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Supplementary Table S1. Comparison of PD-L1 diagnostic assays for patients with NSCLC Supplementary Table S2. Detailed scoring criteria for each assay in the study Supplementary Table S3. Demographics of patients who provided study samples Supplementary Table S4. OPA for pairwise assay comparisons across the full assay range

Purpose: Immunotherapies targeting programmed cell death-1 (PD-1) and programmed cell death ligand-1 (PD-L1) demonstrate encouraging antitumor activity and manageable tolerability in non–small cell lung cancer (NSCLC), especially in patients with high tumor PD-L1 expression, as detected by companion or complementary diagnostic assays developed for individual agents. A laboratory is unlikely to use multiple assay platforms. Furthermore, commercially available diagnostic assays are not standardized, and different assay methods could lead to inappropriate treatment selection. This study establishes the extent of concordance between three validated, commercially available PD-L1 IHC diagnostic assays for NSCLC patients [Ventana SP263 (durvalumab), Dako 22C3 (pembrolizumab), and Dako 28-8 (nivolumab)].Experimental Design: Five hundred formalin-fixed, paraffin-embedded archival NSCLC samples were obtained from commercial sources. Stained slides were read in batches on an assay-by-assay basis by a single pathologist trained in all methods, in a Clinical Laboratory Improvements Amendments program–certified laboratory. An additional pathologist performed an independent review of 200 stained samples for each assay.Results: PD-L1 expression was evaluable with all assays in 493 samples. The three assays showed similar patterns of tumor membrane staining, with high correlation between percent PD-L1 staining. An overall percentage agreement of >90% was achieved between assays at multiple expression cutoffs, including 1%, 10%, 25%, and 50% tumor membrane staining.Conclusions: This study builds optimism that harmonization between assays may be possible, and that the three assays studied could potentially be used interchangeably to identify patients most likely to respond to anti-PD-1/PD-L1 immunotherapies, provided the appropriate clinically defined algorithm and agent are always linked. Clin Cancer Res; 23(14); 3585–91. ©2017 AACR.

SummaryThe discovery of mutated Calreticulin (CALR) in myeloproliferative neoplasms (MPN) has provided proof of clonality, diagnostic importance, and influence on prognosis of this pathology. The identification of this MPN-associated driver mutation -currently based on molecular assays- is represented as a major diagnostic criterion for essential thrombocythaemia (ET), pre-fibrotic myelofibrosis and primary myelofibrosis (PMF) in the updated World Health Organization (WHO) 2008 classification. In the present study, we validated by immunohistochemistry the diagnostic usefulness of the monoclonal CAL2 antibody. Cases of acute myeloid leukaemia (AML) and myelodysplastic/ myeloproliferative neoplasms (MDS/MPN) have been also investigated to assess the specificity of CAL2 antibody. For this purpose, the result of the CAL2 immunostaining was compared with the result of molecular assays. Additionally, we investigated by double staining whether expression of mutated CALR can also be demonstrated on cells of the erythroid and myeloid lineage. We confirmed the usefulness of the CAL2 monoclonal antibody in successfully detecting mutant CALR in bone marrow biopsies. We showed that the immune-reactivity of CAL2 was absolutely restricted to the presence of CALR mutations, which were seen only in ET and MDS/MPN biopsies, but not in AML biopsies (14/14). There was 100% concordance in biopsy specimens with the concomitant molecular results. We applied double staining technique and confirmed that a subpopulation of granulopoietic and erythropoietic cells express mutated CALR as demonstrated with the CAL2 antibody in cases of MPNs. This supports the suggestion that the CALR mutations occur in a multipotent progenitor capable of generating both myeloid and erythroid progeny with preferential expansion of megakaryocytic cell lineage as a result of CALR mutation in an immature hematopoietic stem cell.

Non-urothelial bladder cancer samples included in the analysis (DOCX 14 kb)

OPA, PPA, and NPA between PD-L1 assays in invasive UC samples (NÂ =â 255) (DOCX 16 kb)

