Yuqiang Fang

Intratumoral heterogeneity of receptor tyrosine kinases EGFR and PDGFRA amplification in glioblastoma defines subpopulations with distinct growth factor response

Glioblastoma (GBM) is distinguished by a high degree of intratumoral heterogeneity, which extends to the pattern of expression and amplification of receptor tyrosine kinases (RTKs). Although most GBMs harbor RTK amplifications, clinical trials of small-molecule inhibitors targeting individual RTKs have been disappointing to date. Activation of multiple RTKs within individual GBMs provides a theoretical mechanism of resistance; however, the spectrum of functional RTK dependence among tumor cell subpopulations in actual tumors is unknown. We investigated the pattern of heterogeneity of RTK amplification and functional RTK dependence in GBM tumor cell subpopulations. Analysis of The Cancer Genome Atlas GBM dataset identified 34 of 463 cases showing independent focal amplification of two or more RTKs, most commonly platelet-derived growth factor receptor α (PDGFRA) and epidermal growth factor receptor (EGFR). Dual-color fluorescence in situ hybridization was performed on eight samples with EGFR and PDGFRA amplification, revealing distinct tumor cell subpopulations amplified for only one RTK; in all cases these predominated over cells amplified for both. Cell lines derived from coamplified tumors exhibited genotype selection under RTK-targeted ligand stimulation or pharmacologic inhibition in vitro. Simultaneous inhibition of both EGFR and PDGFR was necessary for abrogation of PI3 kinase pathway activity in the mixed population. DNA sequencing of isolated subpopulations establishes a common clonal origin consistent with late or ongoing divergence of RTK genotype. This phenomenon is especially common among tumors with PDGFRA amplification: overall, 43% of PDGFRA-amplified GBM were found to have amplification of EGFR or the hepatocyte growth factor receptor gene (MET) as well.

Retinoblastoma Has Properties of a Cone Precursor Tumor and Depends Upon Cone-Specific MDM2 Signaling

Retinoblastomas result from the inactivation of the RB1 gene and the loss of Rb protein, yet the cell type in which Rb suppresses retinoblastoma and the circuitry that underlies the need for Rb are undefined. Here, we show that retinoblastoma cells express markers of postmitotic cone precursors but not markers of other retinal cell types. We also demonstrate that human cone precursors prominently express MDM2 and N-Myc, that retinoblastoma cells require both of these proteins for proliferation and survival, and that MDM2 is needed to suppress ARF-induced apoptosis in cultured retinoblastoma cells. Interestingly, retinoblastoma cell MDM2 expression was regulated by the cone-specific RXRgamma transcription factor and a human-specific RXRgamma consensus binding site, and proliferation required RXRgamma, as well as the cone-specific thyroid hormone receptor-beta2. These findings provide support for a cone precursor origin of retinoblastoma and suggest that human cone-specific signaling circuitry sensitizes to the oncogenic effects of RB1 mutations.

Distinction of conjunctival melanocytic nevi from melanomas by fluorescence in situ hybridization

A conjunctival melanocytic nevus may on occasion be difficult to distinguish from melanoma both clinically and histopathologically. An unambiguous correct diagnosis is critical because of major differences in management and prognosis. We evaluated a fluorescence in situ hybridization (FISH) assay, which has previously been shown to be of value for the diagnosis of melanocytic nevi and melanomas of the skin, using probes targeting 6p25 (RREB1), 6q23 (MYB), 11q13 (CCND1) and centromere 6 (CEP6), for its potential to assist in the distinction of conjunctival melanocytic nevi from melanomas. Four melanocytic nevi and eight melanomas of the conjunctiva were analyzed. Two of the melanomas were diagnostically problematic because of suboptimal histopathology. None of the conjunctival melanocytic nevi showed a level of chromosomal aberrations that met FISH criteria for a diagnosis of melanoma. All eight conjunctival melanomas (six unequivocal and two suspicious lesions) met FISH criteria for melanoma. Thus, results from FISH assay targeting 6p25, 6q23, 11q13 and centromere 6 correlated well with the histopathologic diagnoses and supported the histopathologic suspicion in two problem cases. The findings encourage further exploration of this technique as an ancillary method for the work‐up of conjunctival melanocytic proliferations.

