carcinoma (ESCC) following radiochemotherapy.Materials and methodsTwenty-six patients with histologically proven intrathoracic, locally advanced ESCC (cT3, cN0/+, cM0) underwent preoperative, combined simultaneous radiochemotherapy followed by R0-transthoracic esophagectomy. Because R0 resection is the strongest known independent prognostic factor in this tumor entity, only R0-resected patients were included in this study. Pre-therapeutically taken, formalin-fixed, and paraffin-embedded tumor biopsies were used for laser-assisted microdissection of tumor cells and RNA extraction and subjected to real-time (TaqMan) quantitative reverse transcriptase-polymerase chain reaction (Q-RT-PCR).ResultsNo significant correlation between clinical or histopathological parameters and the relative gene expression of TS, TP, DPD, or Her-2/neu was observed. However, patients with relative cyclin D1 levels below the median gene expression did not reach median survival compared to the 19.9xa0months seen in patients with relative cyclin D1 gene expression above the median (Pu2009=u20090.02). Patients with low cyclin D1 levels experienced significantly less frequent recurrence of the tumor (20% versus 63%; Pu2009=u20090.006), and there was a significant difference in the recurrence-free interval (Pu2009=u20090.003).ConclusionsDespite the small number of investigated patients, our data seem to show that high levels of cyclin D1 measured by real-time Q-RT-PCR before neoadjuvant radiochemotherapy correlate significantly with patient survival, tumor recurrence, and recurrence-free-interval. Cyclin D1 might be useful in identifying patients at high risk of poor prognosis and suffering from recurrence after neoadjuvant radiochemotherapy treatment and R0 resection. Further investigations with a larger cohort are warranted.

Molecular diagnosis of a mycobacterium chelonae infection

A 23-year-old female presented with enlarged cervical lymph nodes, and a diagnosis of nonspecific lymphadenitis with formation of pyogranulomas was rendered. Despite an initial oral antibiosis and subsequent long-term intravenous and oral antibiosis under hospitalized conditions, the symptoms progressed. The lymph nodes became larger and then affected the cervical region bilaterally. Her general condition worsened, and an exanthema of the extremities accompanied by a reactive arthritis occurred. Serological assays of various viral and bacterial markers and blood cultures were negative. Application of a polymerase chain reaction (PCR) protocol allowing specific amplification of mycobacterial DNA revealed DNA of Mycobacterium chelonea in formalin-fixed, paraffin-embedded lymph node tissue. Sequencing of the PCR product showed a 97% homology with the known Mycobacterium chelonae sequence. Modification of the antibiotic therapy with clarithromycin, imipenem and amikacin resulted in a rapid regression of the symptoms. The clinical course, in combination with the difficulties in detecting the infectious agent, supports the usefulness of molecular pathological analyses specific for nontuberculous mycobacteria (NTM).