Dual Targeting of EGFR and IGF1R in the TNFAIP8 Knockdown Non-Small Cell Lung Cancer Cells.

Aberrant regulation of EGFR is common in non-small cell lung carcinomas (NSCLC), and tumor resistance to targeted therapies has been attributed to emergence of other co-occurring oncogenic events, parallel bypass receptor tyrosine kinase pathways including IGF1R, and TNFα-driven adaptive response via NF-κB. TNFAIP8, TNFα-inducible protein 8, is an NF-κB-activated prosurvival and oncogenic molecule. TNFAIP8 expression protects NF-κB-null cells from TNFα-induced cell death by inhibiting caspase-8 activity. Here, we demonstrate that knockdown of TNFAIP8 inhibited EGF and IGF-1-stimulated migration in NSCLC cells. TNFAIP8 knockdown cells showed decreased level of EGFR and increased expression of sorting nexin 1 (SNX1), a key regulator of the EGFR trafficking through the endosomal compartments, and treatment with SNX1 siRNA partially restored EGFR expression in these cells. TNFAIP8 knockdown cells also exhibited downregulation of IGF-1-induced pIGF1R and pAKT, and increased expression of IGF-1-binding protein 3 (IGFBP3), a negative regulator of the IGF-1/IGF1R signaling. Consistently, treatment of TNFAIP8 knockdown cells with IGFBP3 siRNA restored pIGF1R and pAKT levels. TNFAIP8 knockdown cells had enhanced sensitivities to inhibitors of EGFR, PI3K, and AKT. Furthermore, IHC expression of TNFAIP8 was associated with poor prognosis in NSCLC. These findings demonstrate TNFAIP8-mediated regulation of EGFR and IGF1R via SNX1 and IGFBP3, respectively. We posit that TNFAIP8 is a viable, multipronged target downstream of the TNFα/NF-κB axis, and silencing TNFAIP8 may overcome adaptive response in NSCLC. IMPLICATIONS: TNFAIP8 and its effectors SNX1 and IGFBP3 may be exploited to improve the efficacy of molecular-targeted therapies in NSCLC and other cancers.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/5/1207/F1.large.jpg.

Image Processing and Analysis of Mucosal Calretinin Staining to Define the Transition Zone in Hirschsprung Disease: A Pilot Study.

PURPOSE: Quantification of calretinin-stained mucosal nerve fibers by image processing and analysis (IPA) may objectively define the transition zone (TZ) of Hirschsprung disease (HD). We tested the utility of IPA as an adjunctive tool in HD. MATERIALS AND METHODS: Calretinin immunostain was performed on 15 HD pull-through specimens, and multiple images were captured from the proximal aganglionic zone, TZ, and probable normal zone (NZ). Pixel count (PC), defined as the percentage of brown-stained pixels in the mucosa, was quantified and plotted against distance from the rectal distal end. To validate the method, PCs from 45 images were compared with three-tiered visual scoring by five pathologists. Results were correlated against pertinent variables, which were retrieved from the clinical record. RESULTS: The PC gradually increased in the TZ toward the proximal resection margin in 10/13 (77%) cases. The PC variation in the probable NZ and around the circumference was substantial by the coefficient of variation. The mean PC of images with a visual score of 1 was less than scores of 2 and 3 by all five (100%) pathologists (p < 0.01). One patient had possible TZ pull-through that was clinically confirmed. CONCLUSION: While the mucosal calretinin staining gradually increases in the TZ, for now, the boundaries of the TZ cannot be accurately defined by mucosal biopsies given the substantial variation of staining around the circumference at the same distance and in the NZ. However, the IPA technique does provide a continuous variable and warrants further utility in HD studies.

Novel Finding of Paired Box 5 (PAX5) Cytoplasmic Staining in Well-differentiated Rectal Neuroendocrine Tumors (Carcinoids) and Its Diagnostic and Potentially Prognostic Utility.

