Kara A. Lombardo

Medullary carcinoma of the colon: a distinct morphology reveals a distinctive immunoregulatory microenvironment

Medullary carcinoma of the colon is a unique histologic subtype of microsatellite unstable colorectal carcinoma but little is known regarding its tumor-immunoregulatory microenvironment. The aims of this study were to characterize the immune environment of medullary carcinoma and compare it with other microsatellite unstable and microsatellite stable colorectal carcinomas. An initial gene expression microarray analysis of six cases of medullary carcinoma was used to detect potentially differentially expressed genes. We extended this analysis utilizing genomic data from the Cancer Genome Atlas to compare eight cases of medullary carcinoma with other microsatellite unstable and stable carcinomas. Finally, we evaluated expression of key immune pathway proteins and lymphocyte subsets via immunohistochemistry of a large group of medullary carcinomas (n=105) and compared these findings with three other groups: poorly differentiated, microsatellite unstable well-differentiated and microsatellite stable well-differentiated carcinomas. Microarray and the Cancer Genome Atlas data analysis identified significant upregulation of several immunoregulatory genes induced by IFNγ including IDO-1, WARS (tRNA(trp)), GBP1, GBP4, GBP5, PDCD1 (PD-1), and CD274 (PD-L1) in medullary carcinoma compared with other microsatellite unstable and microsatellite stable tumors. By immunohistochemistry, IDO-1 was expressed in 64% of medullary carcinomas compared with 19% (9/47) of poorly differentiated carcinomas, 14% (3/22) of microsatellite unstable, and 7% (2/30) of the microsatellite stable well-differentiated carcinomas (P<0.0001). tRNA(trp) was overexpressed in 81% (84/104) of medullary carcinomas, 19% (9/47) of poorly differentiated, 32% (7/22) of microsatellite unstable, and 3% (1/30) of microsatellite stable well-differentiated carcinomas (P<0.0001). Medullary carcinoma had higher mean CD8+ and PD-L1+ tumor-infiltrating lymphocytes compared with all other groups (P<0.0001). This study demonstrates overexpression of several immunoregulatory genes in microsatellite unstable colorectal carcinomas and that expression of these genes and proteins is more prevalent in the medullary carcinoma subtype, which may be of use both diagnostically and therapeutically.

Oncogenic ALK Fusion in Rare and Aggressive Subtype of Colorectal Adenocarcinoma as a Potential Therapeutic Target

Purpose: Chromosomal translocations in the anaplastic lymphoma kinase (ALK) gene have been identified as oncogenic drivers in lung adenocarcinomas and other tumors, recently including rare cases of colorectal carcinoma. We identified a patient with refractory metastatic colorectal carcinoma harboring a STRN–ALK gene fusion who achieved an exceptional clinical benefit to the ALK inhibitor ceritinib. Our goal was to further define the clinicopathologic features of ALK-rearranged colorectal carcinoma in a large cohort. Experimental Design: Clinical cases of colorectal carcinoma evaluated by comprehensive genomic profiling (CGP) or by ALK immunohistochemistry (IHC) were reviewed retrospectively. FISH and microsatellite instability (MSI) analyses were performed. Results: Nine colorectal carcinoma cases harbored ALK gene fusions. Six cases were identified by CGP of 3,157 colorectal carcinoma (0.2%) and three by IHC of 2,980 colorectal carcinoma (0.1%). The ALK fusions involved known ALK partners EML4, C2orf44, CAD, and the novel STRN, PPP1R21, SENPF, MAPRE3, and PRKAP1B partners. These advanced-stage colorectal carcinomas lacked mutations in other oncogenic drivers, predominantly involved the proximal colon, and often exhibited MSI and mucinous phenotype. The index patient was treated with the ALK inhibitor ceritinib, resulting in a marked decrease in size of a skin metastasis, and resolution by computerized tomography of all contrast enhancing tumor. After 9 months of treatment, biopsy of progressive disease demonstrated a KRAS mutation, consistent with acquired resistance to ceritinib. Conclusions: Colorectal carcinoma harboring ALK fusions represent a rare aggressive subtype of colorectal carcinoma with distinct clinicopathologic features. This report provides the first clinical evidence that such patients may benefit from targeted monotherapy with ALK inhibitors. Clin Cancer Res; 22(15); 3831–40. ©2016 AACR.

Primary Undifferentiated Sarcoma of the Kidney Harboring a Novel Variant of CIC-DUX4 Gene Fusion.

