Evgeny Yakirevich

Intratumoral CD8+ T Lymphocytes as a Prognostic Factor of Survival in Endometrial Carcinoma

Purpose: CTLs are a prominent immune component infiltrating many solid tumors. These cells are considered to be a manifestation of host-immune response to the tumor; however, their prognostic significance remains a subject of considerable debate. The objective of this study was to evaluate the distribution pattern and prognostic value of CD8+ T cells in endometrial carcinoma. Experimental Design: We studied 90 cases of endometrial carcinoma, including 75 endometrioid and 15 papillary serous carcinomas. Immunohistochemical staining for CD8 and granzyme B was performed on paraffin-embedded sections. The number of immunohistochemically staining CD8+ T cells was enumerated in the following four regions: lymphocytes infiltrating the tumor epithelium at the invasive border, within the underlying tumor stroma, within the superficial tumor epithelium, and in the perivascular areas of the myometrium. Results: Patients with >10 CD8+ T lymphocytes/high-power field within the tumor epithelium at the invasive border displayed improved overall survival compared with patients with fewer intraepithelial CD8+ T lymphocytes (87 and 50%, respectively; P = 0.027). Multivariate analysis revealed that stage, vascular invasion, grade, and the number of intraepithelial CD8+ T lymphocytes at the invasive border were the only independent predictors of survival (P < 0.0001, P = 0.001, P = 0.011, and P = 0.025, respectively). Granzyme B+ cytoplasmatic granules were detected in a high proportion of CTLs, confirming their activated cytotoxic phenotype. Conclusions: Our study demonstrates for the first time that increased numbers of CTLs at the invasive border may be a reliable independent prognostic factor of survival in patients with endometrial carcinoma.

Succinate Dehydrogenase (SDH)-deficient Renal Carcinoma: A Morphologically Distinct Entity: A Clinicopathologic Series of 36 Tumors From 27 Patients

Succinate dehydrogenase (SDH)-deficient renal carcinoma has been accepted as a provisional entity in the 2013 International Society of Urological Pathology Vancouver Classification. To further define its morphologic and clinical features, we studied a multi-institutional cohort of 36 SDH-deficient renal carcinomas from 27 patients, including 21 previously unreported cases. We estimate that 0.05% to 0.2% of all renal carcinomas are SDH deficient. Mean patient age at presentation was 37 years (range, 14 to 76 y), with a slight male predominance (M:F=1.7:1). Bilateral tumors were observed in 26% of patients. Thirty-four (94%) tumors demonstrated the previously reported morphology at least focally, which included: solid or focally cystic growth, uniform cytology with eosinophilic flocculent cytoplasm, intracytoplasmic vacuolations and inclusions, and round to oval low-grade nuclei. All 17 patients who underwent genetic testing for mutation in the SDH subunits demonstrated germline mutations (16 in SDHB and 1 in SDHC). Nine of 27 (33%) patients developed metastatic disease, 2 of them after prolonged follow-up (5.5 and 30 y). Seven of 10 patients (70%) with high-grade nuclei metastasized as did all 4 patients with coagulative necrosis. Two of 17 (12%) patients with low-grade nuclei metastasized, and both had unbiopsied contralateral tumors, which may have been the origin of the metastatic disease. In conclusion, SDH-deficient renal carcinoma is a rare and unique type of renal carcinoma, exhibiting stereotypical morphologic features in the great majority of cases and showing a strong relationship with SDH germline mutation. Although this tumor may undergo dedifferentiation and metastasize, sometimes after a prolonged delay, metastatic disease is rare in the absence of high-grade nuclear atypia or coagulative necrosis.

