PD-L1 expression and CD8-positive T cells are associated with favorable survival in HER2-positive invasive breast cancer.

Programmed cell death 1 (PD-1) and its ligand (PD-L1) are key physiologic suppressors of the cytotoxic immune reaction. However, to date, the combination of PD1/PD-L1 expression and tumor-infiltrating lymphocytes (TILs) and antigen-presenting cells has been only minimally reported in breast carcinoma, in particular in relation to HER2-positive cases. The goal of this study was to evaluate both cellular tumoral immune reaction and PD-L1/PD1 distribution in HER2-positive cases, as well as any associations with clinical outcome using conventional chemotherapy combined with HER2 blocking. Multicolor immunohistochemical multiplex assays simultaneously demonstrating PD1, PD-L1, and CD8 or PD-L1, CD3, and CD163 were performed on tissue microarrays (TMA) representing 216 pretreatment cases of HER2-positive invasive breast carcinoma. PD-L1 expression was identified in 38 cases (18%), including 12 cases (6%) with PD-L1 labeling of tumor cells and 26 cases (12%) with PD-L1 labeling of immune cells only. Ten of 12 cases with PD-L1 staining of tumor cells showed staining of associated immune cells as well. With this assay method, PD1 was detectable in many fewer cases (6 cases or 3%). PD-L1 expression was positively associated with high Nottingham grade, negative ER and PR, the absence of lymph node metastasis, and high levels of CD8+ cells. The overall survival by univariate analysis was positively associated with lower tumor stage, the absence of lymph node metastasis, PD-L1 expression, and high levels of CD8+ cells. Therefore, our data suggest cytotoxic immune reaction mediated by CD8-positive T cells and PD-L1 expression may predict a better outcome in patients with HER2-positive breast carcinoma managed with conventional chemotherapy and HER2-blocking therapy. These findings recommend clinical trials utilizing checkpoint blocking immunotherapy in some form for HER2-positive breast cancer.

Interpathologist Diagnostic Agreement for Non-Small Cell Lung Carcinomas Using Current and Recent Classifications.

CONTEXT.­: Measurement of interpathologist diagnostic agreement (IPDA) should allow pathologists to improve current diagnostic criteria and disease classifications. OBJECTIVES.­: To determine how IPDA for pathologists' diagnoses of non-small cell lung carcinoma (NSCLC) is affected by the addition of a set of mucin and immunohistochemical (IHC) stains to hematoxylin-eosin (H&E) alone, by recent NSCLC reclassifications, by simplification of these classifications, and by pathologists' practice location, pulmonary pathology expertise, practice duration, and lung carcinoma case exposure. DESIGN.­: We used a Web-based survey to present core images of 54 NSCLC cases to 22 practicing pathologists for diagnosis, initially as H&E only, then as H&E plus mucin and 4 IHC stains. Each case was diagnosed according to published 2004, 2011, and 2015 NSCLC classifications. Cohen's kappa was calculated for the 231 pathologist pairs as a measure of IPDA. RESULTS.­: Twenty-two pathologists diagnosed 54 NSCLC cases by using 4 published classifications. IPDA is significantly higher for H&E/mucin/IHC diagnoses than for H&E-only diagnoses. IPDA for H&E/mucin/IHC diagnoses is highest with the 2015 classification. IPDA is estimated higher after collapse of stated diagnoses into subhead or dichotomized classes. IPDA for H&E/mucin/IHC diagnoses with the 2015 World Health Organization classification is similar for community and academic pathologists, and is higher when pathologists have pulmonary pathology expertise, have more than 6 years of practice experience, or diagnose more than 100 new lung carcinoma cases per year. CONCLUSIONS.­: Higher IPDA is associated with use of mucin and IHC stains, with the 2015 NSCLC classification, and with pathologists' pulmonary pathology expertise, practice duration, and frequency of lung carcinoma cases.

Evaluation of Immune Reaction and PD-L1 Expression Using Multiplex Immunohistochemistry in HER2-Positive Breast Cancer: The Association With Response to Anti-HER2 Neoadjuvant Therapy.

