Ilan Weinreb

Mammary analogue secretory carcinoma of salivary glands, containing the ETV6-NTRK3 fusion gene: a hitherto undescribed salivary gland tumor entity.

We present a series of 16 salivary gland tumors with histomorphologic and immunohistochemical features reminiscent of secretory carcinoma of the breast. This is a hitherto undescribed and distinctive salivary gland neoplasm, with features resembling both salivary acinic cell carcinoma (AciCC) and low-grade cystadenocarcinoma, and displaying strong similarities to breast secretory carcinoma. Microscopically, the tumors have a lobulated growth pattern and are composed of microcystic and glandular spaces with abundant eosinophilic homogenous or bubbly secretory material positive for periodic acid-Schiff, mucicarmine, MUC1, MUC4, and mammaglobin. The neoplasms also show strong vimentin, S-100 protein, and STAT5a positivity. For this tumor, we propose a designation mammary analogue secretory carcinoma of salivary glands (MASC). The 16 patients comprised 9 men and 7 women, with a mean age of 46 years (range 21 to 75). Thirteen cases occurred in the parotid gland, and one each in the minor salivary glands of the buccal mucosa, upper lip, and palate. The mean size of the tumors was 2.1 cm (range 0.7 to 5.5 cm). The duration of symptoms was recorded in 11 cases and ranged from 2 months to 30 years. Clinical follow-up was available in 13 cases, and ranged from 3 months to 10 years. Four patients suffered local recurrences. Two patients died, 1 of them owing to multiple local recurrences with extension to the temporal bone, and another owing to metastatic dissemination to cervical lymph nodes, pleura, pericardium, and lungs. We have shown a t(12;15) (p13;q25) ETV6-NTRK3 translocation in all but one case of MASC suitable for analysis. One case was not analyzable and another was not available for testing. This translocation was not found in any conventional salivary AciCC (12 cases), nor in other tumor types including pleomorphic adenoma (1 case) and low-grade cribriform cystadenocarcinoma (1 case), whereas ETV6-NTRK3 gene rearrangements were proven in all 3 tested cases of mammary secretory carcinoma. Thus, our results strongly support the concept that MASC and AciCC are different entities.

Deintensification Candidate Subgroups in Human Papillomavirus-Related Oropharyngeal Cancer According to Minimal Risk of Distant Metastasis

PURPOSE To define human papillomavirus (HPV) -positive oropharyngeal cancers (OPC) suitable for treatment deintensification according to low risk of distant metastasis (DM). PATIENTS AND METHODS OPC treated with radiotherapy (RT) or chemoradiotherapy (CRT) from 2001 to 2009 were included. Outcomes were compared for HPV-positive versus HPV-negative patients. Univariate and multivariate analyses identified outcome predictors. Recursive partitioning analysis (RPA) stratified the DM risk. RESULTS HPV status was ascertained in 505 (56%) of 899 consecutive OPCs. Median follow-up was 3.9 years. HPV-positive patients (n = 382), compared with HPV-negative patients (n = 123), had higher local (94% v 80%, respectively, at 3 years; P < .01) and regional control (95% v 82%, respectively; P < .01) but similar distant control (DC; 90% v 86%, respectively; P = .53). Multivariate analysis identified that HPV negativity (hazard ratio [HR], 2.9; 95% CI, 2.0 to 5.0), N2b-N3 (HR, 2.9; 95% CI, 1.8 to 4.9), T4 (HR, 1.8; 95% CI, 1.2 to 2.9), and RT alone (HR, 1.8; 95% CI, 1.1 to 2.5) predicted a lower recurrence-free survival (RFS; all P < .01). Smoking pack-years > 10 reduced overall survival (HR, 1.72; 95% CI, 1.1 to 2.7; P = .03) but did not impact RFS (HR, 1.1; 95% CI, 0.7 to 1.9; P = .65). RPA segregated HPV-positive patients into low (T1-3N0-2c; DC, 93%) and high DM risk (N3 or T4; DC, 76%) groups and HPV-negative patients into different low (T1-2N0-2c; DC, 93%) and high DM risk (T3-4N3; DC, 72%) groups. The DC rates for HPV-positive, low-risk N0-2a or less than 10 pack-year N2b patients were similar for RT alone and CRT, but the rate was lower in the N2c subset managed by RT alone (73% v 92% for CRT; P = .02). CONCLUSION HPV-positive T1-3N0-2c patients have a low DM risk, but N2c patients from this group have a reduced DC when treated with RT alone and seem less suited for deintensification strategies that omit chemotherapy.