Pathology of Gastric Cancer and Its Precursor Lesions

Gastric cancers are a histologically heterogenous group of neoplasms arising from unique epidemiologic and molecular backgrounds. There is accumulating evidence that the intestinal type of gastric adenocarcinoma develops through a multistep process beginning with chronic gastritis triggered primarily by Helicobacter pylori and progressing through atrophy, intestinal metaplasia, and dysplasia (intraepithelial neoplasia) to carcinoma. Loss of E-cadherin expression resulting from CDH1 gene alterations is the primary carcinogenetic event in hereditary diffuse gastric cancer. Proximal gastric adenocarcinomas likely result from either gastroesophageal reflux or H pylori gastritis. This article provides an update of the histologic, immunohistochemical, and molecular pathways of gastric cancer and its precursors.

Cloning of a Glucose Phosphate Isomerase/Neuroleukin-Like Sperm Antigen Involved in Sperm Agglutination

Abstract The mouse monoclonal antibody (mAb) A36 produced by us and shown to induce extensive, “tangled” sperm agglutination was used to isolate cDNAs encoding its cognate antigen. Three overlapping cDNA clones specifically recognized by the mAb were isolated from a human testis cDNA expression library in λgt11. Sequencing of these cDNAs yielded the complete nucleotide sequence of a 3-kilobase cDNA that encodes the mAb-related polypeptide, designated sperm antigen-36 (SA-36), composed of 558 deduced amino acids. SA-36 cDNA contained a 5′ untranslated region of 234 nucleotides (nt), an open reading frame of 1674 nt, and a 3′ untranslated region of 1138 nt. SA-36 cDNA displayed > 99% homology to glucose phosphate isomerase (GPI)/neuroleukin (NLK) mRNA. This surprising homology was confirmed in Western blots demonstrating that mAb A36 reacted specifically with GPI obtained from rabbit muscle and from bakers yeast. Moreover, polyclonal, monospecific antibodies produced against β-galactosidase/SA-36-3 fusion protein stained human spermatozoa and caused intensive agglutination of these cells in a manner similar to that with the mAb. Taken together, the data presented here demonstrated that mAb A36 cognate sperm surface antigen, encoded by SA-36 cDNA, is a GPI/NLK-like protein involved in sperm agglutination.

Claudin Expression in High Grade Invasive Ductal Carcinoma of the Breast: Correlation with the Molecular Subtype

Claudin proteins are a major component of the tight junctions. Dysregulation of claudin protein expression has been described in a number of malignancies. Gene expression profiling has stratified breast cancers into distinct molecular subtypes: luminal, HER2 positive (HER2+), and basal-like. Recently, a novel claudin-low molecular subtype has been described. In this study, we correlated the expression patterns of claudins with the molecular subtypes of breast cancer. On the basis of immunohistochemical expression, 226 grade 3 invasive ductal carcinomas were stratified into 65 luminal (estrogen receptor positive (ER+)), 65 HER2+, 86 basal-like, including 14 metaplastic carcinomas (ER−, HER2−, CK5/6, and/or epidermal growth factor receptor positive), and 10 unclassified. Tissue microarrays were analyzed for the expression of claudins 1, 3, 4, 7, and 8 by immunohistochemistry and scored semiquantitatively. High levels of expression were detected in 17% of all cases for claudin 1, 32% claudin 3, 41% claudin 4, 44% claudin 7, and 40% claudin 8. Luminal cancers exhibited increased claudins 7 and 8; basal-like tumors demonstrated increased expression of claudins 1 and 4. Low expression of all five claudins was detected in 30 of 226 cases (13%) and this group was designated ‘claudin-low’. The majority of the claudin-low subgroup were basal-like cancers (23 of 30, 77%). In contrast, only 1 of 30 (3%) claudin-low tumors was of the luminal phenotype and 6 of 30 cases (20%) were HER2+ (P<0.001). Within the basal-like subgroup, 64% of the metaplastic and 19% of the non-metaplastic tumors were claudin-low. The claudin-low group was strongly associated with disease recurrence (P=0.0093). In conclusion, this study is the first to examine comprehensively the differential expression of claudins 1, 3, 4, 7, and 8 in the molecular subtypes of high-grade breast cancer. Claudin-low subtype is a frequent phenomenon in metaplastic and basal-like breast cancer and appears to be a strong predictor of disease recurrence.