Although nuclear immunostaining for paired box protein (PAX5) is widely used in practice, its cytoplasmic localization has not been evaluated. Recently we encountered cytoplasmic granular PAX5 staining in rectal well-differentiated neuroendocrine tumor (WD-NET) in the absence of nuclear staining. We investigated the specificity of this staining pattern for rectal NET (n=21) in comparison with 108 NETs, 1 WD rectal NET with elevated proliferation (WD-NET G3), and 40 poorly differentiated neuroendocrine carcinomas from the gastrointestinal and pancreatobiliary tract and liver. Representative tumor sections were subject to immunohistochemical stain for PAX5 antibody. Immunohistochemistry for 3 L-cell markers, glucagon-like peptide 1 and 2, and peptide YY, was performed on all rectal and appendiceal NETs and all other NETs with cytoplasmic PAX5 staining. Cytoplasmic PAX5 staining was observed in 90% (19/21) of rectal NET, 27% (3/11) of appendiceal, 14% (2/14) of pancreatic, 7% (2/29) of lung, 25% (3/12) metastatic NET in the liver, and 100% (1/1) of renal NET. No PAX5 cytoplasmic staining was seen in all grades of NET in other organs, rectal WD-NET G3, and all neuroendocrine carcinoma. L-cell marker staining was observed in all 21 (100%) rectal, in 3 of 3 (100%) PAX5-positive, and 1 of 7 (14%) PAX5-negative appendiceal NET. Cytoplasmic PAX5 staining is specific for rectal carcinoids. The sensitivity and specificity of PAX5 to detect L-cell type rectal carcinoids is 90% (19/21) and 100% (21/21), respectively. Cytoplasmic localization of the PAX5 protein may be utilized as a surrogate marker to detect L-cell type rectal carcinoids.

The expression of TTF1, CDX2 and ISL1 in 74 poorly differentiated neuroendocrine carcinomas.

BACKGROUND: The expression profile of immunohistochemical markers of origin in poorly differentiated neuroendocrine carcinoma (PDNEC) is not well studied. MATERIALS AND METHODS: Seventy-four PDNECs from gastroenteropancreatic (GEP) organs and the lung, including 48 large cell NEC (LCNEC) and 26 small cell carcinomas (SmCC), were subject to immunohistochemical staining for CDX2, TTF1 and ISL1. The staining intensity (1 to 3) and percentage of positive tumor cells [0 (negative), 1 (<50%) and 2 (≥50%)] were assessed. The multiplicative index (maximum 6) was calculated and the average total score (aTS) was determined for each primary site and histologic subtype. RESULTS: In the 38 GEP and 36 lung PDNECs, CDX2, TTF1 and ISL1 staining was observed in 71% (aTS 2.8), 16% (aTS 0.4), 63% (aTS 1.9), and 22% (aTS 0.6), 72% (aTS 2.9) and 92% (aTS 3.8), respectively. GEP PDNECs showed a higher aTS for CDX2 and lower aTS for TTF1 and ISL1, compared to that of lung PDNECs (Student's t-test, p < 0.001). SmCC had a higher aTS for TTF1 and ISL1 (p < 0.001) and lower aTS for CDX2 (p < 0.002) than that of LCNEC. CONCLUSIONS: CDX2 and TTF1 demonstrate potential utility in suggesting the primary site of PDNEC. In addition, CDX2 may be useful in supporting the diagnosis of LCNEC in cases with overlapping or borderline morphology. Utility of ISL1 as an adjunctive diagnostic marker of SmCC remains to be studied.

Paired Box 5 (PAX5) Expression in Poorly Differentiated Neuroendocrine Carcinoma of the Gastrointestinal and Pancreatobiliary Tract: Diagnostic and Potentially Therapeutic Implications.

Paired Box 5 (PAX5), a well-established B-cell marker, is preferentially expressed in small cell lung carcinoma and regulates the transcription of c-Met, offering a potential for therapeutic target. Its expression in poorly differentiated neuroendocrine carcinoma (PDNEC) of the digestive system has not been systemically evaluated. Archived pathology materials from 38 PDNEC in the gastrointestinal (GI) and pancreatobiliary (PB) tract were reviewed. Representative tumor sections were subject to immunohistochemical stain for PAX5, c-Met, and CD20. The extent of the staining [focal (<10%), patchy (10% to 50%), and diffuse (>50%)] and intensity (1+ to 3+) was evaluated. In total, 38 cases of well-differentiated neuroendocrine tumors from GI/PB tract served as controls. Nuclear PAX5 staining was observed in 16 (42%) cases in total, in 46% (11/24) of large cell neuroendocrine carcinoma, 67% (4/6) of small cell neuroendocrine carcinoma, and 13% (1/8) of mixed adenoneuroendocrine carcinoma, with diffuse (8), patchy (4), or focal (4) staining. The intensity was 3+ (2), 2+ (6), and 1+ (8). PAX5 expression was common in ampullary (4/5) and gastroesophageal junctional/esophageal (5/9) PDNEC. Two (5%) of 38 well-differentiated neuroendocrine tumors were positive for PAX5. Three PAX5 positive PDNEC showed weak cytoplasmic c-Met immunolabeling. CD20 was negative in all tumors. Our data show that PAX5 is commonly expressed in PDNEC of the GI/PB tract including small cell neuroendocrine carcinoma. This observation warrants a cautious approach when interpreting small biopsy of poorly differentiated neoplasms, especially when lymphoma is considered in the differentials. Further study of PAX5/c-Met signaling pathway and its potential therapeutic value in GI/PB PDNEC is warranted.