NF2 Loss Promotes Oncogenic RAS-Induced Thyroid Cancers via YAP-Dependent Transactivation of RAS Proteins and Sensitizes Them to MEK Inhibition

UNLABELLED Ch22q LOH is preferentially associated with RAS mutations in papillary and in poorly differentiated thyroid cancer (PDTC). The 22q tumor suppressor NF2, encoding merlin, is implicated in this interaction because of its frequent loss of function in human thyroid cancer cell lines. Nf2 deletion or Hras mutation is insufficient for transformation, whereas their combined disruption leads to murine PDTC with increased MAPK signaling. Merlin loss induces RAS signaling in part through inactivation of Hippo, which activates a YAP-TEAD transcriptional program. We find that the three RAS genes are themselves YAP-TEAD1 transcriptional targets, providing a novel mechanism of promotion of RAS-induced tumorigenesis. Moreover, pharmacologic disruption of YAP-TEAD with verteporfin blocks RAS transcription and signaling and inhibits cell growth. The increased MAPK output generated by NF2 loss in RAS-mutant cancers may inform therapeutic strategies, as it generates greater dependency on the MAPK pathway for viability. SIGNIFICANCE Intensification of mutant RAS signaling through copy-number imbalances is commonly associated with transformation. We show that NF2/merlin inactivation augments mutant RAS signaling by promoting YAP/TEAD-driven transcription of oncogenic and wild-type RAS, resulting in greater MAPK output and increased sensitivity to MEK inhibitors.

Two cases of multiple Spitz nevi: Correlating clinical, histologic, and fluorescence in situ hybridization findings

BACKGROUND The occurrence of multiple Spitz nevi is rare, especially the disseminated variant. Multiple Spitz nevi may be confused with, and must be differentiated from, primary spitzoid melanoma and cutaneous melanoma metastases. Over the past decade, fluorescence in situ hybridization (FISH) has emerged as a tool for studying melanocytic neoplasms, helping to differentiate between melanoma and benign melanocytic nevi. We describe 2 cases of patients with multiple Spitz nevi and their FISH results. OBSERVATIONS One case of disseminated Spitz nevi, in a 17-year-old female, showed balanced tetraploidy using FISH, while the other case, in a 51-year-old female with multiple Spitz nevi, showed normal diploid cells without significant gains or losses in chromosomes 6 or 11. CONCLUSIONS Patients may present with multiple, even disseminated, Spitz nevi. This phenotype should not be confused with melanoma and/or cutaneous metastasis. The use of FISH studies in context with careful correlation of clinical features and dermoscopic and histologic findings can assist in the diagnostic workup.

Fluorescence in situ hybridization (FISH) analysis of melanocytic nevi and melanomas: sensitivity, specificity, and lack of association with sentinel node status.

A 4-color fluorescence in situ hybridization (FISH) assay, using probes to chromosomes 11q, 6p, 6q, and 6 cent, has recently been proposed as an ancillary tool for the diagnosis of melanoma. The authors report herein their experience with this assay. To determine the sensitivity and specificity of the assay for histopathologically unequivocal cases, they analyzed 50 melanocytic nevi, 50 primary melanomas, and 15 metastatic melanomas. Of 50 melanocytic nevi, 47 were FISH negative on initial readout (test sensitivity, 94%); 49 were FISH negative after correction for tetraploidy (test specificity, 98%). Of 50 primary melanomas, 41 were FISH positive (test sensitivity, 82%). Of 15 metastatic lesions, 13 were FISH positive (test sensitivity, 85%). Of the 9 FISH-negative melanomas, 6 metastasized. The tumors of the 5 patients who had survived thick primary melanoma for more than 5 years without recurrence were all FISH positive. Half of the patients whose primary melanoma was tested by FISH had undergone sentinel lymph node (SLN) biopsy. When the authors compared the FISH results of those 25 melanomas with the SLN status, no statistically significant correlation was found. These findings document limitations of the current FISH assay. A rare nevus may be FISH positive. Some primary metastasizing melanomas are FISH negative. Even metastatic melanomas can be FISH negative. Awareness of the limitations in test sensitivity and specificity of the FISH assay is important to avoid an erroneous diagnosis by overreliance on cytogenetic findings. Correlation with clinical and histopathological findings is paramount for accurate diagnosis.

Melanoma arising in a large plaque-type blue nevus with subcutaneous cellular nodules.

Melanoma may arise in association with and/or simulate the appearance of a cellular blue nevus. The distinction of a cellular blue nevus with atypical features from blue nevus-like melanoma can be difficult. One rare setting, in which one may face this diagnostic challenge, is nodule formation in a large plaque-type blue nevus. We have previously reported 2 patients with cellular blue nevus-like subcutaneous nodules without overt malignant features and indolent clinical behavior. Herein we report a patient who developed malignant melanoma in association with a large plaque-type blue nevus. A 46-year-old woman with a history of “cellular blue nevus” of the breast that developed 7 years earlier during pregnancy had soft tissue nodules at the prior surgical site. She was found to have melanoma associated with a large plaque-type blue nevus, which involved the skin and subcutis of the chest wall and extended into breast parenchyma. Ten years after mastectomy and axillary node dissection with negative lymph nodes, the melanoma recurred as a subcutaneous chest wall nodule. Cytogenetic analysis revealed a number of aberrations, including deletion of 6q and gains of 6p and 8q. The patient then developed visceral metastases, first to the liver, and died of widely metastatic melanoma. In contrast, the cytogenetic findings of a previously reported cellular blue nevus-like nodule in a patient with a large plaque-type blue nevus were normal. That patient is still alive with no evidence of melanoma 16 years after the surgical excision of the subcutaneous cellular blue nevus-like nodule.