The diagnostic and prognostic utility of claudin expression in renal cell neoplasms

This study evaluated the expression patterns of claudins 1, 3, 4, 7, and 8 in human renal cell carcinomas and oncocytomas and correlated expression with patient prognosis. Tissue microarrays were created from paraffin-embedded tissue samples from 141 patients with renal cell carcinomas or oncocytoma (90 clear cell, 22 papillary, 17 chromophobe renal cell carcinomas, and 12 oncocytomas). The staining pattern for claudins 3, 4, 7, and 8 was membranous and/or cytoplasmic, whereas claudin 1 was predominantly membranous in both nonneoplastic renal tissue and tumors. Negative to weak claudin 3 staining was predominantly detected in Fuhrmans grade 1 and 2 clear cell renal cell carcinomas (78%; P=0.016), suggesting that upregulation of claudin 3 potentially occurs concomitantly with increasing grade of clear cell renal cell carcinomas. In addition, Kaplan–Meier univariate analysis showed a significant inverse correlation between moderate to strong claudin 3 and 4 expression with overall survival in clear cell renal cell carcinomas (P=0.038 and P=0.031). Moderate to strong claudin 7 expression was significantly more common in chromophobe renal cell carcinomas (94%) than in oncocytomas (55%; P=0.041). Claudin 8 staining was moderate to strong in 92% of oncocytomas, which differentiated them from papillary and clear cell renal cell carcinomas (14 and 12%; both P<0.0001). Only negative to weak claudin 8 staining was detected in all chromophobe renal cell carcinomas, whereas there were no claudin 8 negative oncocytomas and 8% exhibited a weak staining pattern (P<0.0001). Due to their distinctive expression patterns, claudins 7 and 8 can be used as useful immunohistochemical markers for the separation of chromophobe renal cell carcinomas from oncocytomas, whereas claudins 3 and 4 may serve as indicators of prognosis in clear cell renal cell carcinomas.

Histone Deacetylase 6 (HDAC6) Deacetylates Survivin for Its Nuclear Export in Breast Cancer

Background: Survivin is an oncogenic protein that is acetylated by CBP, which restricts its location to the nuclear compartment and blocks its anti-apoptotic effect. Results: HDAC6 deacetylates survivin to promote its nuclear exit in estrogen receptor-positive breast cancer cells. Conclusion: Cross-talk between estrogen, CBP, and HDAC6 regulate the amount of nuclear acetylated survivin. Significance: Understanding how estrogen regulates survivin nuclear export may influence breast cancer treatment. Survivin is an oncogenic protein that is highly expressed in breast cancer and has a dual function that is dependent on its subcellular localization. In the cytosol, survivin blocks programmed cell death by inactivating caspase proteins; however, in the nucleus it facilitates cell division by regulating chromosomal movement and cytokinesis. In prior work, we showed that survivin is acetylated by CREB-binding protein (CBP), which restricts its localization to the nuclear compartment and thereby inhibits its anti-apoptotic function. Here, we identify histone deacetylase 6 (HDAC6) as responsible for abrogating CBP-mediated survivin acetylation in the estrogen receptor (ER)-positive breast cancer cell line, MCF-7. HDAC6 directly binds survivin, an interaction that is enhanced by CBP. In quiescent breast cancer cells in culture and in malignant tissue sections from ER+ breast tumors, HDAC6 localizes to a perinuclear region of the cell, undergoing transport to the nucleus following CBP activation where it then deacetylates survivin. Genetically modified mouse embryonic fibroblasts that lack mhdac6 localize survivin predominantly to the nuclear compartment, whereas wild-type mouse embryonic fibroblasts localize survivin to distinct cytoplasmic structures. Together, these data imply that HDAC6 deacetylates survivin to regulate its nuclear export, a feature that may provide a novel target for patients with ER+ breast cancer.

