Membrane cholesterol is essential for triterpenoid saponin augmentation of a saporin-based immunotoxin directed against CD19 on human lymphoma cells.

Triterpenoid saponins from Saponinum Album (SA) exert potent lytic effects on eukaryotic cell plasma membranes and, when used at sub-lytic concentrations, significantly augment the cytotoxicity of saporin-based immunotoxins (IT). To help elucidate the mechanism(s) behind these two phenomena we investigated the role of cholesterol to both. Human Daudi lymphoma cells were lipid deprived using a combination of three different approaches. Following treatment, the total cellular lipid content was analyzed by electrospray ionization mass spectrometry (ESI-MS) and plasma membrane (PM) cholesterol content measured using the lipophilic fluorescent probe NR12S. Maximal lipid deprivation of cells resulted in a complete loss of sensitivity to lysis by SA. Similarly augmentation of the anti-CD19 immunotoxin (IT) BU12-SAPORIN by SA was lost but without a concomitant loss of intrinsic IT cytotoxicity. The lytic activity of SA was restored following incubation of lipid deprived Daudi cells with Synthecol or LDL. The augmentative effect of SA on IT cytotoxicity for Daudi cells was restored following repletion of PM cholesterol levels with LDL. NR12S fluorescence and ESI-MS analysis of cellular lipids demonstrated that restoration of SA lytic activity by Synthecol was entirely due to increased PM cholesterol levels. Restoration of cellular and PM cholesterol levels by LDL also restored the augmentative effect of SA for IT, an effect associated with repletion of PM cholesterol with minor changes in some phospholipid species. These results indicate that the lytic and IT augmentative properties of SA are cholesterol-dependent in contrast to intrinsic IT cytotoxicity that is at least partially cholesterol independent.

Antibacterial and Cytotoxic Phenolic Metabolites from the Fruits of Amorpha fruticosa.

Fourteen new natural products, namely, 2-[(Z)-styryl]-5-geranylresorcin-1-carboxylic acid (1), amorfrutin D (2), 4-O-demethylamorfrutin D (3), 8-geranyl-3,5,7-trihydroxyflavanone (4), 8-geranyl-5,7,3'-trihydroxy-4'-methoxyisoflavone (5), 6-geranyl-5,7,3'-trihydroxy-4'-methoxyisoflavone (6), 8-geranyl-7,3'-dihydroxy-4'-methoxyisoflavone (7), 3-O-demethyldalbinol (8), 6a,12a-dehydro-3-O-demethylamorphigenin (9), (6aR,12aR,5'R)-amorphigenin (10), amorphispironones B and C (11 and 12), resokaempferol 3-O-ß-d-glucopyranosyl-(1→2)-ß-d-glucopyranoside-7-O-α-l-rhamnopyranoside (13), and daidzein 7-O-ß-d-glucopyranosyl-(1→2)-ß-d-glucopyranoside (14), together with 40 known compounds, were isolated from the fruits of Amorpha fruticosa. The structures of the new compounds were elucidated by 1D and 2D NMR spectroscopic analysis as well as from the mass spectrometry data. ECD calculations were performed to determine the absolute configurations of 11 and 15. Compounds 1, 4-6, and 16-23 showed potent to moderate antibacterial activities against several Gram-positive bacteria with MIC values ranging from 3.1 to 100 µM. In addition, compounds 11 and 24-33 were significantly cytotoxic against the L5178Y mouse lymphoma cell line and exhibited IC50 values from 0.2 to 10.2 µM.

Hematolymphoid lesions of the breast.

Hematolymphoid malignancies of the breast are most commonly neoplasms of mature B-lymphocytes, and may arise as a primary disease or by secondary involvement of a systemic disease. Primary breast lymphomas (PBL) account for 0.04-0.5% of breast malignancies, less than 1% of all non-Hodgkin's lymphomas (NHL), and less than 5% of extranodal lymphomas (Lakhani et al., 2012; Swerdlow et al., 2008; Joks et al., 2011; Barista et al., 2000; Giardini et al., 1992; Brogi and Harris, 1999; Topalovski et al., 1999).1-7 Secondary breast lymphomas (SBL) are also rare, with an estimated annual incidence of 0.07% (Domchek et al., 2002; Talwalkar et al., 2008).8,9 Recognition of breast lesions as hematolymphoid is critical to distinguish them from other entities that can occur in the breast.

Hematolymphoid lesions of the sinonasal tract.