To the Editor: In recent issues of this journal, Yoshida et al1 reported on CIC-rearranged sarcomas, and Falzarano et al2 described renal cell carcinomas (RCCs) in patients previously treated for neuroblastoma (NB). Herein, we describe a primary renal CIC-DUX4 sarcoma, originally diagnosed as Wilms tumor (WT), in a patient previously treated for contralateral stage 4 NB. CIC-DUX4 sarcoma was retrospectively suspected after WT1 nuclear staining was reported in 95% and 70% of CIC-rearranged sarcomas.1,3 CIC-DUX4 undifferentiated sarcomas are a group of EWSR1negative primitive tumors or Ewinglike sarcomas with fusion of the human homologue of the Drosophila capicua (CIC) gene on chromosome 19 and the double-homeobox 4 (DUX4) retrogene on chromosome 4 or 10.1,3 Occasional peripheral tumors harbor variant translocation in which the CIC gene is fused with the forkhead box O4 (FOXO4) gene on the X chromosome.4 Yoshida et al1 reported that the majority of their CIC-rearranged sarcomas demonstrated nuclear immunoreactivity for WT1 protein, are positive for calretinin, and negative for NKX2.2. The vast majority of these sarcomas occur primarily in peripheral soft tissues, but visceral cases including the brain are reported.1,5,6

Quantification of three beta-lactam antibiotics in breast milk and human plasma by hydrophilic interaction liquid chromatography/positive-ion electrospray ionization mass spectrometry

The use of cephalosporins during breast feeding raises several issues, including the risk of drug exposure through breast milk for the infant. In this paper, a hydrophilic interaction liquid chromatography/positive ion electrospray mass spectrometric assay (HILIC/ESI-MS) was developed for the quantitation of cefuroxime, cefoxitin, and cefazolin in breast milk and human plasma. The assay was based on the use of small sample size, 25 μL of biological samples, following acetonitrile precipitation of proteins and filtration that enabled injection into the HILIC/ESI-MS system. All analytes and the internal standard, alfuzosin, were separated by using a ZIC®-HILIC analytical column (150.0 × 2.1 mm i.d., particle size 3.5 µm, 200 Å) with isocratic elution. The mobile phase was composed of a 6% 12.5 mM ammonium acetate water solution in acetonitrile and pumped at a flow rate of 0.25 mL min-1 . The assay was linear over a concentration range of 0.2 to 5 µg mL-1 and 0.4 to 20 µg mL-1 for all the analytes in breast milk and in human plasma, respectively. Intermediate precision was found to be less than 4.2% over the tested concentration ranges. A run time of less than 12 min for each sample made it possible to analyze a large number of biological samples per day. The method is the first reported application of HILIC in the analysis of antibiotics in breast milk and human plasma and it can be used to support a wide range of clinical studies. Copyright

Differentiating tumor heterogeneity in formalin‐fixed paraffin‐embedded (FFPE) prostate adenocarcinoma tissues using principal component analysis of matrix‐assisted laser desorption/ionization imaging mass spectral data

RATIONALE Many patients with adenocarcinoma of the prostate present with advanced and metastatic cancer at the time of diagnosis. There is an urgent need to detect biomarkers that will improve the diagnosis and prognosis of this disease. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is playing a key role in cancer research and it can be useful to unravel the molecular profile of prostate cancer biopsies. METHODS MALDI imaging data sets are highly complex and their interpretation requires the use of multivariate statistical methods. In this study, MALDI-IMS technology, sequential principal component analysis (PCA) and two-dimensional (2-D) peak distribution tests were employed to investigate tumor heterogeneity in formalin-fixed paraffin-embedded (FFPE) prostate cancer biopsies. RESULTS Multivariate statistics revealed a number of mass ion peaks obtained from different tumor regions that were distinguishable from the adjacent normal regions within a given specimen. These ion peaks have been used to generate ion images and visualize the difference between tumor and normal regions. Mass peaks at m/z 3370, 3441, 3447 and 3707 exhibited stronger ion signals in the tumor regions. CONCLUSIONS This study reports statistically significant mass ion peaks unique to tumor regions in adenocarcinoma of the prostate and adds to the clinical utility of MALDI-IMS for analysis of FFPE tissue at a molecular level that supersedes all other standard histopathologic techniques for diagnostic purposes used in the current clinical practice. Copyright

Differentiating tumor heterogeneity in FFPE prostate adenocarcinoma tissues using PCA analysis of MALDI IMS spectral data

RATIONALE Many patients with adenocarcinoma of the prostate present with advanced and metastatic cancer at the time of diagnosis. There is an urgent need to detect biomarkers that will improve the diagnosis and prognosis of this disease. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is playing a key role in cancer research and it can be useful to unravel the molecular profile of prostate cancer biopsies. METHODS MALDI imaging data sets are highly complex and their interpretation requires the use of multivariate statistical methods. In this study, MALDI-IMS technology, sequential principal component analysis (PCA) and two-dimensional (2-D) peak distribution tests were employed to investigate tumor heterogeneity in formalin-fixed paraffin-embedded (FFPE) prostate cancer biopsies. RESULTS Multivariate statistics revealed a number of mass ion peaks obtained from different tumor regions that were distinguishable from the adjacent normal regions within a given specimen. These ion peaks have been used to generate ion images and visualize the difference between tumor and normal regions. Mass peaks at m/z 3370, 3441, 3447 and 3707 exhibited stronger ion signals in the tumor regions. CONCLUSIONS This study reports statistically significant mass ion peaks unique to tumor regions in adenocarcinoma of the prostate and adds to the clinical utility of MALDI-IMS for analysis of FFPE tissue at a molecular level that supersedes all other standard histopathologic techniques for diagnostic purposes used in the current clinical practice. Copyright