Purpose: Immunotherapies targeting programmed cell death-1 (PD-1) and programmed cell death ligand-1 (PD-L1) demonstrate encouraging antitumor activity and manageable tolerability in non–small cell lung cancer (NSCLC), especially in patients with high tumor PD-L1 expression, as detected by companion or complementary diagnostic assays developed for individual agents. A laboratory is unlikely to use multiple assay platforms. Furthermore, commercially available diagnostic assays are not standardized, and different assay methods could lead to inappropriate treatment selection. This study establishes the extent of concordance between three validated, commercially available PD-L1 IHC diagnostic assays for NSCLC patients [Ventana SP263 (durvalumab), Dako 22C3 (pembrolizumab), and Dako 28-8 (nivolumab)]. Experimental Design: Five hundred formalin-fixed, paraffin-embedded archival NSCLC samples were obtained from commercial sources. Stained slides were read in batches on an assay-by-assay basis by a single pathologist trained in all methods, in a Clinical Laboratory Improvements Amendments program–certified laboratory. An additional pathologist performed an independent review of 200 stained samples for each assay. Results: PD-L1 expression was evaluable with all assays in 493 samples. The three assays showed similar patterns of tumor membrane staining, with high correlation between percent PD-L1 staining. An overall percentage agreement of >90% was achieved between assays at multiple expression cutoffs, including 1%, 10%, 25%, and 50% tumor membrane staining. Conclusions: This study builds optimism that harmonization between assays may be possible, and that the three assays studied could potentially be used interchangeably to identify patients most likely to respond to anti-PD-1/PD-L1 immunotherapies, provided the appropriate clinically defined algorithm and agent are always linked. Clin Cancer Res; 23(14); 3585–91. ©2017 AACR.

A 57-year-old female with a history of diffuse large B-cell lymphoma (DLBCL) presented with a renal mass. Microscopic analysis of the nephrectomy specimen revealed DLBCL, confirmed by immunohistochemical analysis (CD45, CD20, CD3, pan-keratin) and polymerase chain reaction (PCR). Multiple spicules of metaplastic bone were identified within the tumor mass, but not within the uninvolved kidney parenchyma. No evidence of bone or dystrophic calcification was detected on the pre-nephrectomy computerized tomography (CT) scan. To our knowledge, this is the first incidence of osseous metaplasia occurring within DLBCL. The pathogenesis of osseous metaplasia is briefly discussed.

T-cell prolymphocytic leukemia (T-PLL) is an aggressive lymphoproliferative disorder with post-thymic T cell phenotype and prolymphocytoid morphology. T-PLL is characterized by an increased WBC cou...

Heat shock protein 90 (HSP90) is required for structural folding and maintenance of conformational integrity of various proteins, including several associated with cellular signaling. Recent studies utilizing 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90, demonstrated an antitumor effect in solid tumors. To test whether HSP90 could be targeted in multiple myeloma (MM) patients, we first investigated expression of HSP90 by immunofluorescence and flow cytometric analysis in a myeloma cell line (U266) and primary myeloma cells. Following demonstration of HSP90 expression in myeloma cells, archival samples of 32 MM patients were analysed by immunoperoxidase staining. Myeloma cells in all patients showed strong cytoplasmic expression of HSP90 in all samples and 55% also demonstrated concurrent nuclear immunopositivity. Treatment of U266 and primary MM cells with 17AAG resulted in significantly increased apoptosis compared to untreated control cells. Analysis of anti-apoptotic BCL2 family proteins and akt in MM cells incubated with 17-AAG revealed down-regulation of BCL-2, BCL-XL, MCL-1 and akt. Furthermore, although a low concentration of bortezomib resulted in no cell death, a combination of 17AAG and bortezomib treatment revealed a synergistic apoptotic effect on the U266 cell line. These data suggest that targeted inhibition of HSP90 may prove to be a valid and innovative strategy for the development of future therapeutic options for MM patients.

The diagnosis of atypia in breast fine-needle aspiration (FNA) continues to be an area of debate in cytology practice. The aim of this study was to assess the clinical significance of this term and to evaluate potential morphological criteria, which would determine the patient's outcome. A computer-based search was carried out to retrieve breast FNAs performed between 1990 and 2000 that were diagnosed as atypical. Cases followed by surgical resection were reexamined for the presence of morphological features potentially differentiating benign and malignant lesions. Out of 1,568 breast FNAs, there were 64 cases (4%) with a diagnosis of atypia. Thirty-eight cases had surgical follow-up material that revealed malignancy in 14 cases (37%) and benign lesions in 24 cases (63%). The benign diagnostic categories included fibrocystic change (12/24), fibroadenoma (3/24), tubular adenoma (2/24), and nonspecific findings (7/24). The malignant diagnoses included ductal carcinoma (9/14), lobular carcinoma (3/14), ductal carcinoma in situ (DCIS; 1/14), and tubular carcinoma (1/14). The evaluation of cytological criteria used to differentiate benign from malignant lesions (i.e., cellularity, loss of cohesion, myoepithelial cells, nuclear enlargement, nuclear overlap, prominent nucleoli) revealed significant overlap between benign and malignant cases, particularly in cases of fibroadenoma, tubular adenoma, and proliferative breast disease. The surgical follow-up of four hypocellular cases revealed lobular carcinoma in two cases and ductal carcinoma in the remaining two cases. Our study confirmed that the diagnosis of atypia is clinically significant because it is associated with a high probability of malignancy. No morphological criterion is able to reliably differentiate benign and malignant lesions in cases diagnosed with atypia. Diagnosis of atypia is particularly significant in hypocellular cases. We recommended that breast FNAs with a diagnosis of atypia be evaluated further histologically.