Refining American Joint Committee on Cancer/Union for International Cancer Control TNM Stage and Prognostic Groups for Human Papillomavirus–Related Oropharyngeal Carcinomas

PURPOSE To refine stage and prognostic group for human papillomavirus (HPV) -related nonmetastatic (M0) oropharyngeal cancer (OPC). METHODS All patients with nonmetastatic (M0) p16-confirmed OPC treated with radiotherapy with or without chemotherapy from 2000 to 2010 were included. Overall survival (OS) was compared among TNM stages for patients with HPV-related and HPV-unrelated OPC separately. For HPV-related OPC, recursive partitioning analysis (RPA) derived new RPA stages objectively. Cox regression was used to calculate adjusted hazard ratios (AHRs) to derive AHR stages. The performance of survival prediction of RPA stage and AHR stage was assessed against the current seventh edition TNM stages. Prognostic groups were derived by RPA, combining RPA stage and nonanatomic factors. RESULTS The cohort comprised 573 patients with HPV-related OPC and 237 patients with HPV-unrelated OPC, with a median follow-up of 5.1 years. Lower 5-year OS with higher TNM stage was evident for patients with HPV-unrelated OPC (stage I, II, III, and IV 5-year OS: 70%, 58%, 50%, and 30%, respectively; P = .004) but not for patients with HPV-related OPC (stage I, II, III, and IV 5-year OS: 88%, 78%, 71%, and 74%, respectively; P = .56). RPA divided HPV-related OPC into RPA-I (T1-3N0-2b), RPA-II (T1-3N2c), and RPA-III (T4 or N3; 5-year OS: 82%, 76%, and 54%, respectively; P < .001). AHR also yielded a valid classification, but RPA stage demonstrated better survival prediction. A further RPA (including RPA stage, age, and smoking pack-years [PYs]) derived the following four valid prognostic groups for survival: group I (T1-3N0-N2c_≤ 20 PY), group II (T1-3N0-N2c_> 20 PY), group III (T4 or N3_age ≤ 70), and group IVA (T4 or N3_age > 70; 5-year OS: 89%, 64%, 57%, and 40%, respectively; P < .001). CONCLUSION An RPA-based TNM stage grouping (stage I/II/III: T1-3N0-N2b/T1-3N2c/T4 or N3, with M1 as stage IV) is proposed for HPV-related OPC as a result of significantly improved survival prediction compared with the seventh edition TNM, and prognostication is further improved by an RPA-based prognostic grouping within the American Joint Committee on Cancer/Union for International Cancer Control TNM framework for HPV-related OPC.

EWSR1‐ATF1 fusion is a novel and consistent finding in hyalinizing clear‐cell carcinoma of salivary gland

Hyalinizing clear‐cell carcinoma (HCCC) is a rare, low‐grade salivary gland tumor with distinctive clear‐cell morphology and pattern of hyalinization as well as focal mucinous differentiation. However, histological overlap exists with other salivary gland tumors, such as epithelial–myoepithelial carcinoma (EMCa), salivary myoepithelial carcinoma, and mucoepidermoid carcinoma (MEC). The potential relationship between HCCC and its morphological mimics has not been yet investigated at the genetic level. In this study, we conducted a molecular analysis for the presence of rearrangements in MAML2, commonly seen in MECs, and EWSR1, involved in “soft tissue myoepithelial tumors” (SMET) by fusion with POU5F1, PBX1, or ZNF444. Fluorescence in situ hybridization (FISH) was performed on 23 HCCC cases for abnormalities in MAML2, EWSR1, FUS, POU5F1, PBX1, and ZNF444. FISH for MAML2 was negative in all cases (0 of 14), including those with mucinous differentiation (0 of 7). An EWSR1 rearrangement was identified in 18 of 22 HCCCs (82%), while no break‐apart signals were seen in FUS, POU5F1, PBX1, or ZNF444. 3′RACE on an EWSR1 rearranged HCCC identified an EWSR1‐ATF1 fusion, which was confirmed by RT‐PCR. ATF1 involvement was further confirmed by FISH analysis in 13 of 14 EWSR1‐rearranged HCCC cases (93%). In contrast, all control cases tested, including among others 5 EMCa and 3 MEC with clear cells, were negative for EWSR1 and ATF1 rearrangements. The presence of EWSR1‐ATF1 fusion in most HCCCs reliably separates these tumors from its histological mimics. The distinction from MEC is particularly important, as conventional MEC grading schemes overgrade these indolent HCCCs, potentially impacting on treatment.