Various hematolymphoid lesions involve the sinonasal tract, including aggressive B, T, and NK-cell neoplasms; myeloid sarcoma; low-grade lymphomas; indolent T-lymphoblastic proliferations; and Rosai-Dorfman disease. Differentiating aggressive lymphomas from non-hematopoietic neoplasms such as poorly differentiated squamous cell carcinoma, olfactory neuroblastoma, or sinonasal undifferentiated carcinoma may pose diagnostic challenges. In addition, the necrosis, vascular damage, and inflammatory infiltrates that are associated with some hematolymphoid disorders can result in misdiagnosis as infectious, autoimmune, or inflammatory conditions. Here, we review hematolymphoid disorders involving the sinonasal tract including their key clinical and histopathologic features.

Primary CNS lymphoma.

PURPOSE OF REVIEW: Despite recent progress, the diagnosis of primary CNS lymphoma (PCNSL) remains a challenge and is often delayed by several months. Treatment options are still debated and the prognosis of PCNSL lymphoma is poor for most patients. This review will describe recent progress and future orientations for diagnosis of PCNSL and report on the recent trends regarding therapeutic options. RECENT FINDINGS: PCNSL must be suspected in cases of chronic posterior uveitis, especially in patients over 50 years old. Diagnosis is based on cytology and molecular analysis of clonality of vitreous samples. Intraocular interleukin (IL)-10 level has proved to be a valuable tool for screening purposes in cases where there is a suspicion of primary vitreoretinal lymphoma. Intraocular cytokine dosage could also be a useful marker to follow the therapeutic response of patients with PCNSL. Treatment of PCNSL remains under debate. SUMMARY: Diagnosis of PCNSL is challenging. Suspicion relies on clinical history and on IL-10 and IL-6 levels in ocular fluid samples. Definite diagnosis is based on cytology and molecular analysis of clonality. New diagnostic and prognostic markers are currently evaluated. Whether isolated vitreoretinal lymphoma should be treated locally or with systemic treatment remains highly controversial.

A highly curable lymphoma occurs preferentially in the proximal tibia of young patients.

The presentation of two 19-year-old male subjects with stage I non-Hodgkin lymphoma in the proximal tibia prompted an extensive review of institutional and national databases to assess whether there is any statistical evidence that these reflected a previously overlooked syndromic pattern of presentation. The institutional records of a single institution were reviewed for presentation of non-Hodgkin lymphoma in the bone. The records of two additional institutions were reviewed for all reports of non-Hodgkin lymphoma in the tibia. Analysis was performed on data from Surveillance, Epidemiology, and End Results (SEER) dichotomized to bone presentation in the lower extremity versus other bones. Institutional databases included 20 patients with tibial presentation of lymphoma with a median age of 22.5 years (versus 42 for all bone lymphomas; P<0.001). Eighteen out of twenty patients had diffuse large B-cell lymphoma, and all patients aged ≤40 achieved remission and apparent cure. Distinctive and unusual features were a tendency for bilateral involvement of the tibia and sclerotic changes on X-ray. SEER data included 808 cases of bone lymphoma; the fraction of cases presenting in the lower extremity versus other bone sites is higher at ages ≤40 years (38% versus 19%; P<0.0001). Presentation in the lower extremity, as compared with other bone sites, confers 97% overall survival in patients aged ≤40 (versus 82%; P=0.01). This survival effect was independent of stage. In contrast, no significant difference in overall survival was identified for lower extremity versus non-lower extremity site for age >40. These data show a previously undescribed syndromic pattern of disease presentation: bone lymphoma in young patients is likely to present in the lower extremity-specifically the proximal tibia-has atypical sclerotic features on X-ray, is often bilateral, and has an excellent prognosis compared with bone lymphomas at other sites matched for stage and age.

Association of renal tubular hyaline droplets with lymphoma in CD-1 mice.

In mice, hyaline droplets in renal proximal tubules have been associated with histiocytic sarcoma but have not been reported with lymphoma. Tissues from CD-1 mice in a 2-year carcinogenicity bioassay were examined microscopically. Twenty-five mice with hyaline droplets in renal tubules were identified. Immunohistochemistry to detect IgA, IgG, IgM, lysozyme, albumin, CD3, and CD79a was performed on kidneys of 21 affected mice. Hyaline droplets were present in the kidneys of 11 mice with lymphoma (1 male, 10 female), of which 1 female also had histiocytic sarcoma. Hyaline droplets were also present in 7 other mice with histiocytic sarcoma, 2 with chronic progressive nephropathy, 3 with renal cortical tubular necrosis, and 2 with granulocytic leukemia. Five of the 11 lymphomas were CD3+, indicating a T lymphocyte origin. Hyaline droplets in mice with lymphoma did not stain for IgA, IgG, or IgM, except in one questionable case. Results of other immunohistochemical stains were inconclusive. Although the droplet composition could not be determined immunohistochemically, the study findings indicate that renal tubular hyaline droplets may be associated with lymphoma in mice.

