Pten-NOLC1 fusion promotes cancers involving MET and EGFR signalings.

Inactivation of Pten gene through deletions and mutations leading to excessive pro-growth signaling pathway activations frequently occurs in cancers. Here, we report a Pten derived pro-cancer growth gene fusion Pten-NOLC1 originated from a chr10 genome rearrangement and identified through a transcriptome sequencing analysis of human cancers. Pten-NOLC1 fusion is present in primary human cancer samples and cancer cell lines from different organs. The product of Pten-NOLC1 is a nuclear protein that interacts and activates promoters of EGFR, c-MET, and their signaling molecules. Pten-NOLC1 promotes cancer proliferation, growth, invasion, and metastasis, and reduces the survival of animals xenografted with Pten-NOLC1-expressing cancer cells. Genomic disruption of Pten-NOLC1 induces cancer cell death, while genomic integration of this fusion gene into the liver coupled with somatic Pten deletion produces spontaneous liver cancers in mice. Our studies indicate that Pten-NOLC1 gene fusion is a driver for human cancers.

Donor mucosal immunocytes perpetuate refractory GVHD after intestinal transplantation without engrafting in recipient bone marrow: Case report and review of the literature.

GVHD as a complication of SOT presents both a diagnostic and therapeutic challenge. Typically affecting the skin, gastrointestinal tract, and liver, GVHD occurs when donor lymphocytes engrafted in recipient tissues are activated by host antigen-presenting cells resulting in cytokine release and donor cell-mediated cytotoxicity to host tissue. Here, we describe a 5-year-old girl who developed fatal, refractory GVHD after isolated intestinal transplantation when recipient immune cells failed to repopulate the allograft in the setting of CMV viremia. Persistence of the donor immune cells in the allograft mucosa, rather than engraftment in the recipient bone marrow, likely perpetuated this refractory GVHD. Early diagnosis and intervention are critical to reduce morbidity and mortality. Thus, periodic monitoring of peripheral blood and allograft mucosal chimerism with sensitive detection methods may allow early detection and potentially curative enterectomy in similar cases of refractory GVHD.

Correlation of Classic and Molecular Cytogenetic Alterations in Soft-Tissue Sarcomas: Analysis of 46 Tumors With Emphasis on Adipocytic Tumors and Synovial Sarcoma.

INTRODUCTION: Sarcomas are heterogeneous, and their treatment and prognosis are driven by the morphologic subtype and the clinical stage. Classic cytogenetics and fluorescence in situ hybridization (FISH) analysis play an important role in their diagnostic work up. MATERIALS AND METHODS: Forty-six cases of soft-tissue sarcoma were reviewed that underwent karyotyping and simultaneous FISH analysis at initial diagnosis. They included 10 dedifferentiated liposarcomas, 10 myxoid liposarcomas, and 14 synovial sarcomas. Six tumors were investigated for EWSR1 rearrangement. Six high-grade miscellaneous sarcomas were also examined. RESULTS: The dedifferentiated liposarcoma had complex karyotypes and MDM2 amplification by FISH, and of these, 5 tumors with myxoid changes also had complex signals for DDIT3. All but 4 myxoid liposarcomas had complex karyotypes, in addition to the characteristic translocation. FISH analysis displayed DD1T3 rearrangement. All synovial sarcomas except 1 recurrence had a t(X;18) translocation by karyotyping and FISH. The EWSR1 rearrangement was present in all extraskeletal myxoid chondrosarcomas, angiomatoid fibrous histiocytoma, atypical Ewing sarcoma, and a clear-cell sarcoma, all of which had characteristic karyotypes. Seven high-grade sarcomas had no specific karyotype or rearrangements for DDIT3, SS18, and EWSR1 by FISH. CONCLUSIONS: There is good correlation between karyotyping and FISH. Complex FISH signals found in dedifferentiated liposarcomas may be related to an increased chromosome 12 copy number and ploidy. Karyotyping is an important baseline standard for the quality assurance of newly developed FISH probes. It also provides a global view of chromosomal changes and the opportunity to investigate the role of other genetic alterations and potential therapeutic targets.