BACKGROUND: Immune reaction with tumor-infiltrating lymphocytes (TILs) has been extensively investigated in breast cancer. Programmed cell death 1 and its ligand (PD-L1) are key physiologic suppressors of cytotoxic immune reaction. However, the combination of TILs with PD-L1 expression has not been well studied in breast cancer. PATIENTS AND METHODS: A multi-color immunohistochemical multiplex assay simultaneously detecting PD-L1, CD8, and CD163 was performed on biopsy whole sections from 123 HER2-positive (HER2+) breast cancers, including 64 treated with anti-HER2 neoadjuvant therapy and subsequent resection. RESULTS: PD-L1 expression was identified in 88 cases (72%) including 21 (17%) in tumor cells and 67 (55%) in immune cells. PD-L1 expression was positively associated with high Nottingham grade, high nuclear grade, and a high level of CD8+ and CD163+ cells. Among the 64 patients who received neoadjuvant therapy, 39 had pathologic complete remission (pCR) and 25 had incomplete response. Multivariate analysis showed progesterone receptor negativity, HER2/chromosome 17 centromere (CEN17) ratio and intratumoral CD8+ cells were significantly associated with pCR. Furthermore, all patients with intratumoral CD8+ cells but no PD-L1 expression achieved pCR. CONCLUSION: Our data have shown that examination of intratumoral CD8+ cells together with PD-L1 expression proves useful in predicting response to anti-HER2 targeted therapy in patients with HER2+ breast cancer.

HER2 intratumoral heterogeneity is independently associated with incomplete response to anti-HER2 neoadjuvant chemotherapy in HER2-positive breast carcinoma.

PURPOSE: Anti-HER2 neoadjuvant chemotherapy has been widely used in HER2-positive breast cancer patients; however, pathologic complete response (pCR) is achieved in only 40-50% of patients. The aim of this study was to investigate the association of HER2 intratumoral heterogeneity (ITH) with response to anti-HER2 neoadjuvant chemotherapy. METHODS: Assessment of HER2 ITH was performed on whole tissue sections of pre-treatment samples from a cohort of 64 invasive breast carcinoma cases originally considered positive for HER2 and treated with anti-HER2 neoadjuvant chemotherapy. Both HER2 gene signal and protein expression were simultaneously evaluated by means of a single-slide dual assay, designated as a HER2 gene-protein assay (GPA). HER2 GPA was carried out as well on surgical resection tissues from 25 cases with incomplete therapeutic response. RESULTS: Nineteen of 64 cases (30%) showed HER2 ITH. Significantly more cases with HER2 ITH were found in the incomplete response group (56%, 14/25) than in the pCR group (13%, 5/39). Patients without ITH detectable by GPA had a 76% pCR outcome (34/45), as compared to 26% (5/19) for those with detectable ITH. Multivariate analysis demonstrated HER2 ITH, progesterone receptor positivity, and relatively low HER2/chromosome 17 centromere ratio to be significantly associated with incomplete response. CONCLUSIONS: HER2 ITH analyses conducted with GPA method revealed that HER2 ITH is an independent factor predicting incomplete response to anti-HER2 neoadjuvant chemotherapy.

Comparison of Dual-ISH (DISH) With Fluorescence In Situ Hybridization (FISH) and Correlation With Immunohistochemical Findings for HER2/Neu Status in Breast Carcinoma.

INTRODUCTION: The most widely used methods for determination of HER2/neu status in breast carcinoma are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Both techniques are associated with technical and interpretive difficulties. Alternative methods exist including quantitative PCR and the newly developed chromogenic dual in situ hybridization (DISH). METHODS: We evaluated HER2 DISH as an alternative to FISH and report our findings from 101 cases. In addition, we correlated HER2 DISH and FISH results with HercepTest and 4B5 immunohistochemistry. RESULTS: Eight cases failed FISH analysis and none failed DISH analysis. A 95% (88/93) concordance was found between DISH and FISH for all cases in the series. When only 2+ IHC cases were evaluated, the concordance was 94% for DISH and FISH. Using the 2013 ASCO/CAP recommendations, none of the tested cases were equivocal by FISH or DISH despite 66% of cases being 2+ by HercepTest and 32% by the 4B5 antibody. COMMENT: Our study, which utilizes a majority of IHC equivocal cases, demonstrates that HER2 FISH and DISH are concordant methodologies. HER2 DISH is therefore an acceptable alternative to FISH.