PD-L1 protein expression in tumour cells and immune cells in mismatch repair protein-deficient and -proficient colorectal cancer: the foundation study using the SP142 antibody and whole section immunohistochemistry.

AIMS: Routine application of PD-L1 immunohistochemistry (IHC) in colorectal cancer (CRC) is limited due to lack of standardized scoring criteria, antibody clones, and intratumoral staining heterogeneity. We assessed PD-L1 protein expression on full face CRC tissue sections and applied two algorithms based on the published clinical trials that support the recent FDA approval for immune checkpoint inhibitors (ICPI) therapy in non-small cell lung cancer (NSCLC). METHODS: PD-L1/CD274 IHC (Roche/Ventana, clone SP142) was performed on representative tumour blocks from 52 mismatch repair-deficient (MMR-D) and 52 MMR-proficient (MMR-P) CRCs. Membranous PD-L1 expression was scored for the tumour cell (TC) and tumour-infiltrating immune cell (IC) components. PD-L1 positivity status was determined based on the published NSCLC clinical trials that utilized the Ventana SP142 assay. Hybrid capture-based comprehensive genomic profiling (CGP) was performed on a separate set of 2268 clinically advanced CRCs and the frequency of PD-L1/PD-L2 amplification was determined. RESULTS: PD-L1 expression in the TC and IC correlated with MMR-D (p=0.013, p<0.0001), T stage (p=0.036, p=0.0036) and clinical stage (p=0.022, p=0.0037). PD-L1 positivity status correlated with MMR-D by two algorithms. Five of 2268 (<1%) advansced CRCs demonstrated amplification of either the PD-L1 or PD-L2 genes by CGP. CONCLUSIONS: PD-L1 expression in TC and IC is associated with advanced stage and MMR-D. PD-L1 positivity status by the published algorithm is associated with MMR-D. PD-L1 amplification is extremely uncommon in CRC. Evaluation of whole tissue section and incorporation of IC staining enhance the sensitivity to screen patients who may benefit from ICPI therapy.

ALK Fusions in a Wide Variety of Tumor Types Respond to Anti-ALK Targeted Therapy.

BACKGROUND: Genomic fusions of the anaplastic lymphoma kinase gene (ALK) are a well-established therapy target in non-small cell lung cancer (NSCLC). From a survey of 114,200 clinical cases, we determined the prevalence of ALK rearrangements (rALK) in non-NSCLC tumors and report their responsiveness to therapies targeting ALK. MATERIALS AND METHODS: Comprehensive genomic profiling of 114,200 relapsed and metastatic malignancies, including both solid tumors and hematolymphoid cancers, was performed using a hybrid-capture, adaptor ligation-based next-generation sequencing assay. RESULTS: Of 114,200 clinical samples, 21,522 (18.8%) were NSCLC and 92,678 (81.2%) were other tumor types. Of the 876 (0.8%) cases with ALK fusions (fALK) or rALK, 675 (77.1%) were NSCLC and 201 (22.9%) were other tumor types. ALK fusions were significantly more frequent in NSCLC (3.1%) than non-NSCLC (0.2%; p < .0001). Patients with non-NSCLC tumors harboring fALK were significantly younger (p < .0001) and more often female (p < .0001) than patients with fALK-positive NSCLC. EML4 was more often the fusion partner in NSCLC (83.5%) versus non-NSCLC tumors (30.9%; p < .0001). CONCLUSION: ALK rearrangements can be identified in a wide variety of epithelial and mesenchymal malignancies beyond NSCLC. Anti-ALK therapies can be effective in non-NSCLC tumors driven by fALK, and further study of therapies targeting ALK in clinical trials involving a wider variety of cancer types appears warranted. IMPLICATIONS FOR PRACTICE: Rearrangements involving the ALK gene have been detected in dozens of cancer types using next-generation sequencing. Patients whose tumors harbor ALK rearrangements or fusions respond to treatment with crizotinib and alectinib, including tumors not normally associated with ALK mutations, such as non-Langerhans cell histiocytosis or renal cell carcinoma. Comprehensive genomic profiling using next-generation sequencing can detect targetable ALK fusions irrespective of tumor type or fusions partner.