Validation and utilization of a TFE3 break-apart FISH assay for Xp11.2 translocation renal cell carcinoma and alveolar soft part sarcoma.

BACKGROUND: Xp11.2 or TFE3 translocation renal cell carcinomas (RCC) and alveolar soft part sarcoma (ASPS) are characterized by chromosome translocations involving the Xp11.2 breakpoint resulting in transcription factor TFE3 gene fusions. The most common translocations documented in TFE3 RCCs are t(X;1) (p11.2;q21) and t(X;17) (p11.2;q25) which leads to fusion of TFE3 gene on Xp11.2 with PRCC or ASPL respectively. TFE3 immunohistochemistry (IHC) has been inconsistent over time due to background staining problems in part related to fixation issues. Karyotyping to detect TFE3 gene rearrangement requires typically unavailable fresh tissue. Reverse transcriptase-polymerase chain reaction (RT-PCR) is generally very challenging due to degradation of RNA in archival material. The study objective was to develop and validate a TFE3 break-apart fluorescence in situ hybridization (FISH) assay to confirm Xp11 translocation RCCs and ASPS. METHODS: Representative sections of formalin-fixed paraffin-embedded tissue blocks were selected in 40 possible cases. Approximately 60 tumor cells were analyzed in the targeted region. The validation of TFE3 FISH was done with 11 negative and two positive cases. Cut off for a positive result was validated as >7.15 % positive nuclei with any pattern of break-apart signals. FISH evaluation was done blinded of the immunohistochemical or karyotype data. RESULTS: Three out of forty cases were positive for the TFE3 break-apart signals by FISH. The negative cases were reported as clear cell RCC with papillary features (10), clear cell RCC with sarcomatoid areas (2), Papillary RCC with clear cell areas (9), Chromophobe RCC (2), RCC, unclassified type (3) and renal medullary carcinoma (1). 3 of the negative cases were consultation cases for renal tumor with unknown histology. Seven negative cases were soft tissue tumor suspicious for ASPS. CONCLUSION: Our study validates the utility of TFE3 break-apart FISH on formalin-fixed paraffin-embedded tissue sections for diagnosis and confirmation of Xp11.2 translocation RCCs and ASPS.

Novel fusion transcripts associate with progressive prostate cancer.

The mechanisms underlying the potential for aggressive behavior of prostate cancer (PCa) remain elusive. In this study, whole genome and/or transcriptome sequencing was performed on 19 specimens of PCa, matched adjacent benign prostate tissues, matched blood specimens, and organ donor prostates. A set of novel fusion transcripts was discovered in PCa. Eight of these fusion transcripts were validated through multiple approaches. The occurrence of these fusion transcripts was then analyzed in 289 prostate samples from three institutes, with clinical follow-up ranging from 1 to 15 years. The analyses indicated that most patients [69 (91%) of 76] positive for any of these fusion transcripts (TRMT11-GRIK2, SLC45A2-AMACR, MTOR-TP53BP1, LRRC59-FLJ60017, TMEM135-CCDC67, KDM4-AC011523.2, MAN2A1-FER, and CCNH-C5orf30) experienced PCa recurrence, metastases, and/or PCa-specific death after radical prostatectomy. These outcomes occurred in only 37% (58/157) of patients without carrying those fusion transcripts. Three fusion transcripts occurred exclusively in PCa samples from patients who experienced recurrence or PCaerelated death. The formation of these fusion transcripts may be the result of genome recombination. A combination of these fusion transcripts in PCa with Gleason's grading or with nomogram significantly improves the prediction rate of PCa recurrence. Our analyses suggest that formation of these fusion transcripts may underlie the aggressive behavior of PCa.

A validation study of quantum dot multispectral imaging to evaluate hormone receptor status in ductal carcinoma in situ of the breast.