Short telomeres initiate telomere recombination in primary and tumor cells.

Human tumors that lack telomerase maintain telomeres by alternative lengthening mechanisms. Tumors can also form in telomerase-deficient mice; however, the genetic mechanism responsible for tumor growth without telomerase is unknown. In yeast, several different recombination pathways maintain telomeres in the absence of telomerase-some result in telomere maintenance with minimal effects on telomere length. To examine non-telomerase mechanisms for telomere maintenance in mammalian cells, we used primary cells and lymphomas from telomerase-deficient mice (mTR-/- and Emumyc+mTR-/-) and CAST/EiJ mouse embryonic fibroblast cells. These cells were analyzed using pq-ratio analysis, telomere length distribution outliers, CO-FISH, Q-FISH, and multicolor FISH to detect subtelomeric recombination. Telomere length was maintained during long-term growth in vivo and in vitro. Long telomeres, characteristic of human ALT cells, were not observed in either late passage or mTR-/- tumor cells; instead, we observed only minimal changes in telomere length. Telomere length variation and subtelomeric recombination were frequent in cells with short telomeres, indicating that length maintenance is due to telomeric recombination. We also detected telomere length changes in primary mTR-/- cells that had short telomeres. Using mouse mTR+/- and human hTERT+/- primary cells with short telomeres, we found frequent length changes indicative of recombination. We conclude that telomere maintenance by non-telomerase mechanisms, including recombination, occurs in primary cells and is initiated by short telomeres, even in the presence of telomerase. Most intriguing, our data indicate that some non-telomerase telomere maintenance mechanisms occur without a significant increase in telomere length.

[Second expert review on lymphoma: assessment of a one year activity in a general hospital]. / Expertise systématique des lymphomes diagnostiqués au cours d'une année d'activité d'un centre hospitalier : évaluation.

A standardized second histological review for lymphomas was established by the French National Cancer Institute in 2010. The objective of our study was to assess the clinical impact of this process between a general hospital (reader 1) and an expert (reader 2). This prospective study was conducted between April 1st 2010 and April 1st 2011. Fifty-four cases of lymphoma were subjected to an expert review following the "LYMPHOPATH" recommendations and diagnoses of readers 1 and 2 were compared according to the WHO 2008 classification of lymphomas. We distinguished serious discrepancies (lymphoma versus other malignancy) from subtyping disagreement with or without impact on therapeutic strategy. We also determined the delays between the initial reception of the sample and reader 1's (period A) and reader 2's (period B) reports, respectively. Any additional analysis performed by second reader was also reported. Our study revealed one case of subtyping discordance (1.85%). The mean delays were 7 days for period A and 20 days for period B, respectively. Additional immunohistochemical techniques were requested by reader 2 in 11 cases (20.4%). These data provide evidence to suggest that in our department, a second review targeted on difficult diagnoses, rare lymphomas or when further analyses are required would be more relevant than a standardized second review.

Molecular biology and cytopathology. Principles and applications.

Some of the main applications of molecular techniques using cellular materials obtained from tumors by means of non-gynecological exfoliative cytology or fine-needle aspiration are briefly described in this review.

STAT6 and phosphorylated STAT6 are differentially expressed in lymphomas.

BACKGROUND: The STAT6 pathway is implicated in the pathogenesis of various lymphomas; however, its immunohistochemical expression has not been fully investigated. Thus, the aim of this study was to investigate the immunohistochemical expression of the two forms of STAT6, phosphorylated or not, in a series of systemic lymphomas. MATERIALS AND METHODS: Immunohistochemical expression of two antibodies, STAT6 (clone YE361) and pSTAT6 (clone Y641), which recognise the phosphorylated form of the molecule was studied in 60 lymphomas. RESULTS: STAT6YE361 expression was cytoplasmic, with 23.3% of the cases showing high expression. pSTAT6Y641 expression was mostly nuclear and found in 45% of the cases. pSTAT6Y641 nuclear expression was associated with the lymphoma type (p < 0.0001), as it was seen mostly in follicular, Hodgkin and angioimmunoblastic T cell lymphomas. STAT6YE361 cytoplasmic expression was also associated with lymphoma type (p = 0.001), as it was mostly found in diffuse large B cell and marginal B cell lymphomas. No association with PD-L1 expression, other clinicopathological data or prognosis was found. CONCLUSION: The two STAT6 clones are differentially expressed between lymphoma types.

