SHP2 regulates the development of intestinal epithelium by modifying OSTERIX+ crypt stem cell self-renewal and proliferation.

The protein tyrosine phosphatase SHP2, encoded by PTPN11, is ubiquitously expressed and essential for the development and/or maintenance of multiple tissues and organs. SHP2 is involved in gastrointestinal (GI) epithelium development and homeostasis, but the underlying mechanisms remain elusive. While studying SHP2's role in skeletal development, we made osteoblast-specific SHP2 deficient mice using Osterix (Osx)-Cre as a driver to excise Ptpn11 floxed alleles. Phenotypic characterization of these SHP2 mutants unexpectedly revealed a critical role of SHP2 in GI biology. Mice lacking SHP2 in Osx+ cells developed a fatal GI pathology with dramatic villus hypoplasia. OSTERIX, an OB-specific zinc finger-containing transcription factor is for the first time found to be expressed in GI crypt cells, and SHP2 expression in the crypt Osx+ cells is critical for self-renewal and proliferation. Further, immunostaining revealed the colocalization of OSTERIX with OLFM4 and LGR5, two bona fide GI stem cell markers, at the crypt cells. Furthermore, OSTERIX expression is found to be associated with GI malignancies. Knockdown of SHP2 expression had no apparent influence on the relative numbers of enterocytes, goblet cells or Paneth cells. Given SHP2's key regulatory role in OB differentiation, our studies suggest that OSTERIX and SHP2 are indispensable for gut homeostasis, analogous to SOX9's dual role as a master regulator of cartilage and an important regulator of crypt stem cell biology. Our findings also provide a foundation for new avenues of inquiry into GI stem cell biology and of OSTERIX's therapeutic and diagnostic potential.

Aggressive CD4/CD8 Double-Negative Primary Cutaneous T-Cell Lymphoma With Dural Invasion: A Rare Presentation of Mycosis Fungoides?

ABSTRACT: Mycosis fungoides (MF) is primarily characterized by epidermotropic CD3+/CD4+/CD45RO+ memory T cells. CD4/CD8 double-negative MF is an uncommon variant with no presumed prognostic significance. Despite the variability in the clinical course and presentation of MF, most cases behave indolently. About 5% of patients, however, advance to stage IV with visceral organ involvement. Central nervous system metastasis in MF is rare with no known cases of direct central nervous system invasion by MF to date. We report an exceedingly rare locally aggressive case of CD4/CD8 double-negative MF with direct dural invasion and underline pertinent diagnostic challenges encountered in our case.

Analysis of Intestinal Metaplasia Without Dysplasia in the Urinary Bladder Reveal Only Rare Mutations Associated With Colorectal Adenocarcinoma.

Intestinal metaplasia (IM) is a rare finding in urinary bladder specimens. It is unclear whether IM without dysplasia is a precursor of malignancy in the urinary system. We retrospectively selected 9 cases of IM of bladder (1 case harboring high-grade dysplasia), and performed mutation analysis for genes frequently mutated in colon cancer including BRAF, APC, KRAS, MET, NRAS, PIK3CA, CTNNB1, FBXW7, and TP53 using validated clinical tests. Control groups included 7 colonic tubular adenomas, 10 high-grade papillary urothelial carcinomas. One IM case revealed an APC mutation and another showed an NRAS mutation. Among the tubular adenomas cases, 6 of 7 (85.7%) harbored KRAS mutations and 3 of 7 (42%) APC mutations. Among urothelial carcinomas cases, 1 revealed a KRAS mutation, 2 had PIK3CA mutations, and all cases were negative for APC mutations. Clinical follow-up for the IM patients was available with a median follow-up of 70 months. One patient-without any mutation in the genes investigated-developed invasive bladder adenocarcinoma with intestinal differentiation with metastasis to the liver and lung. Neither of the 2 patients harboring mutations developed any malignancy. In conclusion, a minority of cases with IM without dysplasia bear mutations in the genes commonly associated with colonic adenocarcinoma, suggesting a premalignant potential for such lesions possibly following the classic multistep chromosomal instability pathway of carcinogenesis. A larger cohort of patients with longer follow-up is needed to better establish whether close follow-up is warranted for mutation-harboring IM of the bladder.

Endometrial Adenocarcinomas With Significant Mucinous Differentiation: A Characterization of Intratumoral Heterogeneity of KRAS Mutations in Mucinous and Endometrioid Histologic Components.