The assessment of hormone receptors, including estrogen receptor and progesterone receptor, has become a standard practice in breast cancer management. However, the need for multiple sections to evaluate each receptor individually by conventional immunohistochemistry may preclude the analysis on some core biopsies with a limited amount of tumors. The aim of the study was to validate the quantitative analysis of nuclear markers estrogen receptor and progesterone receptor by quantum dot-based immunohistochemistry using a multispectral imaging system in ductal carcinoma in situ of the breast. Consecutive sections from a total of 17 cases of ductal carcinoma in situ with excisional biopsies or mastectomies were stained with conventional immunohistochemistry and quantum dot-based, single- and double-labeled immunohistochemistry for estrogen receptor and progesterone receptor. The semiquantitative results from double-labeled, quantum dot-based immunohistochemistry were compared with those from single-labeled, quantum dot-based immunohistochemistry as well as from conventional immunohistochemistry. There was good concordance between double- and single-labeled quantum dot-based immunohistochemistry, and quantum dot-based immunohistochemistry correlated well with conventional immunohistochemistry (Spearman correlation coefficient range from 0.884 to 0.958, P < .001). The findings proved the validity and accuracy of quantum dot-based multiplex, multispectral technique in detecting 2 tumor markers in the same cellular compartment simultaneously on a single slide. This technique may enhance our ability to assess multiple breast tumor markers in specimens with limited available tissue. However, several technical and logistic issues await significant improvement before this novel technique can be justified for routine clinical application.

Sex-determining region Y-box 2 amplification in preneoplastic squamous lesions of the lung.

Sex-determining region Y-box 2 gene at 3q26.33 has been identified as oncogene in squamous cell carcinoma occurring at different anatomical sites including the lung. Sex-determining region Y-box 2 protein expression and gene amplification have been found in preinvasive squamous cell lesions such as dysplasia and carcinoma in situ. We sought to evaluate sex-determining region Y-box 2 expression and amplification in a spectrum of premalignant squamous lesions ranging from squamous metaplasia to low- and high-grade dysplasia to in situ and invasive squamous cell carcinoma of the lung. Each lesion was taken from 1 of 3 study groups: 18 patients with concurrent squamous cell carcinoma, 17 patients with prior squamous cell carcinoma undergoing surveillance biopsies, and 11 patients with no history of squamous cell carcinoma. Sex-determining region Y-box 2 amplification occurred only in a subset of invasive squamous cell carcinoma (3/5; 60%) and their associated high-grade dysplasia (3/4; 75%), but not in any of the low-grade dysplasias (0/1; 0%) or metaplasias (0/9; 0%). No sex-determining region Y-box 2 amplification was observed in squamous preneoplastic lesions in surveillance biopsies without current evidence of squamous cell carcinoma or in benign lungs. Sex-determining region Y-box 2 protein expression was seen in all squamous lesions regardless of presence or degree of dysplasia. Our results suggest that sex-determining region Y-box 2 amplification is not an early event in squamous carcinogenesis and is important for progression in a subset of squamous cell carcinoma. It appears that sex-determining region Y-box 2 gene amplification in lung squamous carcinogenesis is not the only regulator of sex-determining region Y-box 2 protein expression.

Chronic lymphocytic leukemia/small lymphocytic lymphoma with cyclin D1 positive proliferation centers do not have CCND1 translocations or gains and lack SOX11 expression.

Cyclin D1 expression, usually absent in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), has been described in the proliferation centers (PC) of some CLL/SLL. The prevalence of this finding is uncertain, as is the explanation for its occurrence and whether these cases have any other unique features. Cyclin D1 immunohistochemical staining was therefore investigated in 57 extramedullary CLL/SLL biopsies. In 6 cases, cyclin D1 immunofluorescence followed by CCND1 fluorescence in situ hybridization (FISH) and PC targeted analysis was performed using a Bioview Duet system. Excluding the prospectively selected cases that had the targeted FISH studies, cyclin D1+ PC were identified in 20% of cases. The cyclin D1+ CLL did not appear pathologically or phenotypically distinctive, though 46% had an interfollicular growth pattern. The cyclin D1+ PCs were SOX11- and lacked CCND1 translocations and gains in 5 of 5 informative cases. The recognition of cyclin D1 expression in PC of a significant minority of CLL/SLL can be a diagnostic aid and should not lead to the diagnosis of focal mantle cell lymphoma.

Paradoxical relationship between the degree of EGFR amplification and outcome in glioblastomas.