Attempting to distinguish between endogenous and contaminating cytokeratins in a corneal proteomic study.

BACKGROUND: The observation of cytokeratins (CK's) in mass spectrometry based studies raises the question of whether the identified CK is a true endogenous protein from the sample or simply represents a contaminant. This issue is especially important in proteomic studies of the corneal epithelium where several CK's have previously been reported to mark the stages of differentiation from corneal epithelial stem cell to the differentiated cell. METHODS: Here we describe a method to distinguish very likely endogenous from uncertain endogenous CK's in a mass spectrometry based proteomic study. In this study the CK identifications from 102 human corneal samples were compared with the number of human CK identifications found in 102 murine thymic lymphoma samples. RESULTS: It was anticipated that the CK's that were identified with a frequency of <5%, i.e. in less than one spot for every 20 spots analysed, are very likely to be endogenous and thereby represent a 'biologically significant' identification. CK's observed with a frequency >5% are uncertain endogenous since they may represent true endogenous CK's but the probability of contamination is high and therefore needs careful consideration. This was confirmed by comparison with a study of mouse samples where all identified human CK's are contaminants. CONCLUSIONS: CK's 3, 4, 7, 8, 11, 12, 13, 15, 17, 18, 19, 20 and 23 are very likely to be endogenous proteins if identified in a corneal study, whilst CK's 1, 2e, 5, 6A, 9, 10, 14 and 16 may be endogenous although some are likely to be contaminants in a proteomic study. Further immunohistochemical analysis and a search of the current literature largely supported the distinction.

Immunocytochemistry and the diagnosis of cutaneous lymphoma.

Cutaneous lymphoid infiltrates may pose some of the most difficult diagnostic problems in dermatopathology. Immunocytochemistry is often employed in an effort to determine whether an infiltrate is neoplastic or, in the case of clearly malignant infiltrates, to provide a specific diagnosis. The rarity of these disorders and the variant immunocytochemical profiles they may present further thwart understanding and sometimes prevent an accurate diagnosis. In this review the common immunocytochemical profiles of various cutaneous lymphomas are presented and potential pitfalls and problems considered. Immunocytochemistry is not a diagnostic test but, as in other areas of histopathology, is a highly valuable tool that requires critical interpretation within a context: so applied, it is an indispensable part of the pathologist's arsenal in evaluating lymphoid infiltrates and defining different lymphomas.

Cutaneous metastases: a study of 78 biopsies from 69 patients.

A retrospective study of all cases of cutaneous metastases received in the Department of Pathology during the last 13 years was performed. Seventy-eight cases from 69 patients were examined. The majority of the patients were between 60 and 80 years of age. The most frequent anatomical location for the metastases was the abdomen. Regarding the primary tumor, breast carcinoma was the most common. In females breast carcinoma was the most common, while lymphomas predominated in males. In 14 patients (17.95%), the origin of the primary tumor was unknown. In all these cases, there was no clinical suspicion of metastasis. Epidermotropism was found in only 9 out of 78 cases (11.54%). Contrary to this, vascular invasion was a common feature, being present in 49 out of 78 cases (62.82%). Necrosis was evident in 38 cases (48.72%), but it was either moderate or prominent only in 8 cases (10.26%). Inflammatory infiltrate was absent in 3 cases (3.85%), mild in 49 (62.82%), moderate in 20 (25.64%), and prominent (graded either 3 of 4 or 4 out of 4) in 5 cases (6.41%). The metastases involved exclusively the dermis in 26 cases (33.33%), hypodermis in 7 cases (8.97%), and dermis and hypodermis in 44 cases (56.41%). We concluded that vascular invasion is a common feature in cutaneous metastases, especially in nonhematologic neoplasms, and because sometimes is focal, it should be carefully searched.

As the world turns, evolving lymphoma classifications-past, present and future.