Background. Transitional cell carcinoma (TCC) has recently been acknowledged as a distinct histologic pattern of the uncommon primary fallopian tube carcinoma. However, rare cases of TCC that are closely associated to the extraluminal portion of the tube remain widely unrecognized. Case. We present a left adnexal high-grade TCC in a 56-year-old postmenopausal woman with an elevated serum CA-125 level. The tumor was attached by a small stalk to the serosal surface of the left fallopian tube and was completely separate from the uninvolved uterus and ipsilateral ovary. Histologic examination of the tubal lumen epithelium revealed neither atypia nor involvement by the neoplasm. Immunohistochemistry showed the tumor cells to be positive for pankeratin, calretinin, progesterone and estrogen receptors, and cytokeratin 7, and negative for cytokeratin 20, consistent with a Mullerian derivation. Conclusion. Our case represents the fourth reported instance of a primary paratubal TCC. Perhaps, this entity falls under a previously unrecognized category of carcinomas that collectively may arise from Walthard's rest, paratubal cyst, or directly from the tubal serosa.

e14503 Background: PD-1/PD-L1 directed antibodies are emerging as effective oncology therapeutics with multiple diagnostic PD-L1 tests available for patient segmentation. It is essential to compare these assays to understand the risk if assays are used interchangeably. Previous data showed strong concordance (OPA >85%) of TC PD-L1 expression at multiple clinically relevant cutoffs with the Ventana SP263, Dako 28-8 and 22C3 assays (Ratcliffe et al. Clin Cancer Res 2017; Ratcliffe et al. ESMO 2016 (955PD)). This work supplements previous data with concordance of IC scoring and data from Ventana SP142. Methods: IC PD-L1 IHC expression data were generated from 493 clinical NSCLC biopsy samples stained with Ventana SP263 and Dako 22C3 and 28-8 assays. In addition, consecutive sections from 200 blocks of the 493 samples were later sectioned and stained with SP142 and SP263. Concordance at matched cutoffs between the 4 assays is reported for IC, and concordance between SP263 and SP142 for TC. Results: Strong agreement in IC scoring between the SP263 and 22C3 and 28-8 assays is observed. The SP142 assay showed lower IC concordance and reduced TC PD-L1 staining/concordance vs SP263 (Table). Conclusions: Analytical similarity is shown between the SP263, 28-8 or 22C3 assays when measuring TC or IC PD-L1 expression, with high concordance between patient classification at matching TC cutoffs up to ≥50% or IC up to ≥10%, strengthening the evidence that these 3 assays could be used interchangeably at relevant clinical cutoffs. The SP142 assay shows clear analytical differences in both IC and TC staining. [Table: see text]

7 Background: PD-1/PD-L1 directed antibodies are emerging as effective therapeutics in multiple oncology settings. Multiple diagnostic PD-L1 tests are being used to understand how PD-L1 expression predicts response to these therapies. It is vital to compare these assays to understand the risk if assays are used interchangeably. Tumor cell (TC) PD-L1 expression by the Ventana SP263 and the Dako 28-8 and 22C3 assays has proven concordant at multiple clinically relevant cut offs.(Ratcliffe et al AACR 2016 [LB-094]); (Ratcliffe et al ESMO 2016 [955PD]). The current study builds on the previous study by inclusion of immune cell (IC) scoring and the Ventana SP142 assay. Methods: 493 tumor biopsy samples from NSCLC patients stained with the Ventana SP263 and Dako 22C3 and 28-8 were scored for IC PD-L1 expression, calculated as % of infiltrating IC expressing PD-L1 at any intensity. A subset of 200 tumor blocks from this cohort were sectioned and stained with SP142 and serial sections restained with SP263 to ensure an appropriate comparison. Concordance between IC and TC between these 2 assays is reported. Results: There was strong agreement in IC scoring with the Ventana SP263 and Dako 22C3 and 28-8 assays. The SP142 assay showed lower concordance for IC and reduced TC PD-L1 staining/concordance vs SP263 (Table). Conclusions: This study shows analytical similarity between the Ventana SP263, Dako 28-8 or 22C3 assays when measuring TC or IC PD-L1 expression, and high concordance between patient classification at matching TC or IC cut offs. This strengthens the evidence that these 3 assays could be used interchangeably. The Ventana SP142 assay shows clear analytical differences for both IC and TC staining, suggesting it is not appropriate to interchange with the other assays. [Table: see text]