OBJECTIVE: KRAS mutations are frequently seen in malignancies with mucinous morphology. In our previous study, mucinous endometrial carcinomas were associated with a significantly higher frequency of KRAS mutations as compared with matched conventional endometrioid carcinomas. This study expands our previous report by exploring possible intratumoral heterogeneity for KRAS gene mutations in the mucinous components of mucinous carcinomas (MCs) and endometrioid carcinomas with significant mucinous differentiation (ECSMD) versus their associated "usual" endometrioid components. MATERIALS AND METHODS: KRAS-positive cases from our previous report were studied, including 10 MCs and 10 ECSMDs. The specimens were microscopically dissected to separately isolate morphologically mucinous and endometrioid components. Direct DNA sequencing for KRAS mutations at codons 12 and 13 using capillary electrophoresis were performed. RESULTS: KRAS mutations were detected in the endometrioid components of 8 (80%) of 10 MCs and 3 (30%) of 10 ECSMDs. The endometrioid component of the ECSMD group was less frequently associated with KRAS mutation than the endometrioid component of the MC group, even when the mucinous component of the same tumor contained a mutation; the difference is statistically significant (P < 0.05). CONCLUSIONS: Our current study shows that intratumoral heterogeneity for KRAS gene mutation was associated with ECSMD, but less frequently with MC. It is possible that when the mucinous component predominates, qualifying for an MC, KRAS mutations appear to be widespread, irrespective of the mucinous or nonmucinous differentiation of the tumor cells. The findings suggest that multiple samples for KRAS tests may be useful, especially in endometrioid carcinoma with significant mucinous differentiation.

The clinical significance of K-ras mutation in endometrial "surface epithelial changes" and their associated endometrial adenocarcinoma.

OBJECTIVES: The entity of 'surface epithelial changes' (SECs) was first described in 1995 [1]. Morphologically, SECs usually arise from malignant glands at the superficial aspect of well differentiated (WD) endometrioid carcinomas (ECs) and impart the appearance of a 'maturational' phenomenon at the surface of the cancer. Exhibiting a paradoxically bland histologic appearance, SECs typically show morphologic features that mimic benign entities, particularly endocervical microglandular hyperplasia (MGH). SECs have been associated with approximately half of WD endometrioid carcinomas many of which showed focal mucinous differentiation. Despite their morphologically benign histology, some have questioned whether the presence of SECs represents a 'marker' for an underlying malignancy, especially in postmenopausal women with endocervical or MGH-type SECs in their endometrial sampling. Since the biologic nature of SECs is unknown, we aimed to study the prevalence of KRAS gene mutations in SECs and the underlying WD endometrioid adenocarcinomas (EC) from which they directly arise. METHODS: 24 cases with biopsy proven SECs and ECs in their subsequent hysterectomy were retrieved. Genomic DNA was extracted from formalin-fixed paraffin-embedded tissue. PCR amplification for KRAS codons 12 and 13 was performed, followed by sequencing using capillary electrophoresis. RESULTS: KRAS codons 12 and 13 mutations were detected in 19 of 24 (79%) SECs, and 19 of 24 (79%) ECs. All SECs had the same KRAS mutation as the underlying EC. CONCLUSIONS: Our results suggest that SECs are of neoplastic origin and that KRAS mutations play an important role in the tumorigenesis of ECs and SECs.

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.

KRAS Mutations in Mucinous Lesions of the Uterus.

OBJECTIVES: The current study examined the KRAS mutation status in a spectrum of mucinous lesions of the uterus, including mucinous metaplasia (MM), atypical mucinous proliferation (AMP), endocervical mucosa, and microglandular hyperplasia (MGH). METHODS: Thirty-nine cases, including 15 AMPs, nine MMs, nine MGHs, and six normal endocervical mucosas, were selected from the departmental archive. All AMP cases with follow-up biopsies or hysterectomies were reviewed. Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissue and KRAS codons 12 and 13 sequence analyzed. RESULTS: KRAS codon 12 and 13 mutations were detected in 10 (67%) of the 15 AMP cases. No KRAS mutations were identified in MMs, MGHs, and endocervical mucosas (P = .002, AMP vs MM or MGH, Fisher exact test). Most women with AMP were postmenopausal (13/15 [86.7%]) and presented with dysfunctional uterine bleeding. Among the 10 cases of AMP harboring KRAS mutations, six (60%) cases were subsequently diagnosed with carcinoma, one with atypical complex hyperplasia, and two with AMP within endometrial polyps. CONCLUSIONS: The results suggest a possible association between KRAS mutations and mucinous differentiation in endometrial carcinogenesis. KRAS status can help in assessing benign from precursor or malignant mucinous lesions as well as differentiate endometrial lesions from those of cervical origin.

Comparison of tyrosine kinase receptors HER2, EGFR, and VEGFR expression in micropapillary urothelial carcinoma with invasive urothelial carcinoma.

Invasive micropapillary urothelial carcinomas (MPUC) emerge at higher stages and follow a more aggressive course than conventional invasive urothelial carcinomas (UC). Little is known about the target therapies using tyrosine kinase inhibitors in MPUC. This study is to investigate potential effectiveness of tyrosine kinase receptor inhibitors by determining expression of epidermal growth factor receptor (EGFR), human epidermal receptor 2 (HER2), and vascular endothelial growth factor receptor 2 (VEGFR) proteins in MPUC and UC. 16 cases of MPUC and 16 stage-matched UC were identified. Immunohistochemistry for EGFR, HER2, and VEGFR2 and HER2 gene amplification by fluorescence in situ hybridization (FISH) were performed. HER2 and EGFR proteins were expressed in MPUC and UC, with significantly higher HER2 expression in MPUC (ratio 1.82, p < 0.01). HER2 gene amplification was identified in 4 of 16 MPUC (25 %). Amplification was limited to cases with 3+ HER2 expression (100% concordance). EGFR expression in MPUC was slightly higher than UC but not statistically significant (ratio 1.57, p = 0.19). EGFR and HER2 coexpression was noted in 75% of MPUC and 37.5% of UC. No VEGFR expression was identified in the urothelium. Strong VEGFR expression was noted in stromal vessels in both MPUC and UC. In conclusion, EGFR and HER2 are potential targets for neoadjuvant chemotherapy in MPUC and UC. There is no direct anti-tumor effect expected for VEGFR inhibitors.