The last century and a half has seen first the recognition of lymphomas, and then the publication of one lymphoma classification after another often together with highly critical comments about preceding classifications or a welcome that was less than warm. The introduction of HUMAN PATHOLOGY in 1970 came just before one of the very acrimonious periods in lymphoma classification, as we were learning more about the normal immune system and with the proposed functional lymphoma classifications of Lukes/Collins and Kiel in 1974 relating the lymphomas to their normal B-cell or T-cell 'counterparts'. Those difficult times were followed by the regressive strictly morphologic NCI Working Formulation in 1982, with the REAL classification in 1994 putting us back on a rational path, once again grouping the lymphoid neoplasms first into those of B-cell and T- and putative NK-cell origin, and then using multiple parameters to define specific entities. Planning for the first modern WHO lymphoma classification began soon afterward, with concordance and collegiality leading to the 2001 WHO classification, which then evolved with publication of the 2008 and 2016 WHO classifications. While this review looks at these important past developments which have gotten us to where we are today, it also concentrates on where we are now, what has been learned since the most recent WHO classification and 'Blue Book' were published and on some of the unanswered questions that remain as we look to the future.

Orbital soft tissue composite lymphoma presenting as recurrence of a nodal lymphoma with mantle and follicular cell components: A case report, literature review and guideline for the treatment of patients.

Composite lymphoma with mantle and follicular cell components is a challenging diagnosis. Flow cytometry, immunohistochemistry and molecular genetics are required to distinguish the two components, as often the more aggressive one is predominant and masks the other. A 58-year-old man with history of nodal composite lymphoma presented with right exophthalmos and diplopia. A head CT scan showed an orbital tumor. A biopsy of the tumor revealed a mantle cell lymphoma predominating over a follicular lymphoma. Immunoglobulin heavy chain and light chain rearrangements analysis by PCR proved that both components of the orbital tumor were recurrences of the same nodal composite lymphoma diagnosed two years earlier. The nodal lymphoma was composed of a follicular lymphoma and an in situ mantle cell neoplasia. Consensus view is that dominant lymphoma should be treated when needed but taking into account if the mantle cell lymphoma is an in situ neoplasia and if it expresses CD5 and SOX11.

Using an RNA aptamer probe for flow cytometry detection of CD30-expressing lymphoma cells.

Aptamers are small molecular ligands composed of short oligonucleotides that bind targets with high affinity. In contrast to antibodies, as synthetic oligonucleotides, aptamers have lower production costs and elicit no antigenic reactions. Therefore, aptamers are potential agents for disease diagnosis and treatment. In this study, we validate a fluorescently labeled RNA aptamer, which has been reported to bind specifically to mouse CD30 proteins in solution, for human CD30 protein recognition on intact cells. The aptamer probe was tested with cultured anaplastic large cell lymphoma and Hodgkin's lymphoma cells that express high levels of CD30. Flow cytometry and fluorescence microscopy showed specific and sensitive binding of the aptamer probe to CD30-expressing lymphoma cells at low concentrations (0.3 nM). Studies performed on multiple cell lines and nuclear cells from healthy donors confirmed that the CD30 aptamer and anti-CD30 antibody, the standard clinical probe, recognized the same set of cells. The potential application of multicolor flow cytometry analysis using the CD30 aptamer probe and antibodies was also shown. In conclusion, the developed CD30 aptamer probe could act as a replacement and/or a supplement for antibodies in the diagnosis of the CD30-expressing lymphomas.

Distribution of malignant lymphoma in Japan: analysis of 2260 cases, 2001-2006.

The World Health Organization classification was used to conduct an analysis of geographic, age, sex, and lesion primarily biopsied/resected distribution of 2260 lymphoid neoplasms diagnosed during 2001-2006 throughout Japan. B-cell neoplasms accounted for 65% of all lymphoid neoplasms, T/natural killer (T/NK)-cell neoplasms for 25% and Hodgkin lymphoma for 7%. The most common type was diffuse large B-cell lymphoma (DLBCL, 33%), followed by follicular lymphoma (18%), and adult T-cell leukemia/lymphoma (ATLL, 10%). The high rate of 18% for follicular lymphoma was similar to that in Western countries (11-33%). T/NK-cell neoplasms accounted for a higher percentage of lymphoid neoplasms in Kyushu (30%) and Okinawa (38%) compared with other areas of Japan (18-20%). Among T/NK-cell neoplasms, ATLL was the most common type in Okinawa (54%) and Kyushu (59%). Extranodal NK/T cell lymphoma was the second most common type of T/NK-cell neoplasms in Okinawa (15%). This epidemiological study shows that the distribution patterns of malignant lymphoma differ especially in Kyushu and Okinawa, the endemic area of human T-cell leukemia/lymphoma virus type 1.