Comprehensive genomic profiling of malignant phyllodes tumors of the breast.

PURPOSE: Malignant phyllodes tumors (MPT) are exceptionally rare, and the genomic drivers of these tumors are still being elucidated. We performed comprehensive genomic profiling (CGP) of MPT to identify genomic alterations that will inform approaches to targeted therapy for patients with MPT, including relapsed, refractory, and metastatic disease. METHODS: DNA was extracted from formalin-fixed, paraffin-embedded samples from 24 consecutive patient cases of MPT. CGP was performed using a hybrid capture, adaptor ligation-based next generation sequencing assay to a high, uniform coverage (mean, 582×). Tumor mutational burden (TMB) was calculated from a minimum of 1.14 Mb of sequenced DNA as previously described and reported as mutations/Mb. The results were analyzed for all classes of genomic alterations, including short variants (SV; base substitutions, small insertions, and deletions), rearrangements, and copy number changes, including amplifications and homozygous deletions. RESULTS: The 24 cases of MPT included 15 patients with localized and 9 with metastatic disease. The median TMB was 2.7 mut/Mb, and no cases had a TMB > 10 mut/Mb. 20 out of 24 cases were evaluable for microsatellite status, and all were microsatellite stable. The most commonly mutated genes were TP53 (58.3%), TERT-promoter (57.9%), NF1 (45.8%), MED12 (45.8%), CDKN2A/B (33.3%), and MLL2 (33.3%). Targetable kinase fusions including KIAA1549-BRAF or FGFR3-TACC3 were identified in 2/24 (8.3%) tumors. CONCLUSIONS: This study identifies clinically relevant genomic alterations that suggest novel targeted therapy approaches for patients with MPT.

Next-Generation Sequencing Reveals Pathway Activations and New Routes to Targeted Therapies in Cutaneous Metastatic Melanoma.

BACKGROUND: Comprehensive genomic profiling of clinical samples by next-generation sequencing (NGS) can identify one or more therapy targets for the treatment of metastatic melanoma (MM) with a single diagnostic test. METHODS: NGS was performed on hybridization-captured, adaptor ligation-based libraries using DNA extracted from 4 formalin-fixed paraffin-embedded sections cut at 10 microns from 30 MM cases. The exons of 182 cancer-related genes were fully sequenced using the Illumina HiSeq 2000 at an average sequencing depth of 1098X and evaluated for genomic alterations (GAs) including point mutations, insertions, deletions, copy number alterations, and select gene fusions/rearrangements. Clinically relevant GAs (CRGAs) were defined as those identifying commercially available targeted therapeutics or therapies in registered clinical trials. RESULTS: The 30 American Joint Committee on Cancer Stage IV MM included 17 (57%) male and 13 (43%) female patients with a mean age of 59.5 years (range 41-83 years). All MM samples had at least 1 GA, and an average of 2.7 GA/sample (range 1-7) was identified. The mean number of GA did not differ based on age or sex; however, on average, significantly more GAs were identified in amelanotic and poorly differentiated MM. GAs were most commonly identified in BRAF (12 cases, 40%), CDKN2A (6 cases, 20%), NF1 (8 cases, 26.7%), and NRAS (6 cases, 20%). CRGAs were identified in all patients, and represented 77% of the GA (64/83) detected. The median and mean CRGAs per tumor were 2 and 2.1, respectively (range 1-7). CONCLUSION: Comprehensive genomic profiling of MM, using a single diagnostic test, uncovers an unexpectedly high number of CRGA that would not be identified by standard of care testing. Moreover, NGS has the potential to influence therapy selection and can direct patients to enter relevant clinical trials evaluating promising targeted therapies.

A novel histologic grading scheme based on poorly differentiated clusters is applicable to treated rectal cancer and is associated with established histopathological prognosticators.