Glioblastoma (GBM) is the most common primary brain tumor in adults and often has amplification of the epidermal growth factor receptor (EGFR) gene. The value of EGFR as a prognostic marker in GBMs is unclear; some studies have shown an adverse correlation, whereas others have indicated a neutral or even favorable association with longer survival. Furthermore, EGFR-amplified GBMs are usually regarded as a single subgroup of tumors, although the range of EGFR copy number varies greatly. In this study, 532 GBMs were analyzed for EGFR amplification via fluorescence in situ hybridization at the time of initial diagnosis. Although there was no difference in survival by EGFR amplification (P = 0.33), stratification by the amount of EGFR amplification showed that, surprisingly, median survival was 39% longer in the high-amplifier group (EGFR:chromosome 7 ratio >20) compared to nonamplified GBMs (P = 0.03) and was 43% longer compared to GBMs with low to moderate EGFR amplification (EGFR:chromosome 7 ratio = 2 to 20; P = 0.0007). Stratifying by postsurgical treatment regimens, this difference was seen only when temozolomide (TMZ) was used; tumors without amplification and with high EGFR amplification both responded better to TMZ than those with low to moderate amplification (P = 0.01), whereas GBMs that had not been treated with adjuvant therapy nor with adjuvant therapy lacking TMZ showed no survival differences (P = 0.63 and 0.91, respectively). These results suggest that GBMs with EGFR amplification are a heterogenous group of tumors and that behavior might differ according to the degree of amplification, although not in a straightforward dose-response manner.

HER2 amplification in gastroesophageal adenocarcinoma: correlation of two antibodies using gastric cancer scoring criteria, H score, and digital image analysis with fluorescence in situ hybridization.

We assessed 103 resected gastroesophageal adenocarcinomas for HER2 amplification by fluorescence in situ hybridization (FISH) and 2 commercial immunohistochemical assays. Of 103, 30 (29%) were FISH-amplified. Both immunohistochemical assays had greater than 95% concordance with FISH. However, as a screening test for FISH amplification, the Ventana Medical Systems (Tucson, AZ) 4B5 antibody demonstrated superior sensitivity (87%) compared with the DAKO (Carpinteria, CA) A0485 (70%). Of the cases, 28 were immunohistochemically 3+ or immunohistochemically 2+/FISH-amplified with the 4B5 assay compared with only 22 cases with the A0485 assay, representing a large potential difference in patient eligibility for anti-HER2 therapy. Cases with low-level FISH amplification (HER2/CEP17, 2.2-4.0) express lower levels of HER2 protein compared with cases with high-level amplification (HER2/CEP17, ≥4.0), raising the possibility of a differential response to anti-HER2 therapy. The H score and digital image analysis may have a limited role in improving HER2 test performance.

DOG1: a novel marker of salivary acinar and intercalated duct differentiation.

Anoctamin-1 (ANO1) (DOG1, TMEM16a) is a calcium-activated chloride channel initially described in gastrointestinal stromal tumors, but now known to be expressed in a variety of normal and tumor tissues including salivary tissue in murine models. We herein perform a comprehensive survey of DOG1 expression in 156 cases containing non-neoplastic human salivary tissues and tumors. ANO1 mRNA levels were significantly higher (8-fold increase, P<0.0001) in normal parotid tissue (n=6) as compared with squamous mucosa (n=15). By immunohistochemistry, DOG1 showed a diffuse moderate (2+) apical membranous staining pattern in normal serous acini, 1+ apical membranous pattern in mucous acini, and variable 1-2+ apical staining of distal intercalated ducts. Myoepithelial cells, striated and excretory ducts were invariably negative. All acinic cell carcinomas (n=28) were DOG1 positive demonstrating a complex mixture of intense (3+) apical membranous, cytoplasmic and complete membranous staining. Most ductal tumor types were negative or only showed a subset of positive cases. Within the biphasic tumor category, adenoid cystic carcinomas (18/24 cases) and epithelial-myoepithelial carcinomas (8/15 cases) were frequently positive, often showing a distinctive combined apical ductal and membranous/cytoplasmic myoepithelial staining profile. Thus, DOG1 staining is a marker of salivary acinar and to a lesser extent intercalated duct differentiation. Strong staining can be used to support the diagnosis of acinic cell carcinoma. DOG1 may also be a marker of a 'transformed' myoepithelial phenotype in a subset of biphasic salivary gland malignancies.