Metastatic carcinoma presenting in a supraclavicular lymph node, especially in a young adult, has a wide differential diagnosis, including metastatic small blue cell tumors (lymphoma, neuroblastoma, rhabdomyosarcoma, Ewings sarcoma, primitive neuroectodermal tumor) as well as other less commonly seen neoplasms in this age group. Immunohistochemical techniques as well as regular cytologic and histologic morphologic features are important in arriving at a correct diagnosis. We report the case of a young adult with small cell carcinoma who presented with metastasis to a supraclavicular lymph node and who was diagnosed by fine-needle aspiration cytology. (The J Histotechnol 25:143, 2002)Submitted: April 10, 2002; Accepted May 15, 2002

Dynamic left ventricular outflow tract (LVOT) obstruction is seen classically in hypertrophic cardiomyopathy. Cardiac amyloidosis can present with asymmetric hypertrophy that resembles hypertrophic cardiomyopathy, and, in some cases, with dynamic LVOT obstruction. The occurrence of syncope in such patients is not uncommon. The syncope is usually thought to be related to mechanisms other than LVOT obstruction, such as arrhythmias, conduction disturbances, orthostatic hypotension, or vasovagal effects associated with neuropathy.Herein, we report the case of a patient who had immunocyte-derived (primary AL-type) cardiac amyloidosis with the echocardiographic appearance of hypertrophic cardiomyopathy and evidence of LVOT obstruction that caused syncope. We were able to provoke and identify dynamic LVOT obstruction that produced presyncopal symptoms similar to those that typically occur in such patients spontaneously. Dynamic LVOT obstruction as a cause of syncope should be considered in patients who have cardiac amyloidosis and echocardiographic evidence of hypertrophic cardiomyopathy.

We report the MRI findings of a large deep intermuscular lipoma in a 2-year-old child with a painless palpable shoulder mass, and its differentiation from liposarcoma and other soft-tissue masses. To our knowledge, the imaging features of deep lipomas in children have not been reported.

A 29-year-old woman with cystic fibrosis presented with abdominal pain, which had worsened over 3 days, together with low-grade fever and chills. The pain, which radiated from the central to the lower abdomen, increased with movement and had been present since bilateral lung transplantation 4months earlier. She had occasional nausea and nonbilious vomiting. The medical history included laparotomy for postoperative small bowel obstruction, diabetes mellitus, and osteoporosis. Medications included metoclopramide, lactulose, prednisone, tacrolimus, azathioprine, valacyclovir, itraconazole, cotrimoxazole, alendronate, lansoprazole, pancreatic enzymes, insulin, calcium, folate, and a multivitamin. Examination was normal except for mild periumbilical tenderness. Laboratory tests revealed mild anemia and a slight nonanion gap acidosis. Two small duodenal ulcers were discovered at EGD. Biopsy specimens revealed mild duodenitis with superficial ulceration. At

Heat Shock Protein 90 (HSP90) is required for structural folding and maintenance of conformational integrity of various proteins, including several associated with cellular signaling. Recent studies utilizing 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90, demonstrated antitumor effect in solid tumors. In order to test whether HSP90 could be targeted in multiple myeloma (MM) patients, we first investigated expression of HSP90 by immunofluorescence and flow cytometric analysis in a myeloma cell line (U266) and primary myeloma cells. Following demonstration of HSP90 expression in myeloma cells, archival samples of 32 MM patients were analyzed by immunoperoxidase staining. Myeloma cells in all patients showed strong cytoplasmic expression of HSP90 in all samples and 55 % also demonstrated concurrent nuclear immunopositivity. Treatment of U266 and primary MM cells with 17AAG resulted in significantly increased apoptosis compared to untreated control cells. Analysis of anti-apoptotic BCL2 family proteins in MM cells incubated with 17-AAG revealed down-regulation of BCL-2, BCL-XL and MCL-1. Furthermore, while low concentration of bortezomib had no cell death, combination of 17AAG and bortezomib treatment revealed a synergistic apoptotic effect on the U266 cell line. These data suggest that targeted inhibition of HSP90 may prove to be a valid and innovative strategy for the development of future therapeutic options for MM patients.