The conventional histologic grading of colorectal cancer (CRC) is less suited for resected rectal cancer following neoadjuvant chemoradiation. Enumeration of poorly differentiated clusters (PDC) is a recently proposed histologic grading scheme. We aimed to apply PDC grading to treated rectal cancer and to test the prognostic significance of this novel approach. Archived hematoxylin and eosin slides of 72 rectal adenocarcinomas resected following neoadjuvant treatment were retrieved. PDC, tumor budding, and tumor regression were assessed. The parameters were correlated with clinicopathological features and survival. PDC was strongly associated with tumor budding, perineural invasion (PNI), metastasis, and low degree of tumor regression. Tumor budding was significantly associated with lymphovascular invasion and PNI, and metastasis. Tumors with a lower degree of regression were more likely to show high pathologic T stage and advanced clinical stage. Local recurrence was associated with poor survival. PDC did not correlate with overall survival. PDC grading is applicable to resected rectal cancer status post neoadjuvant treatment and correlates with established histopathological prognosticators. PDC and tumor budding may represent a histologic spectrum reflective of the same biological significance. Validation and incorporation of these simple histologic grading schemes may strengthen the prognostic power of the histologic parameters that influence the oncologic outcome in treated rectal cancer. Further study to evaluate the significance of PDC as an oncologic prognosticator is warranted.

Genomic profiling of advanced-stage, metaplastic breast carcinoma by next-generation sequencing reveals frequent, targetable genomic abnormalities and potential new treatment options.

CONTEXT: Metastatic metaplastic breast carcinoma (MPBC) is an uncommon, but aggressive, tumor resistant to conventional chemotherapy. OBJECTIVE: To learn whether next-generation sequencing could identify potential targets of therapy for patients with relapsed and metastatic MPBC. DESIGN: Hybridization capture of 3769 exons from 236 cancer-related genes and 47 introns of 19 genes commonly rearranged in cancer was applied to a minimum of 50 ng of DNA extracted from 20 MPBC formalin-fixed, paraffin-embedded specimens and sequenced to high uniform coverage. RESULTS: The 20 patients with MPBC had a median age of 62 years (range, 42-86 years). There were 9 squamous (45%), 9 chondroid (45%), and 2 spindle cell (10%) MPBCs, all of which were high grade. Ninety-three genomic alterations were identified, (range, 1-11) with 19 of the 20 cases (95%) harboring an alteration that could potentially lead to a targeted treatment option. The most-common alterations were in TP53 (n = 69; 75%), PIK3CA (n = 37; 40%), MYC (n = 28; 30%), MLL2 (n = 28; 30%), PTEN (n = 23; 25%), CDKN2A/B (n = 19; 20%), CCND3 (n = 14; 15%), CCNE1 (n = 9; 10%), EGFR (n = 9; 10%), and KDM6A (n = 9; 10%); AKT3, CCND1, CCND2, CDK4, FBXW7, FGFR1, HRAS, NF1, PIK3R1, and SRC were each altered in a single case. All 16 MPBCs (100%) that were negative for ERBB2 (HER2) overexpression by immunohistochemistry and/or ERBB2 (HER2) amplification by fluorescence in situ hybridization were also uniformly (100%) negative for ERBB2 amplification by next-generation sequencing-based copy-number assessment. CONCLUSIONS: Our results indicate that genomic profiling using next-generation sequencing can identify clinically meaningful alterations that have the potential to guide targeted treatment decisions in most patients with metastatic MPBC.

Histological Spectrum of Idiopathic Noncirrhotic Portal Hypertension in Liver Biopsies From Dialysis Patients.

OBJECTIVES: Liver biopsy is performed for various indications in dialysis patients. Being a less-common subset, the hepatic pathology in renal dialysis is not well documented. Idiopathic noncirrhotic portal hypertension (INCPH) is a clinical entity associated with unexplained portal hypertension and/or a spectrum of histopathological vascular changes in the liver. After encountering INCPH and vascular changes of INCPH in 2 renal dialysis patients, we sought to further investigate this noteworthy association. MATERIALS AND METHODS: A random search for patients on hemodialysis or peritoneal dialysis with liver biopsy was performed. Hematoxylin and eosin, reticulin, trichrome, and CK7 stains were performed on formalin-fixed, paraffin-embedded tissue sections. Histopathological features were reviewed, and the results were correlated with clinical findings. RESULTS: In all, 13 liver biopsies were retrieved. The mean cumulative duration of dialysis was 50 months (range = 17 months to 11 years). All patients had multiple comorbidities. Indications for biopsy were a combination of abnormal liver function tests (6), portal hypertension (4), ascites (3), and possible cirrhosis (3). Two patients with portal hypertension underwent multiple liver biopsies for diagnostic purposes. All (100%) biopsies showed some histological features of INCPH, including narrowed portal venous lumen (9), increased portal vascular channels (8), shunt vessels (3), dilated sinusoids (9), regenerative nodule (5), and features of venous outflow obstruction (3). No cirrhosis was identified. CONCLUSION: Liver biopsies from patients on dialysis demonstrate histopathological vascular changes of INCPH. Some (31%) patients present with portal hypertension without cirrhosis. The histological changes may be reflective of underlying risk factors for INCPH in this group.