Novel YAP1-TFE3 fusion defines a distinct subset of epithelioid hemangioendothelioma

Conventional epithelioid hemangioendotheliomas (EHE) have a distinctive morphologic appearance and are characterized by a recurrent t(1;3) translocation, resulting in a WWTR1‐CAMTA1 fusion gene. We have recently encountered a fusion‐negative subset characterized by a somewhat different morphology, including focally well‐formed vasoformative features, which was further investigated for recurrent genetic abnormalities. Based on a case showing strong transcription factor E3 (TFE3) immunoreactivity, fluorescence in situ hybridization (FISH) analysis for TFE3 gene rearrangement was applied to the index case as well as to nine additional cases, selected through negative WWTR1‐CAMTA1 screening. A control group, including 18 epithelioid hemangiomas, nine pseudomyogenic HE, and three epithelioid angiosarcomas, was also tested. TFE3 gene rearrangement was identified in 10 patients, with equal gender distribution and a mean age of 30 years old. The lesions were located in somatic soft tissue in six cases, lung in three and one in bone. One case with available frozen tissue was tested by RNA sequencing and FusionSeq data analysis to detect novel fusions. A YAP1‐TFE3 fusion was thus detected, which was further validated by FISH and reverse transcription polymerase chain reaction (RT‐PCR). YAP1 gene rearrangements were then confirmed in seven of the remaining nine TFE3‐rearranged EHEs by FISH. No TFE3 structural abnormalities were detected in any of the controls. The TFE3‐rearranged EHEs showed similar morphologic features with at least focally, well‐formed vascular channels, in addition to a variably solid architecture. All tumors expressed endothelial markers, as well as strong nuclear TFE3. In summary, we are reporting a novel subset of EHE occurring in young adults, showing a distinct phenotype and YAP1‐TFE3 fusions.

Mammary analog secretory carcinoma of salivary gland origin with the ETV6 gene rearrangement by FISH: expanded morphologic and immunohistochemical spectrum of a recently described entity.

Mammary analog secretory carcinoma (MASC) is a recently described tumor predominantly arising in the parotid gland. These tumors represent locally invasive malignancies with microcystic architecture, low-grade nuclei, and granular pink vacuolated cytoplasm. They display strong vimentin and S100 positivity and harbor an identical t(12;15)(p13;q25) to their breast counterpart, leading to a ETV6-NTRK3 fusion oncogene. These features help exclude the most important differential diagnostic considerations, namely, acinic cell carcinoma (AciCC) and low-grade cystadenocarcinoma, not otherwise specified. Here we present a series of 7 recent examples of MASC, which showed features not previously described. These 7 cases were observed in patients ranging in age from 14 to 77 years (mean, 40 y), occurred almost exclusively in male patients (6:1), and showed >50% (4 of 7 cases) involvement of the oral cavity, with only 2 arising in the parotid. The remaining case is the first reported in the submandibular gland. The tumors showed a variety of patterns including single macrocysts, combined macrocystic and microcystic spaces, and solid architecture. They showed prominent hobnailing in the cystic areas. Secretions within the cysts and tubular areas tended to be positive for periodic acid schiff, periodic acid schiff diastage and mucicarmine, the latter also showing occasional intracytoplasmic mucin droplets, a feature not previously recognized. One case showed prominent mucinous differentiation, which, coupled with high-molecular-weight keratins (HMWK) positivity, mimicked mucoepidermoid carcinoma (MEC). The tumors were generally positive for HMWK (6 of 7), S100 (5 of 7), vimentin, CK19, and other epithelial markers. The finding of duct involvement, proven with an incomplete p63-positive basal layer surrounding a minority of tumor cell nests and cysts, raised the possibility of a ductal epithelial origin for MASC. Alternatively, this could represent secondary ductal involvement by tumor. All cases showed rearrangement of the ETV6 gene by fluorescence in situ hybridization, confirming the diagnosis of MASC. These findings reinforce MASC as a unique low-grade salivary gland tumor entity with morphologic overlap with AciCC, MEC, and cystadenocarcinoma.

Atypical clinical behavior of p16-confirmed HPV-related oropharyngeal squamous cell carcinoma treated with radical radiotherapy.

PURPOSE To report atypical clinical behavior observed in human papillomavirus (HPV)-related oropharyngeal carcinoma (OPC) treated with radiotherapy. METHODS AND MATERIALS A retrospective cohort study was conducted for all newly diagnosed OPC cases treated with radiotherapy on July 1, 2003 to April 30, 2009. HPV positivity was determined by p16 immunostaining in tumors. The incidence of additional malignancies and the pattern of distant metastases (DMs) were compared between the HPV-positive (HPV+) and HPV-negative (HPV-) cohorts. RESULTS HPV status was evaluated in 318 of 613 consecutive OPC cases (52%), showing 236 HPV+ and 82 HPV- patients. Compared with HPV-, HPV+ cases were less likely to have additional malignancies (prior: 11% vs. 20%, p = 0.038; synchronous: 1% vs. 9%, p = 0.001; metachronous: 6% vs. 16%, p = 0.003). Whereas the majority (10 of 12) of HPV- additional head-and-neck (HN) mucosal malignancies were in the oral cavity, there was none (0 of 7) in the HPV+ cohort (p < 0.001). HPV+ synchronous HN second primaries (SPs) were in the supraglottis, post-cricoid, and nasopharynx; metachronous HN SPs were in the glottis, supraglottis, and ethmoid plus glottis/post-cricoid region. All SPs that could be tested were HPV+. There was no difference in DM rate (10% vs. 15%, p = 0.272), but HPV+ DMs were more likely to involve multiple organs (46% vs. 0%, p = 0.005) and unusual sites. CONCLUSIONS This study reports atypical clinical behavior seen in HPV+ OPC, including multicentric lesions in HN mucosa and DM to multiple organs and unusual sites. The frequency of these events is low, but they may have clinical implications. The routine assessment of HPV status for all OPC is warranted.