Regulatory T Lymphocytes: Pivotal Components of the Host Antitumor Response

The role of host immunity in the development and progression of cancer has been a subject of speculation and considerable controversy for more than the last 50 years. Tumor-infiltrating lymphocytes (TILs), the primary immune component infiltrating solid tumors, are considered to be a manifestation of the host antitumor reaction. The report by Gao et al in this issue provides additional evidence for the prognostic significance of TILs in solid tumors in general and hepatocellular carcinoma (HCC) specifically. Historically, tumor-associated lymphoid infiltrates were categorized descriptively as brisk, nonbrisk, or absent, based on morphologic observation alone. The survival advantage of a brisk lymphocytic infiltrate in malignant melanoma has been demonstrated by Clark et al, and confirmed by others. However, there is accumulating evidence that the specific type of immune cells, rather than their sheer quantity, governs the host-versus-tumor immune response. With development of immunohistochemical and flow cytometry techniques, it has been demonstrated that the majority of TILs in solid tumors are of the CD3 T-cell phenotype. CD3 T cells can be stratified further into CD4 helper cells (including the Th1 and Th2 subtypes based on their cytokine profile), CD4 regulatory T cells (Tregs), previously designated as suppressor cells, and CD8 cytotoxic effector cells. Initial studies that set out to characterize the antitumor immune response were directed toward CD8 cytotoxic T lymphocytes (CTLs). CTLs destroy tumor cells via the triggering of apoptosis by means of cytotoxic granule exocytosis and/or the Fas/FasL receptor–mediated pathway. In the mid-1990s, antibodies that recognize cytotoxic molecules, including TIA-1, granzyme B, and perforin, became available for immunohistochemical studies. The number of CD8 CTLs expressing TIA-1 and/or granzyme B cytotoxic granules infiltrating tumors has been shown to be associated with improved prognosis in certain human neoplasms, such as Epstein-Barr virus–associated gastric cancer and carcinomas of the colon, breast, uterus, and esophagus. The role of CD4 T cells in the host antitumor response is an area of considerable debate. This subset may be considered as a double-edged immunologic sword, able to promote or inhibit tumor growth. CD4 T-helper cells are subdivided into Th1 cells, which induce cellular immunity, and Th2 cells, which elicit a humoral immune response. The Th1 response is associated with CTL activation and is considered to be beneficial for antitumor immunity. In contrast, a predominant Th2-like cytokine profile is associated with a metastasis-inclined microenvironment in HCC and other tumors. A subpopulation of CD4 T cells, Tregs, which accounts for 5% to 10% of all CD4 cells, is generating intense interest in tumor immunology, autoimmunity, and infectious disease. Historically, Tregs were first hypothesized as suppressive T cells in early 1971 by Gershon and Kondo, however, subsequent studies were hindered because of a lack of specific molecular markers and difficulties in their isolation and culture. The renaissance of these regulatory cells came a few years ago, when FOXP3, a member of the forkhead family of DNA transcription factors, was cloned. There is now considerable evidence that FOXP3 is a key control molecule for Treg development and function, and is an excellent marker for the study of Tregs. Recent studies have demonstrated that Tregs are beneficial for the prevention of autoimmune disease, such as type I diabetes, graft-versus-host disease, transplant rejection, and tissue destruction during infection. In contrast, Tregs seem to be a detrimental factor in the generation of host-versus-tumor immunity via suppression of tumorspecific effector T-cell responses and development of immune tolerance to neoplastic cells. The classic Treg is a thymus-derived CD4 CD25 FOXP3 T lymphocyte. In addition to these markers, Tregs express cell surface CTL-associated antigen 4 (CTLA-4), and the glucocorticoidinduced tumor necrosis factor alpha receptor and secrete immunosuppressive cytokines such as transforming growth factor beta and interleukin 10 (IL-10). Initially it was demonstrated that the number of CD4 CD25 T-cells were increased in several solid tumors and in certain hematologic malignancies. However, subsequent studies provided evidence that CD25, which is an IL-2 receptor subunit, is induced on activation in all T-cells, not just Tregs. Recently, there has been an sudden increase of immunohistochemical studies using FOXP3 as a more specific marker of Tregs. Increased numbers of FOXP3 Tregs infiltrating tumor cell nests have been demonstrated in ovarian, lung, breast, pancreatic, hepatocellular, head and neck, and anal carcinomas, and lymphomas. In most of the solid tumors studied, accumulation of FOXP3 Tregs predicts a striking reduction of patient survival; however, paradoxically, increased FOXP3 Tregs were found to be associated with improved prognosis in lymphoma patients. JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 25 NUMBER 18 JUNE 2

IgG4(+) plasma cells in sclerosing variant of mucoepidermoid carcinoma.