New routes to targeted therapy of intrahepatic cholangiocarcinomas revealed by next-generation sequencing.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is a subtype of primary liver cancer that is rarely curable by surgery and is rapidly increasing in incidence. Relapsed ICC has a poor prognosis, and current systemic nontargeted therapies are commonly extrapolated from those used in other gastrointestinal malignancies. We hypothesized that genomic profiling of clinical ICC samples would identify genomic alterations that are linked to targeted therapies and that could facilitate a personalized approach to therapy. METHODS: DNA sequencing of hybridization-captured libraries was performed for 3,320 exons of 182 cancer-related genes and 36 introns of 14 genes frequently rearranged in cancer. Sample DNA was isolated from 40 µm of 28 formalin-fixed paraffin-embedded ICC specimens and sequenced to high coverage. RESULTS: The most commonly observed alterations were within ARID1A (36%), IDH1/2 (36%), and TP53 (36%) as well as amplification of MCL1 (21%). Twenty cases (71%) harbored at least one potentially actionable alteration, including FGFR2 (14%), KRAS (11%), PTEN (11%), CDKN2A (7%), CDK6 (7%), ERBB3 (7%), MET (7%), NRAS (7%), BRCA1 (4%), BRCA2 (4%), NF1 (4%), PIK3CA (4%), PTCH1 (4%), and TSC1 (4%). Four (14%) of the ICC cases featured novel gene fusions involving the tyrosine kinases FGFR2 and NTRK1 (FGFR2-KIAA1598, FGFR2-BICC1, FGFR2-TACC3, and RABGAP1L-NTRK1). CONCLUSION: Two thirds of patients in this study harbored genomic alterations that are associated with targeted therapies and that have the potential to personalize therapy selection for to individual patients.

Comprehensive genomic profiling of relapsed and metastatic adenoid cystic carcinomas by next-generation sequencing reveals potential new routes to targeted therapies.

We hypothesized that next-generation sequencing could reveal actionable genomic alterations (GAs) and potentially expand treatment options for patients with advanced adenoid cystic carcinoma (ACC). Genomic profiling using next-generation sequencing was performed on hybridization-captured, adapter ligation libraries derived from 28 relapsed and metastatic formalin-fixed paraffin-embedded ACC. The 3230 exons of 182 cancer-related genes and 37 introns of 14 genes frequently rearranged in cancer were fully sequenced using the Illumina HiSeq 2000. All classes of GAs were evaluated. Actionable GAs were defined as those impacting targeted anticancer therapies on the market or in registered clinical trials. A total of 44 GAs were identified in the 28 ACC tumors, with 12 of 28 (42.9%) of tumors harboring at least 1 potentially actionable GA. The most common nonactionable GAs were identified in KD6MA (5 cases; 18%), ARID1A (4 cases; 14%), RUNX1 (2 cases; 7%), and MYC (2 cases; 7%). Actionable GAs included NOTCH1 (3 cases; 11%), MDM2 (2 cases; 7%), PDGFRA (2 cases; 7%), and CDKN2A/B (p16) (2 cases; 7%). Other potentially actionable GAs identified in a single case included: mutations in AKT1, BAP1, EGFR, and PIK3CA, homozygous deletion of FBXW7, and amplifications of CDK4, FGFR1, IGF1R, KDR, KIT, and MCL1. The frequency of GA in ACC is lower than that seen in the more common solid tumors. Comprehensive genomic profiling of ACC can identify actionable GAs in a subset of patients that could influence therapy for these difficult-to-treat progressive neoplasms.

A high frequency of activating extracellular domain ERBB2 (HER2) mutation in micropapillary urothelial carcinoma.