ARID1A alteration in aggressive urothelial carcinoma and variants of urothelial carcinoma

ARID1A mutation leads to loss of the products of this tumor-suppressor gene. Studies demonstrated ARID1A mutation in 20% of stage IV urothelial carcinomas (UCs) with worse prognosis. The expression of ARID1A in aggressive variants of UC is not studied properly. From 2000 to 2015, 81 variants of UC (29 micropapillary, 33 sarcomatoid, 31 small cell, 2 nested, and 3 plasmacytoid variants) were identified in the archives of Rhode Island Hospital. Immunohistochemistry for anti-ARID1A antibody (Sigma-Aldrich, St Louis, MO) was performed. The staining pattern was semiquantitatively scored, and results were analyzed by Fisher exact test (2 tailed) on contingency tables, survival curve, and log-rank test. Patients were predominantly male (78%) with mean age of 67.9 years. The plasmacytoid variant group occurred in younger ages (mean: 54 years). Half of the specimens contained concurrent conventional UCs. Normal urothelium invariably exhibited strong ARID1A nuclear staining. There was no difference in expression between upper and lower tracts. ARID1A expression was lower in the variants compared with conventional UCs (P<.0001). In micropapillary UCs, an inverse correlation between stage and ARID1A expression was noted, with significant correlation between ARID1A expression and overall survival (P=.0221). Sarcomatoid UCs and small cell CCs showed lower ARID1A expression compared with UCs that was not statistically significant, and neither showed any significant correlation with stage or overall survival. ARID1A expression is significantly decreased in higher stages of UC and its aggressive variants; therefore, ARID1A mutation appears to play an important role in the prognosis of UC and its aggressive variants. This finding may have therapeutic implications.

Application of BRAF V600E mutation analysis for the diagnosis of metanephric adenofibroma.