Dual expression of MYC and BCL2 proteins predicts worse outcomes in diffuse large B-cell lymphoma.

Recent studies suggested that MYC and BCL2 protein co-expression is an independent indicator of poor prognosis in diffuse large B-cell lymphoma. However, the immunohistochemistry protocols for dual-expression staining and the scoring cut-offs vary by study. Sixty-nine cases of diffuse large B-cell lymphoma were evaluated for MYC and BCL2 protein expression using various cut-offs that have been recommended in prior studies. Independent of the International Prognostic Index risk group, cases with dual protein expression of BCL2 and MYC using ≥50%/40% cut-offs and ≥70%/40% had significantly shorter overall survival than cases without. It was verified in this patient population that the use of BCL2 and MYC immunohistochemistry, performed with available in vitro diagnostic-cleared antibodies, provides rapid prognostic information in patients with de novo diffuse large B-cell lymphoma. This study has practical implications for diagnostic laboratories and serves as a guide for implementation in the setting of future clinical trials.

The utility of BRAF V600E mutation-specific antibody VE1 for the diagnosis of hairy cell leukemia.

OBJECTIVES: BRAF V600E mutation is characteristic of hairy cell leukemia (HCL). A V600E mutation-specific antibody, VE1, has been recently developed. We studied the diagnostic utility of this antibody in HCL and compared it with other B-cell neoplasms. METHODS: VE1 activity was assessed using immunohistochemistry in 90 mature B-cell neoplasms, including HCL (n = 17), HCL variant (n = 6), chronic lymphocytic leukemia (CLL) (n = 20), and 47 other B-cell lymphomas. Most (87/90) specimens were formalin-fixed, paraffin-embedded bone marrow (BM) biopsy specimens decalcified in either hydrochloric acid or formic acid. RESULTS: VE1 was positive in 15 (88%) cases of HCL and two (10%) cases of CLL and was negative in all other tumors assessed. The VE1-positive HCL cases showed uniform staining in all tumor cells, but intensity was variable. The two VE1-negative HCL cases had BRAF V600 mutations proven by molecular analysis. The two CLL cases positive with VE1 showed an atypical staining pattern with expression in a minority of lymphoma cells. Immunohistochemistry using the VE1 antibody had a sensitivity of 88% and a specificity of 97% for HCL. CONCLUSIONS: VE1 immunohistochemistry is a useful and convenient surrogate for detecting BRAF V600E mutation in BM biopsy specimens decalcified with hydrochloric or formic acid-based solutions.

The recognition and classification of lymphoproliferative disorders of the gut.

Gastrointestinal lymphomas can be difficult to diagnose, particularly in small samples, when early in development, or when of unusual types. In this review, we describe lymphoid proliferations in the gastrointestinal tract in a location-based manner, including, mouth, esophagus, stomach, small intestine, and large bowel. For the purpose of differential diagnosis, benign mimics of lymphoma are also described. Lymphoma types that are specifically addressed include plasmablastic, extranodal natural killer/T-cell-nasal type, extranodal marginal zone lymphoma (eg, mucosa-associated lymphoid tissue lymphoma), diffuse large B cell, primary follicular of small intestine, enteropathy-associated T cell, and Burkitt and mantle cell. Immunohistochemical markers useful in the diagnostic approach are elaborated in detail.

Validation of interobserver agreement in lung cancer assessment: hematoxylin-eosin diagnostic reproducibility for non-small cell lung cancer: the 2004 World Health Organization classification and therapeutically relevant subsets.