IgG4-related sclerosing disease is a recently described syndrome with unique histologic features characterized by intense lymphoplasmacytic infiltrates with increased IgG4+ plasma cells and dense stromal sclerosis. The disease spectrum frequently includes benign inflammatory diseases, such as autoimmune pancreatitis, cholangitis, and chronic sclerosing sialadenitis (CSS). Mucoepidermoid carcinoma (MEC) is the most common primary malignancy in the salivary gland. The rare sclerosing variant of MEC is characterized by dense stromal sclerosis and lymphoplasmacytic infiltrates. Our goal was to further characterize lymphoplasmacytic infiltrates with respect to IgG4 expression. Six sclerosing MECs from our pathology service over the past 20 years were selected. In addition, 11 regular MECs with lymphoplasmacytic infiltrates, 4 CSS cases, and 12 nonsclerosing chronic sialadenitis cases were evaluated. None of the sclerosing MEC patients had IgG4-related sclerosing disease. The absolute number of IgG4+ plasma cells was significantly increased in sclerosing MEC as compared with the regular type (75 vs. 20 per image field; P<0.05). Furthermore, the proportion of IgG4+/IgG+ plasma cells was markedly elevated in sclerosing MEC as compared with the regular type (46.5% vs. 17%; P<0.05). In CSS, IgG4+/IgG+ ratio was significantly increased as compared with nonsclerosing chronic sialadenitis (54% vs. 6.73%; P<0.01). This study is the first to demonstrate increased IgG4 plasma cells in sclerosing MEC. The association of elevated IgG4+ plasma cells with increased fibrosis in the sclerosing variant of MEC suggests a role of IgG4+ plasma cells in fibrogenesis and may be a new concept related to sclerosis in cancer.

Low-fat diet impairs postresection intestinal adaptation in a rat model of short bowel syndrome

BACKGROUND Low-fat diets (LFD) are utilized frequently in patients with short bowel syndrome (SBS). The purpose of this study was to investigate the effects of LFD on intestinal adaptation, enterocyte proliferation, and enterocyte cell death in a rat model of SBS. METHODS Adult male Sprague-Dawley rats were divided into 3 experimental groups: Sham-NC rats underwent bowel transection and reanastomosis and were fed normal chow (NC), SBS-NC rats underwent 75% small bowel resection and were fed NC, and SBS-rats were fed a low-fat diet (SBS-LFD). Parameters of intestinal adaptation, enterocyte proliferation, and enterocyte apoptosis were determined on day 14 after operation. RESULTS SBS-NC rats showed a significant increase (v Sham-NC) in jejunal and ileal bowel and mucosal weight, mucosal DNA and protein, villus height, and crypt depth. A significant 67% increase in crypt cell proliferation rate and 265% increase in villus enterocyte apoptosis was seen in the ileum of SBS-NC rats compared with control animals (P <.05). SBS-LFD animals showed lower ileal mucosal weight (29%; P <.05), jejunal crypt depth (20%; P <.05), and ileal villus height (21%; P <.05). A significant decrease in villus apoptosis in jejunum (74%; P <.05) and ileum (67%; P <.05) and a decrease in cell proliferation in ileum (35%; P <.05) was seen also after exposure to LFD compared with SBS-NC. CONCLUSIONS In a rat model of SBS, early LFD appears to inhibit parameters of intestinal adaptation. A possible mechanisms for this effect may be decreased cell proliferation. Decreased enterocyte loss via apoptosis, found in this study, may reflect a reduced number of enterocyte.