9p21 gene locus in Spitz nevi of older individuals: absence of cytogenetic and immunohistochemical findings associated with malignancy ☆

The diagnosis of Spitz nevus in an elderly individual is often met with skepticism because the lesion can be difficult to distinguish from melanoma and because the probability of a malignant melanoma is higher in older patients. Recently, increased sensitivity for detection of malignant spitzoid neoplasms using 9p21 fluorescence in situ hybridization (FISH) has been described. In this study, we address the question of whether histopathologically typical Spitz nevi occurring in patients 50 years and older show any abnormalities regarding the 9p21 CDKN2A tumor suppressor gene locus. p16 immunohistochemistry (IHC), as well as dual-color FISH for assessment of diploid or hypodiploid status at 9p21, was performed in 25 classic Spitz nevi from patients 50 years and older and was compared with findings in a younger control population. All cases of typical Spitz nevi occurring in older patients retained p16 expression by immunohistochemistry and showed normal, diploid 9p21 FISH signals. Heterozygous loss of 9p21 by FISH was noted in a control case of a 9-year-old girl and is of unknown significance. These findings indicate that p16 expression by immunohistochemistry in classic Spitz nevi correlates well with absence of malignancy-associated cytogenetic abnormalities at 9p21 by FISH independent of the patients age. Assessment of p16 expression by standard immunohistochemistry may therefore be reassuring in routine clinical practice when the patient is of advanced age, and can be helpful as a screening tool to select IHC-negative cases for extended FISH analysis targeting the 9p21 gene locus.

Use of Fluorescence In Situ Hybridization to Distinguish Metastatic Uveal From Cutaneous Melanoma

Metastatic lesions of malignant melanoma can on occasion be difficult to classify with regard to the primary site of origin. Given the lack of specificity of light microscopic features, ancillary studies are needed. In this study, the authors explored the possibility of distinguishing metastatic tumors derived from uveal primaries from those known to have originated from a cutaneous melanoma by fluorescence in situ hybridization (FISH) using probes for chromosome 3, 8q24, and 1p36. A total of 32 metastatic tumors were analyzed by FISH. Monosomy 3 was detected in 9 out of 16 (56.3%) cases of metastatic uveal melanoma but was not found in any of the 16 metastatic cutaneous melanomas (P < .001). With regard to 1p36, amplifications were found in 8 out of 16 (50%) cases of metastatic cutaneous melanoma but not in any case of uveal melanoma (P < .05). 1p36 was deleted in 3 cases of uveal and 1 case of cutaneous melanoma. Amplifications of 8q were found in 15 out of 16 (94%) cases of uveal melanoma metastases and in 12 out of 16 (75%) cases of cutaneous metastases. The findings suggest that FISH for monosomy 3 is a useful adjunct tool in the differential diagnosis of metastatic uveal versus cutaneous melanoma.

Diagnosis of blue nevus-like metastatic uveal melanoma confirmed by fluorescence in situ hybridization (FISH) for monosomy 3.

Metastatic melanoma can on rare occasion simulate the appearance of a blue nevus clinically and/or histopathologically, which may lead to diagnostic confusion and delay in treatment. Given the known difficulty in recognizing a small dermal blue nevus‐like melanoma metastasis by light microscopic findings alone, recent discoveries of unique cytogenetic aberrations in various types of melanomas have led pathologists to explore cytogenetic techniques as an ancillary diagnostic tool. Herein, we report a case of a 58‐year‐old man with a history of uveal melanoma, in which fluorescence in situ hybridization (FISH) analysis for monosomy 3 helped confirm a diagnosis of blue nevus‐like uveal melanoma metastasis. The patient had presented clinically with a new small 1‐mm dark blue‐gray macule on the forehead. Histopathologically, a small dermal nodule of pigmented epithelioid melanocytes and melanophages was found with a rare mitotic figure. The pathologists suspicion of a blue nevus‐like melanoma metastasis was confirmed by FISH analysis: both the tumor cells of the patients prior uveal melanoma and the melanocytes of the new dermal blue nevus‐like nodule carried only one copy of chromosome 3. Furthermore, deletion of 1p36 and amplifications of 8q32 were also identified.