Analysis of T-cell clonality using laser capture microdissection and high-resolution microcapillary electrophoresis.

Identification of clonal lymphocytic populations by polymerase chain reaction may be difficult in cases with scant cellular infiltrates or those with a heterogeneous population of cells. Here, we assessed the diagnostic utility of laser capture microdissection (LCM) and high-resolution microcapillary electrophoresis in the analysis of clonality of small biopsy specimens. Clonality was determined in 24 cases: five reactive tonsils, five reactive lymph nodes, six inflammatory skin lesions, and eight T-cell lymphomas. CD3-positive T lymphocytes were captured by LCM from paraffinized immunohistochemically stained sections. Genomic DNA was analyzed for T-cell receptor-gamma gene rearrangement by polymerase chain reaction followed by high-resolution microcapillary electrophoresis with the DNA 500 LabChip and the Agilent Bioanalyzer. In the reactive specimens, T-cell receptor-gamma polymerase chain reaction revealed monoclonal bands when 10 to 1000 cells were captured. This pattern changed to polyclonal when higher numbers of cells were microdissected (2000 to 10,000 cells). In contrast, lymphoma cells were consistently monoclonal whether low or high numbers were microdissected. Microcapillary electrophoresis coupled with LCM facilitated clonality analysis in equivocal cases. In two of eight lymphoma cases, LCM revealed diagnostic monoclonal bands, whereas routine T-cell receptor-gamma assessment of whole tissue sections with 10% polyacrylamide gel electrophoresis demonstrated only minor clonal bands. We conclude that clonality determined by LCM is cell number-dependent. Biopsy specimens containing low numbers of reactive polyclonal T cells may produce pseudomonoclonal bands and therefore should be interpreted with great caution.

Postmortem RNA and protein stability in perinatal human lungs.

The availability of fetal and neonatal lung tissue is an invaluable resource to elucidate the molecular regulation of human lung development. In this study, we have investigated the mRNA and protein stability of perinatal lung tissues treated with RNA (Ambion Inc., Austin, TX) or snap frozen in liquid nitrogen (LN ). Lung samples were obtained from 25 consecutive perinatal autopsies of live-born and stillborn infants (median gestational age, 23 weeks) with various clinical presentations. Treatment of lung tissue with RNA yielded more total RNA and protein than LN freezing. The integrity of RNA, assessed by spectrophotometry and gel electrophoresis, was equivalent between both tissue preservation methods, and both methods produced RNA suitable for reverse transcriptase-polymerase chain reaction analysis of representative genes (beta-actin and surfactant protein-B [SP-B]). Similarly, the protein integrity of RNA -treated tissues was equivalent to that of LN -frozen tissues, as judged by Western blot analysis of SP-B/actin protein expression. Although the total yield was similar in live-born, nonmacerated stillborn and macerated stillborn infants, only RNA and protein from live-born or nonmacerated stillborn infants was suitable for subsequent molecular analyses. Within the 41-hour range studied, the duration of the postmortem interval did not affect the yield or integrity of RNA and protein with either tissue preservation method. In summary, high-quality RNA and protein, suitable for routine molecular analyses, can be obtained from postmortem lung tissue from live-born and nonmacerated stillborn infants, even with prolonged postmortem intervals. RNA is equivalent, if not superior, to LN for preservation of postmortem RNA and protein in developing human lungs.

Beta-carotene alters the morphology of NCI-H69 small cell lung cancer cells.

The effect of beta-carotene on the morphology of NCI-H69 small cell lung cancer cells that had undergone beta-carotene-induced growth reduction (P < 0.05) was examined. The cells were grown at 1 x 10(8) cells/L and were cultured with or without 20 micromol/L beta-carotene. The qualitative electron microscopic observations revealed that beta-carotene-treated cells contained more vacuoles than control cells not treated with beta-carotene. The quantitative image analysis showed a significantly smaller (P < 0.05) value of the nuclear roundness factor for treated cells compared with control cells, indicating an irregular nuclear morphology of beta-carotene-treated cells. The major diameter of the cells and the minor diameter of the nuclei were significantly smaller (P < 0.05), and the nuclear perimeter was significantly larger (P < 0.05) in beta-carotene-treated cells. The ratio of nucleus to cytoplasm was significantly less (P < 0.05) in beta-carotene-treated cells compared with control cells, indicating a less malignant growth of the cells. These results demonstrate that the treatment of small cell lung cancer cells with beta-carotene induces morphological changes in the cells concomitant with a reduction in their proliferation. Further investigation is required to show a direct effect of beta-carotene or its intracellular polar metabolites on the morphology of these cells.