To the Editor: In a recent issue of this journal, Udager et al1 demonstrated novel BRAF mutations in metanephric adenoma (MA) in addition to BRAF V600E mutations as had been previously described in approximately 90% of MAs. MA is thought to represent one end of a spectrum of rare metanephric neoplasms that include metanephric adenofibroma (MAF) and metanephric stromal tumor (MST).3,4 MAF is a benign biphasic tumor composed of varying proportion of epithelial and stromal components most commonly seen in children (mean age 10.2 y in patients with typical histologic features). In contrast to MAs, the biological nature and molecular characterization of the epithelial and stromal components of MAF have not been investigated. Currently, our knowledge of MAF and MST is based on pure morphology and limited immunophenotypic characterization. We recently encountered a tumor in an open kidney biopsy from a 10-year-old girl with a history of recurrent urinary tract infections who was found to have a solid and cystic lesion of her right upper pole kidney by imaging studies. She had no systemic manifestation, such as hypertension or polycythemia. A posterior approach was used to obtain 1.1 1.0 0.3 cm lesional tissue that was submitted for frozen section diagnosis with the intention of proceeding to nephrectomy if a diagnosis of Wilms tumor (WT) was rendered. Upon review of the histology, the tumor demonstrated a biphasic growth pattern (Figs. 1A–C). There was a 5mm well-circumscribed highly cellular epithelial nodule composed of packed tubules and papillary structures with calcifications typical of MA (Fig. 1B). There was a dominant moderately cellular stromal component composed of fibroblast-like spindle cells with entrapped native renal tubules surrounded by a loose fibromyxoid collarette (onion-skinning) typical of MST (Fig. 1C). Immunohistochemistry demonstrated positive staining of the epithelial component for WT-1, CD57, and cadherin 17, and negative staining for CK7 and AMACR, whereas the stromal component was focally positive for CD34 and WT-1.3–5 BRAF V600E mutation status was assessed separately in the epithelial (tumor cellularity 95%) and stromal (tumor cellularity 50%) components macrodissected from formalin-fixed paraffin-embedded sections (Fig. 1A). Initial screening by mutant allele–specific real-time polymerase chain reaction (PCR) with SYBR green that was carried out on the LightCycler 2.0 (Roche, Indianapolis, IN) demonstrated mutated BRAF products (as defined by a cycle threshold value [Ct]<35 in mutant PCRs) in both epithelial and stromal components (Fig. 1D). Post-PCR melting curve analysis demonstrated a single narrow melting peak for BRAF mutant– amplified reactions in both epithelial and stromal components (81.37± 0.061C), confirming specific amplification. The results of PCR were confirmed by direct Sanger sequencing (Fig. 1D). In addition, we evaluated 3 cases of MA and 3 cases of epithelial Wilms tumor (e-WT) for BRAF V600E mutation. All MAs harbored V600E mutation, whereas all e-WTs had wildtype BRAF. The immunohistochemical profile and molecular testing supported the morphologic diagnosis of MAF and reassured the clinical team that conservative management was appropriate. Subsequently, a partial nephrectomy was performed rather than complete nephrectomy for WT. The entirely submitted 3.0-cm tumor (Fig. 1E) contained only the stromal component similar to MST. In addition to the findings seen in the biopsy specimen, foci of sclerosis with intermixed cellular epithelioid stromal cells were present at the peripheral aspect of the lesion (Fig. 2A). No angiodysplasia, juxtaglomerular hyperplasia, or heterologous elements were present in the stromal component of the biopsy or excision specimen as has been previously described for MST and MAF. Angiodysplasia in the form of epithelioid transformation of medial smooth muscle has been described in approximately two thirds of MAF and the majority of MSTs but is not a constant finding.3,4 Juxtaglomerular cell hyperplasia is typically seen in pure MST rather than MAF, whereas heterologous elements can be seen in a subset of both tumors.3,4 For confirmation of the biopsy findings, molecular testing was performed on the excisional specimen (Fig. 1E) containing exclusively stromal component and was found to be positive for the BRAF V600E mutation. In contrast, sections composed entirely of non-neoplastic renal parenchyma were negative for the BRAF V600E mutation. The post-PCR melting curve of the stromal component demonstrated a similar peak to the biopsy samples, whereas a robust specific melting peak was absent in the non-neoplastic renal parenchyma. The PCR findings were also confirmed by Sanger sequencing. We subsequently performed BRAF V600E immunohistochemistry using the mutation-specific VE-1 monoclonal antibody (Ventana Medical Systems, Tucson, AZ) to correlate with the molecular findings. A melanoma and MA with a known BRAF V600E mutation were used as positive controls. There was moderate to strong positive staining in over half of the cells of the epithelial component but no significant staining of the spindled stromal component of the biopsy (Fig. 2B). However, sections of the excision specimen demonstrated variable weak to moderate positive staining that was most conspicuous in epithelioid stromal cells of the cellular foci (Figs. 2C, D). Our findings underscore the application of recently reported molecular LETTER TO THE EDITOR

Role of immune microenvironment in gastrointestinal stromal tumours

The immune microenvironment is a prognostic factor for various malignancies. The significance of key players of this immune microenvironment, including tumour‐infiltrating lymphocytes (TILs) and expression of programmed death‐ligand 1 (PD‐L1), indoleamine 2,3‐dioxygenase (IDO) and tryptophanyl‐tRNA synthetase (WARS) in gastrointestinal stromal tumours (GISTs) is largely unknown.

Clinicopathologic and gene expression analysis of initial biopsies from patients with eosinophilic esophagitis refractory to therapy

Some patients with eosinophilic esophagitis (EoE) do not respond to therapy. The clinicopathologic characteristics and gene expression profile at time of presentation could help predict response to therapy. Refractory EoE was defined as persistence of symptoms and biopsies with histologic features of EoE after 6 months of therapy with proton pump inhibitors and topical corticosteroids. Initial biopsies from refractory EoE patients (n=21), responder to therapy (n=8), patients who relapsed (n=6), and reflux controls (n=24) were studied. RNA was isolated from a subset of cases, and gene expression analysis of 285 genes involved in inflammation was performed using NanoString technology. There was no difference in the presenting symptoms among groups. The number of eosinophils/high-power field among nonresponders was higher (66±15) than responders (39±8; P<.0001) and similar to patients who relapsed (62±11). Six genes were expressed by at least 4-fold compared with reflux at a false discovery rate < 0.05, including overexpression of ALOX15, CCL26, FCER2, RTNLB, and RNASE2, and underexpression of DSG1. EoE patients refractory to therapy or who relapsed showed a trend toward higher ALOX15 expression compared with patients with good response to therapy (364.4- and 425-fold change, P=.097 and P=.07). RTNLB was significantly overexpressed in patients who were refractory to therapy versus those who responded favorably (10-fold versus 3-fold; P<.01). In conclusion, the number of eosinophils/high-power field in the initial biopsy inversely correlates with therapy response. Overexpression of RTNLB in refractory-to-therapy patients and overexpression of ALOX15 and CCL26 suggest that they are critical in the EoE pathogenesis.