Tumor epidermal growth factor receptor and EGFR PY1068 are independent prognostic indicators for head and neck squamous cell carcinoma.

PURPOSE: To assess the prognostic value of epidermal growth factor receptor (EGFR) molecular characteristics of head and neck squamous cell carcinoma (HNSCC). PATIENTS AND METHODS: HNSCC tumors from patients prospectively enrolled in either an Early Detection Research Network (EDRN) study and treated with surgery without an EGFR-targeted agent (N = 154) or enrolled in a chemoradiation trial involving the EGFR-targeted antibody cetuximab (N = 39) were evaluated for EGFR gene amplification by FISH and EGFR protein by immunohistochemical staining. Fresh-frozen tumors (EDRN) were also evaluated for EGFR protein and site-specific phosphorylation at Y992 and Y1068 using reverse-phase protein array (n = 67). Tumor (n = 50) EGFR and EGFRvIII mRNA levels were quantified using real-time PCR. RESULTS: EGFR expression by immunohistochemistry (IHC) was significantly higher in the EDRN tumors with EGFR gene amplification (P < 0.001), and a similar trend was noted in the cetuximab-treated cohort. In the EDRN and cetuximab-treated cohorts elevated EGFR by IHC was associated with reduced survival (P = 0.019 and P = 0.06, respectively). Elevated expression of total EGFR and EGFR PY1068 were independently significantly associated with reduced progression-free survival in the EDRN cohort [HR = 2.75; 95% confidence interval (CI) = 1.26-6.00 and HR = 3.29; 95% CI = 1.34-8.14, respectively]. CONCLUSIONS: In two independent HNSCC cohorts treated with or without cetuximab, tumor EGFR levels were indicative of survival. Tumor EGFR PY1068 levels provided prognostic information independent of total EGFR.

The importance of 10q status in an outcomes-based comparison between 1p/19q fluorescence in situ hybridization and polymerase chain reaction-based microsatellite loss of heterozygosity analysis of oligodendrogliomas.

1p/19q codeletion is a favorable prognostic marker of oligodendrogliomas. Although fluorescence in situ hybridization (FISH) and microsatellite-based polymerase chain reaction (PCR) for loss of heterozygosity (LOH) are common methods to test for 1p/19q codeletion, it is unclear which test is better at prognostic stratification. This study analyzed outcomes of 111 oligodendrogliomas with both 1p/19q FISH and LOH done at the time of diagnosis. Overall concordance between the 2 assays was 81.1%. In grade III oligodendrogliomas, LOH was better than FISH at survival stratification (p < 0.0001 for LOH vs p = 0.02 for FISH), although increasing the stringency of FISH interpretation criteria improved concordance and prognostic power. Oligodendrogliomas that were 1p/19q-codeleted by FISH but also had 10q LOH were negative for 1p/19q codeletion by PCR analysis in more than 70% of cases, with very poor survival in the grade III subset. Thus, although PCR-based LOH is a better stratifier of 1p/19q status, FISH still has clinical and prognostic utility, especially if 10q data can be incorporated.

Prostatic adenocarcinoma metastatic to pleomorphic liposarcoma, a "collision phenomenon": report of a case with review of pelvic collision tumors.

"Collision tumor" is an uncommon phenomenon characterized by coexistence of two completely distinct and independent tumors at the same site. Collision tumors have been reported in different sites in the body; however, these are particularly uncommon in the pelvic cavity. A 70-year-old man, with prior history of urothelial and prostate cancer, presented with a large pelvic mass detected on imaging studies. Pathological examination revealed a large liposarcoma with prostatic carcinoma embedded in it. Immunohistochemistry and florescence in situ hybridization studies were performed to reach to a conclusive diagnosis. To the best of our knowledge, this is the second case reported till date. We present the challenges encountered in the diagnosis of this case and review of pelvic collision tumors.

EGFR expression stratifies oligodendroglioma behavior.