Outcomes of HPV-related oropharyngeal cancer patients treated by radiotherapy alone using altered fractionation.

PURPOSE To report outcome of HPV-related [HPV(+)] oropharyngeal cancer (OPC) managed predominantly by altered-fractionation radiotherapy-alone (RT-alone). METHODS OPCs treated with RT-alone (n = 207) or chemoradiotherapy (CRT) (n = 151) from 2001 to 2008 were included. Overall survival (OS), local (LC), regional (RC) and distant (DC) control were compared for HPV(+) vs. HPV-unrelated [HPV(-)], by RT-alone vs. CRT, and by smoking pack-years (≤ 10 vs. >10). Multivariate analysis identified predictors. RESULTS HPV(+) (n = 277) had better OS (81% vs. 44%), LC (93% vs. 76%), RC (94% vs. 79%) (all p < 0.01) but similar DC (89% vs. 86%, p = 0.87) vs. HPV(-) (n = 81). HPV(+) stage IV CRT (n = 125) had better OS (89% vs. 70%, p < 0.01), but similar LC (93% vs. 90%, p = 0.41), RC (94% vs. 90%, p = 0.31) and DC (90% vs. 83%, p = 0.22) vs. RT-alone (n = 96). Both HPV(+) RT-alone (n = 37) and CRT (n = 67) stage IV minimal smokers had favorable OS (86% vs. 88%, p = 0.45), LC (95% vs. 92%, p = 0.52), RC (97% vs. 93%, p = 0.22), and DC (92% vs. 86%, p = 0.37). RT-alone and heavy-smoking were independent predictors for lower OS but not CSS in multivariate analysis. CONCLUSIONS Overall, HPV(+) RT-alone stage IV demonstrated lower survival but comparable disease control vs. CRT, but no difference was apparent among minimal smokers.

Hotspot activating PRKD1 somatic mutations in polymorphous low-grade adenocarcinomas of the salivary glands

Polymorphous low-grade adenocarcinoma (PLGA) is the second most frequent type of malignant tumor of the minor salivary glands. We identified PRKD1 hotspot mutations encoding p.Glu710Asp in 72.9% of PLGAs but not in other salivary gland tumors. Functional studies demonstrated that this kinase-activating alteration likely constitutes a driver of PLGA.

Potentially Prognostic miRNAs in HPV-Associated Oropharyngeal Carcinoma

Purpose: Deregulation of miRNAs is associated with almost all human malignancies. Human papillomavirus (HPV)-associated oropharyngeal carcinoma (OPC) has a significantly more favorable outcome compared with HPV-negative OPCs; however, the underlying mechanisms are not well understood. Hence, the objectives of this study were to determine whether miRNA expression differed as a function of HPV status and to assess whether such miRNAs provide prognostic value beyond HPV status. Methods: Global miRNA profilings were conducted on 88 formalin-fixed and paraffin-embedded (FFPE) OPC biopsies (p16-positive: 56; p16-negative: 32), wherein the expression levels of 365 miRNAs plus 3 endogenous controls were simultaneously measured using quantitative real-time (qRT)-PCR. Seven FFPE specimens of histologically normal tonsils were used as controls. Results: Overall, 224 miRNAs were expressed in more than 80% of the investigated samples, with 128 (57%) being significantly differentially expressed between tumor versus normal tissues (P < 0.05). Upregulated miR-20b, miR-9, and miR-9* were significantly associated with HPV/p16-status. Three miRNA sets were significantly associated with overall survival (miR-107, miR-151, miR-492; P = 0.0002), disease-free survival (miR-20b, miR-107, miR-151, miR-182, miR-361; P = 0.0001), and distant metastasis (miR-151, miR-152, miR-324-5p, miR-361, miR492; P = 0.0087), which retained significance even after adjusting for p16 status. The associated biologic functions of these miRNAs include immune surveillance, treatment resistance, invasion, and metastasis. Conclusion: We have identified several miRNAs, which associate with HPV status in OPC; furthermore, three candidate prognostic sets of miRNAs seem to correlate with clinical outcome, independent of p16 status. Furthermore, evaluations will offer biologic insights into the mechanisms underlying the differences between HPV-positive versus HPV-negative OPC. Clin Cancer Res; 19(8); 2154–62. ©2013 AACR.