CONTEXT: Precise subtype diagnosis of non-small cell lung carcinoma is increasingly relevant, based on the availability of subtype-specific therapies, such as bevacizumab and pemetrexed, and based on the subtype-specific prevalence of activating epidermal growth factor receptor mutations. OBJECTIVES: To establish a baseline measure of interobserver reproducibility for non-small cell lung carcinoma diagnoses with hematoxylin-eosin for the current 2004 World Health Organization classification, to estimate interobserver reproducibility for the therapeutically relevant squamous/nonsquamous subsets, and to examine characteristics that improve interobserver reproducibility. DESIGN: Primary, resected lung cancer specimens were converted to digital (virtual) slides. Based on a single hematoxylin-eosin virtual slide, pathologists were asked to assign a diagnosis using the 2004 World Health Organization classification. Kappa statistics were calculated for each pathologist-pair for each slide and were summarized by classification scheme, pulmonary pathology expertise, diagnostic confidence, and neoplastic grade. RESULTS: The 12 pulmonary pathology experts and the 12 community pathologists each independently diagnosed 48 to 96 single hematoxylin-eosin digital slides derived from 96 cases of non-small cell lung carcinoma resection. Overall agreement improved with simplification from the comprehensive 44 World Health Organization diagnoses (κ  =  0.25) to their 10 major header subtypes (κ  =  0.48) and improved again with simplification into the therapeutically relevant squamous/nonsquamous dichotomy (κ  =  0.55). Multivariate analysis showed that higher diagnostic agreement was associated with better differentiation, better slide quality, higher diagnostic confidence, similar years of pathology experience, and pulmonary pathology expertise. CONCLUSIONS: These data define the baseline diagnostic agreement for hematoxylin-eosin diagnosis of non-small cell lung carcinoma, allowing future studies to test for improved diagnostic agreement with reflex ancillary tests.

In immunocompromised patients, Epstein-Barr virus lymphadenitis can mimic angioimmunoblastic T-cell lymphoma morphologically, immunophenotypically, and genetically: a case report and review of the literature.

The development of lymphomas and solid malignancies in association with immunosuppression is a well-documented occurrence in the medical literature. We report the case of a young man who developed progressive diffuse lymphadenopathy with associated extremely high levels of serum Epstein-Barr virus in the setting of chronic immunosuppressive treatment of glomerulonephritis. Excisional biopsy of a right inguinal node revealed a sclerosing process with the morphologic appearance of angioimmunoblastic T-cell lymphoma with a CD3(+), CD4(+) immunophenotype. In situ hybridization of Epstein-Barr virus-encoded RNA was positive. Molecular probe studies demonstrated a clonal T-cell population. Upon reduction of immunosuppression, the patient's lymphadenopathy and Epstein-Barr virus titer have resolved without recurrence over 2 years time. This case demonstrates that a benign Epstein-Barr virus-associated process can mimic angioimmunoblastic T-cell lymphoma and should be considered particularly in the setting of immunosuppression, emphasizing the need for close communication with the treating physician in the interpretation of lymph node biopsies.

Co-occurrence of Langerhans cell histiocytosis and Rosai-Dorfman disease: possible relationship of two histiocytic disorders in rare cases.

Rosai-Dorfman disease and Langerhans cell histiocytosis are both disorders of accessory immune cells. Two cases have been previously reported of concurrent Langerhans cell histiocytosis and Rosai-Dorfman disease. In this report, we characterize the findings and selected molecular studies in nine additional cases. Histology was reviewed. Immunohistochemical stains were performed on all cases in which slides or blocks were available. A combination of CD1a, S-100, CD3, CD20, langerin, CD68, CD163, CD21, CD35 and CD123 immunohistochemical stains were performed. High-resolution array comparative genomic hybridization was performed on six samples from five cases. In these cases, seven were female and two male, with an average age of 25 years (15 months-59 years). A majority of the cases were identified in lymph node. Areas of Langerhans cell histiocytosis had a typical appearance with the existence of bland 'coffee-bean' nuclei, clear cytoplasm and associated eosinophils. The immunophenotype was typical, including expression of CD1a, S100, CD68 and langerin. In areas of Rosai-Dorfman disease, there was emperipolesis seen in all cases. Cells were intermediate-large in size with large round nuclei and ample clear or pale cytoplasm. The lesional cells were positive for S100, CD68, CD163, without expression of langerin or CD1a. Array comparative genomic hybridization showed gains and/or losses in four of the six samples. One case showed no gains or losses and one additional case showed gains and losses in the Langerhans cell histiocytosis, while no abnormalities were discovered in the Rosai-Dorfman disease component. These findings are comparable to those seen in previous studies of Langerhans cell histiocytosis. We report the clinical and pathologic findings of the combination of Langerhans cell histiocytosis and Rosai-Dorfman disease. Furthermore, we suggest on the basis of evidence from our cases that, when simultaneous, the two entities may be pathophysiologically related.