PURPOSE: Micropapillary urothelial carcinoma (MPUC) is a rare and aggressive form of bladder cancer. We conducted genomic analyses [next-generation sequencing (NGS)] of MPUC and non-micropapillary urothelial bladder carcinomas (non-MPUC) to characterize the genomic landscape and identify targeted treatment options. EXPERIMENTAL DESIGN: DNA was extracted from 40 µm of formalin-fixed paraffin-embedded sections from 15 MPUC and 64 non-MPUC tumors. Sequencing (NGS) was performed on hybridization-captured, adaptor ligation-based libraries to high coverage for 3,230 exons of 182 cancer-related genes plus 37 introns from 14 genes frequently rearranged in cancer. The results were evaluated for all classes of genomic alteration. RESULTS: Mutations in the extracellular domain of ERBB2 were identified in 6 of 15 (40%) of MPUC: S310F (four cases), S310Y (one case), and R157W (one case). All six cases of MPUC with ERBB2 mutation were negative for ERBB2 amplification and Erbb2 overexpression. In contrast, 6 of 64 (9.4%) non-MPUC harbored an ERBB2 alteration, including base substitution (three cases), amplification (two cases), and gene fusion (one case), which is higher than the 2 of 159 (1.3%) protein-changing ERBB2 mutations reported for urinary tract cancer in COSMIC. The enrichment of ERBB2 alterations in MPUC compared with non-MPUC is significant both between this series (P < 0.0084) and for all types of urinary tract cancer in COSMIC (P < 0.001). CONCLUSIONS: NGS of MPUC revealed a high incidence of mutation in the extracellular domain of ERBB2, a gene for which there are five approved targeted therapies. NGS can identify genomic alteration, which inform treatment options for the majority of MPUC patients.

Advanced urothelial carcinoma: next-generation sequencing reveals diverse genomic alterations and targets of therapy.

Although urothelial carcinoma (UC) of the urinary bladder generally portends a favorable prognosis, metastatic tumors often follow an aggressive clinical course. DNA was extracted from 40 µm of formalin-fixed, paraffin-embedded (FFPE) sections from 35 stage IV UCs that had relapsed and progressed after primary surgery and conventional chemotherapy. Next-generation sequencing (NGS) was performed on hybridization-captured, adaptor ligation-based libraries for 3320 exons of 182 cancer-related genes plus 37 introns from 14 genes frequently rearranged in cancer to at an average sequencing depth of 1164 × and evaluated for all classes of genomic alterations (GAs). Actionable GAs were defined as those impacting the selection of targeted anticancer therapies on the market or in registered clinical trials. A total of 139 GAs were identified, with an average of 4.0 GAs per tumor (range 0-10), of which 78 (56%) were considered actionable, with an average of 2.2 per tumor (range 0-7). Twenty-nine (83%) cases harbored at least one actionable GA including: PIK3CA (9 cases; 26%); CDKN2A/B (8 cases; 23%); CCND1 (5 cases; 14%); FGFR1 (5 cases; 14%); CCND3 (4 cases; 11%); FGFR3 (4 cases; 11%); MCL1 (4 cases; 11%); MDM2 (4 cases; 11%); EGFR (2 cases, 6%); ERBB2 (HER2/neu) (2 cases, 6%); NF1 (2 cases, 6%) and TSC1 (2 cases, 6%). Notable additional alterations included TP53 (19 cases, 54%) and RB1 (6 cases; 17%). Genes involved in chromatin modification were altered by nonsense mutation, splice site mutation or frameshift indel in a mutually exclusive manner in nearly half of all cases including KDM6A (10 cases; 29%) and ARID1A (7 cases; 20%). Comprehensive NGS of 35 UCs of the bladder revealed a diverse spectrum of actionable GAs in 83% of cases, which has the potential to inform treatment decisions for patients with relapsed and metastatic disease.

Clinical next-generation sequencing successfully applied to fine-needle aspirations of pulmonary and pancreatic neoplasms.

BACKGROUND: Next-generation sequencing was performed on pulmonary and pancreatic fine-needle aspirations (FNAs) and on paired FNAs and resected primary tumors from the same patient. METHODS: DNA was isolated in formalin-fixed, paraffin-embedded cell blocks from 16 pulmonary FNAs, 23 pancreatic FNAs, and 5 resected pancreatic primary tumors. Next-generation sequencing was performed for 4561 exons of 287 cancer-related genes and for 47 introns of 19 genes on indexed, adaptor-ligated, hybridization-captured libraries using a proprietary sequencing system (the Illumina HiSeq 2000). RESULTS: Genomic profiles were generated successfully from 16 of 16 (100%) pulmonary FNAs, which included 14 nonsmall cell lung cancers (NSCLCs) and 2 small cell lung cancers (SCLCs). The NSCLC group included 6 adenocarcinomas, 5 squamous cell carcinomas, and 3 NSCLCs not otherwise specified. Genomic profiles were successfully obtained from 23 of 23 (100%) pancreatic FNAs and from 5 of 5 (100%) matched pancreatic primary tumors, which included 17 ductal adenocarcinomas, 3 mucinous adenocarcinomas, 2 adenocarcinomas NOS, and 1 neuroendocrine tumor. Eighty-one genomic alterations were identified in the 16 pulmonary FNAs (average, 5.1 genomic alterations per patient); and the most common genomic alterations were TP53, RB1, SOX2, PIK3CA, and KRAS. Eighty-seven genomic alterations were identified in the 23 pancreatic tumor FNAs (average, 3.8 genomic alterations per patient); and the most common genomic alterations were KRAS, TP53, CDKN2A/B, SMAD4, and PTEN. Among the pancreatic tumors, there was 100% concordance of 20 genomic alterations that were identified in 5 patient-matched FNA and surgical primary tumor pairs. CONCLUSIONS: The authors were able to perform next-generation sequencing reliably on FNAs of pulmonary and pancreatic tumors, and the genomic alterations discovered correlated well with those identified in matched resected pancreatic tumors.