Epidermal growth factor receptor (EGFR) expression and signaling contribute to glioma biological features and, thus, are a target for new drug development. The role, if any, of EGFR in routine surgical neuropathological diagnostics is less clear. Herein, we describe prospective EGFR IHC analysis in an adult cohort comprising 750 infiltrative gliomas. EGFR expression increased with World Health Organization grade but did not significantly differ between grade-matched astrocytic and oligodendroglial tumors. Survival did not significantly differ by EGFR expression among astrocytic tumors adjusted for World Health Organization grade. However, grade II oligodendrogliomas with strong EGFR expression and 1p/19q codeletion showed reduced survival, compared with their codeleted counterparts with weaker EGFR expression. Surprisingly, an inverse phenomenon was found with grade III anaplastic oligodendrogliomas, in which stronger EGFR expression was a favorable marker for survival. Among all gliomas, the likelihood of EGFR amplification, as viewed by fluorescence in situ hybridization, increased with the strength of EGFR expression, and <1% of cases with weak or no EGFR immunostaining showed amplification. These data suggest that EGFR IHC is useful in certain circumstances (ie, it may help supplement 1p/19q prognostic information in oligodendroglial tumors and screen out cases that would not benefit from more costly EGFR fluorescence in situ hybridization analysis).

Fluorescence in situ hybridization for detection of MAML2 rearrangements in oncocytic mucoepidermoid carcinomas: utility as a diagnostic test.

Oncocytic mucoepidermoid carcinoma poses diagnostic challenge because of its histologic overlap with other oncocytic salivary gland lesions, including Warthin tumor. Although the prognostic value of the t(11;19) MECT1-MAML2 fusion gene has been established in mucoepidermoid carcinoma, its diagnostic use in discriminating oncocytic mucoepidermoid carcinoma from histologic mimics is unexplored. We evaluated the translocation status in 14 cases of oncocytic mucoepidermoid carcinoma using a MAML2-11q21 break-apart probe spanning the entire chromosome region of the MAML2 gene and correlated these findings with clinicopathologic parameters including age, sex, stage, predominant growth pattern, grade, and p63 immunostaining pattern. All oncocytic mucoepidermoid carcinomas were parotid tumors with a mean patient age of 54.6 years (range, 9-85) and a female to male ratio of 5:2. Grade distribution was as follows: low grade, 9; intermediate grade, 2; and high grade, 3. The histologic patterns observed were as follows: solid, 4; cystic, 8 (of these, 5 had Warthin-like lymphoid stroma); and mixed, 2. Solid oncocytic mucoepidermoid carcinomas showed a diffuse p63 staining pattern, whereas cystic oncocytic mucoepidermoid carcinomas showed staining of the outer layer of intermediate cells ranging from a bilayer to areas of complex multilayering and plaque-like proliferation. Ten (71%) of the 14 cases showed a MAML2 rearrangement by fluorescence in situ hybridization. No correlation was seen between rearrangement status and histologic grade, growth pattern, or p63 staining pattern. However, we demonstrate that the presence of MAML2 rearrangement can be used as supportive evidence to distinguish oncocytic mucoepidermoid carcinoma from other oncocytic lesions.

Adenosquamous carcinoma of the lung: a microdissection study of KRAS and EGFR mutational and amplification status in a western patient population.

Molecular testing of pulmonary adenocarcinomas for EGFR and KRAS mutations is becoming more common as tyrosine kinase inhibitor therapy is used for EGFR-mutated adenocarcinomas. Adenosquamous carcinomas represent a hybrid carcinoma, and there is no literature addressing the frequency of EGFR and KRAS mutations in this subset of lung carcinomas in Western populations. For this study, 23 adenosquamous carcinomas were microdissected with the glandular and squamous components analyzed for EGFR and KRAS mutations and EGFR amplification. In 3 cases (13%), there were EGFR mutations, with 2 having the identical mutation in the glandular and squamous elements. In 3 cases (13%), there were KRAS mutations in both histologic elements. Great heterogeneity existed in the rates of EGFR amplification in the 2 histologic components. Amplification was most common in both glandular and squamous components (11/23 [48%]). EGFR mutations occur in adenosquamous carcinoma in the same percentages as in conventional adenocarcinoma in the Western population. KRAS mutations are less common.