Altered aquaporin 9 expression and localization in human hepatocellular carcinoma.

BACKGROUND: In addition to the biochemical components secreted in bile, aquaporin (AQP) water channels exist in hepatocyte membranes to form conduits for water movement between the sinusoid and the bile canaliculus. The aim of the current study was to analyse AQP 9 expression and localization in human hepatocellular carcinoma (HCC) and non-tumourigenic liver (NTL) tissue from patients undergoing hepatic resection. METHODS: Archived tissue from 17 patients was sectioned and analysis performed using an antibody raised against AQP 9. Slides were blind-scored to determine AQP 9 distribution within HCC and NTL tissue. RESULTS: Aquaporin 9 was predominantly expressed in the membranes of hepatocytes and demonstrated zonal distribution relative to hepatic sinusoid structure in normal liver. In HCC arising in the absence of cirrhosis AQP 9 remained membrane-localized with zonal distribution in the majority of NTL. By contrast, AQP 9 expression was significantly decreased in the HCC mass vs. pair-matched NTL. In HCC in the presence of cirrhosis, NTL was characterized by extensive AQP 9 staining in the membrane in the absence of zonal distribution and AQP 9 staining in NTL was significantly greater than that observed in the tumour mass. CONCLUSIONS: These data demonstrate that human HCC is characterized by altered AQP 9 expression and AQP 9 localization in the NTL mass is dependent on underlying liver pathology. Given the central role of AQPs in normal liver function and the potential role of AQPs during transformation and progression, these data may prove valuable in future diagnostic and/or therapeutic strategies.

Risk factors for lymphoproliferative disorders after allogeneic hematopoietic cell transplantation.

We evaluated 26 901 patients who underwent allogeneic hematopoietic cell transplantation (HCT) at 271 centers worldwide to define patterns of posttransplantation lymphoproliferative disorders (PTLDs). PTLDs developed in 127 recipients, with 105 (83%) cases occurring within 1 year after transplantation. In multivariate analyses, we confirmed that PTLD risks were strongly associated (P < .001) with T-cell depletion of the donor marrow, antithymocyte globulin (ATG) use, and unrelated or HLA-mismatched grafts (URD/HLA mismatch). Significant associations were also confirmed for acute and chronic graft-versus-host disease. The increased risk associated with URD/HLA-mismatched donors (RR = 3.8) was limited to patients with T-cell depletion or ATG use (P = .004). New findings were elevated risks for age 50 years or older at transplantation (RR = 5.1; P < .001) and second transplantation (RR = 3.5; P < .001). Lower risks were found for T-cell depletion methods that remove both T and B cells (alemtuzumab and elutriation, RR = 3.1; P = .025) compared with other methods (RR = 9.4; P = .005 for difference). The cumulative incidence of PTLDs was low (0.2%) among 21 686 patients with no major risk factors, but increased to 1.1%, 3.6%, and 8.1% with 1, 2, and more than 3 major risk factors, respectively. Our findings identify subgroups of patients who underwent allogeneic HCT at elevated risk of PTLDs for whom prospective monitoring of Epstein-Barr virus activation and early treatment intervention may be particularly beneficial.

Castleman disease of the hyaline-vascular type confined to the kidney.

The case is a 38-year-old man in whom a solitary subcapsular left renal cortical mass was successfully resected. Comorbidities included a benign epididymal cyst and a history of nephrolithiasis. Computed tomographic imaging demonstrated a 1.8-cm enhancing mass in the anterior midregion of the kidney. An open partial nephrectomy was performed, and histopathologic examination established a diagnosis of the hyaline-vascular type of Castleman disease (CD). The patient had an uneventful postoperative course and has experienced no local or metastatic recurrence in the 10 months since surgery. CD localized in the kidney is an exceptionally rare occurrence but should be included in the complete differential diagnosis of solitary renal cortical mass lesions.

Brief communication: severe hepatotoxicity of telithromycin: three case reports and literature review.