Relapsed classic E-cadherin (CDH1)-mutated invasive lobular breast cancer shows a high frequency of HER2 (ERBB2) gene mutations.

PURPOSE: We queried whether comprehensive genomic profiling using a next-generation sequencing-based assay could identify novel and unanticipated targets of therapy for patients with relapsed invasive lobular carcinoma (ILC). EXPERIMENTAL DESIGN: DNA sequencing (Illumina HiSeq 2000) was conducted for 3,320 exons of 182 cancer-related genes and 37 introns of 14 genes frequently rearranged in cancer on indexed, adaptor-ligated, hybridization-captured libraries using DNA isolated from formalin-fixed paraffin-embedded sections from 22 histologically verified ILC. RESULTS: A total of 75 genomic alterations were identified with an average of 3.4 alterations per tumor (range, 1-6), of which 35 were actionable for an average of 1.59 actionable alterations per patient (range, 0-3). Nineteen of 22 (86%) of the ILC samples harbored at least one actionable alteration. Six (27%) cases featured alterations in ERRB2 including 4 (18%) with ERBB2 mutation, 1 (5%) with an ERBB2 gene fusion, and 1 (5%) with an ERBB2 copy number gain (amplification). The enrichment of ERBB2 mutations/fusion in CDH1-mutated ILC (5 of 22, 23%) compared with the 5 ERBB2 mutations in a series of 286 non-CDH1-mutated breast cancers from which the ILC cases were obtained (5 of 286, 2%) was significant (P = 0.0006). CONCLUSIONS: Comprehensive genomic profiling of relapsed CDH1-mutated ILC revealed actionable genomic alterations in 86% of cases, featured a high incidence of ERBB2 alterations, and can reveal actionable alterations that can inform treatment decisions for patients with ILC.

The significance of TNFAIP8 in prostate cancer response to radiation and docetaxel and disease recurrence.

TNFAIP8 is a NF-κB-inducible, oncogenic molecule. Previous "promoter array" studies have identified differential methylation and regulation of TNFAIP8 in prostate epithelial and cancer cell lines. Here we demonstrate that TNFAIP8 expression is induced by androgen in hormone-responsive LNCaP prostate cancer cells. In athymic mice bearing hormone-refractory PC-3 prostate tumor xenografts, intravenous treatment with a liposomal formulation of TNFAIP8 antisense oligonucleotide (LE-AS5) caused reduced expression of TNFAIP8 in tumor tissues, and a combination of LE-AS5 and radiation or docetaxel treatment resulted in significant inhibition of PC-3 tumor growth as compared to single agents. The immunohistochemical evaluation of TNFAIP8 expression revealed correlation of both cytoplasmic and nuclear TNFAIP8 overexpression with high grade prostatic adenocarcinomas, while nuclear overexpression was found to be an independent predictor of disease recurrence controlling for tumor grade. Increased nuclear TNFAIP8 expression was statistically significantly associated with a 2.44 fold (95 % confidence interval: 1.01-5.91) higher risk of prostate cancer recurrence. Mechanistically, TNFAIP8 seems to function as a scaffold (or adaptor) protein. In the antibody microarray analysis of proteins associated with the TNFAIP8 immune-complex, we have identified Karyopherin alpha2 as a novel binding partner of nuclear TNFAIP8 in PC-3 cells. The Ingenuity Pathway Analysis of the TNFAIP8 interacting proteins suggested that TNFAIP8 influences cancer progression pathways and networks involving integrins and matrix metalloproteinases. Taken together, present studies demonstrate that TNFAIP8 is a novel therapeutic target in prostate cancer, and indicate a potential relationship of the nuclear trafficking of TNFAIP8 with adverse outcomes in a subset of prostate cancer patients.