BACKGROUND: Telithromycin is a ketolide antibiotic approved by the U.S. Food and Drug Administration for acute bacterial infections causing sinusitis, bronchitis, and community-acquired pneumonia. OBJECTIVE: To describe 3 cases of severe hepatotoxicity in patients receiving telithromycin. DESIGN: Case reports. SETTING: A tertiary care medical center. PATIENTS: 3 previously healthy patients who had recently taken telithromycin and took no other prescription medications. MEASUREMENTS: Serologic, histologic, and liver function tests. RESULTS: Within a few days of receiving telithromycin, the patients presented with acute hepatitis. All had jaundice and markedly abnormal results on liver function tests. Results of viral serologic tests were negative. One patient spontaneously recovered, 1 required orthotopic liver transplantation, and 1 died. Histologic examination in the latter 2 patients showed massive hepatic necrosis. LIMITATIONS: Two patients had some history of alcohol use. The frequency of severe telithromycin-related hepatotoxicity cannot be established with case reports. CONCLUSIONS: Telithromycin can cause severe hepatotoxicity. Caution is advised in prescribing this drug pending additional postmarketing surveillance data.

Langerhans cell sarcoma.

Proliferations of Langerhans cells can be histologically divided into cytologically benign Langerhans cell proliferations, which include the clinical syndromes of Langerhans cell histiocytosis, and cytologically malignant Langerhans cell sarcoma. We report a Langerhans cell sarcoma in a 33-year-old male that arose on the posterior thigh with subsequent regional lymph node involvement. Conventional microscopic, immunohistochemical, and ultrastructural analysis confirmed Langerhans cell differentiation. Aberrant CD31 expression, similar to that described previously in Langerhans cell histiocytosis, was prominent in this tumor, possibly enhancing its migratory capabilities.

Acute panmyelosis with myelofibrosis: an entity distinct from acute megakaryoblastic leukemia.

The WHO criteria for diagnosing acute panmyelosis with myelofibrosis are somewhat distinct from those for acute megakaryoblastic leukemia. However, clinical and hematopathologic findings partially overlap. This has raised questions as to whether these are indeed separate, definable entities. To determine the potential importance of bone marrow biopsy supplemented by immunohistochemistry in distinguishing between these two conditions, we studied 17 bone marrow biopsies of well-characterized cases of acute panmyelosis with myelofibrosis (six cases) and acute megakaryoblastic leukemia (11 cases). We compared blast frequency, reticulin content, CD34 expression, and the degree of megakaryocytic differentiation of the blast cells in these two conditions. Our results demonstrate important differences. Acute panmyelosis with myelofibrosis is characterized by a multilineage myeloid proliferation with a less numerous population of blasts than acute megakaryoblastic leukemia (P<0.01). In the former condition, blasts are always positive with CD34, while in acute megakaryoblastic leukemia they express CD34 in 60% of the cases. The blasts in acute panmyelosis with myelofibrosis only rarely express megakaryocytic antigens. By contrast, acute megakaryoblastic leukemia has a significantly higher proportion of blasts expressing megakaryocytic antigens (P<0.01 with CD42b). Our results confirm that histology supplemented by immunohistochemistry permits the distinction of these conditions in routinely processed bone marrow biopsies.

Human herpesvirus-6-associated acute lymphadenitis in immunocompetent adults.

In contrast to other causes of herpetic lymphadenitis, the histological features associated with human herpesvirus-6 (HHV-6) infection have remained elusive since its discovery in 1986. We describe the histologic and phenotypic changes associated with acute HHV-6 lymphadenitis in two immunocompetent adults who presented with fever, fatigue, generalized lymphadenopathy, and elevated liver enzymes. Serologic tests for human immunodeficiency virus, acute Epstein-Barr virus, and cytomegalovirus infection were negative. Lymph node biopsies were consistent with viral lymphadenitis. Intranuclear and cytoplasmic inclusions were identified in CD4-positive T lymphocytes in expanded paracortical areas. Immunohistochemical staining with monoclonal antibody to the HHV-6 gp60/110 kDa envelope glycoprotein showed that the inclusions were positive for viral antigen. Electron microscopy demonstrated numerous viral particles in the cytoplasm and nucleus, characteristic of Herpesviridae family. Clustering of viral particles was observed, which has previously been reported only in infected tissue culture cells. PCR followed by sequencing of DNA extracted from the lymph nodes identified the virus as HHV-6, type B. This is the first report that documents distinctive histologic features of HHV-6 lymphadenitis and demonstrates that the cells harboring the virus in vivo are CD4-positive T lymphocytes.