Prognostic factors of metastatic neuroendocrine carcinoma under first-line treatment with platinum etoposide with a focus on NEC score and Rb expression: Results from the multicentre RBNEC study of the Groupe d'Etude des Tumeurs Endocrines (GTE) and the ENDOCAN-RENATEN network.

INTRODUCTION AND AIM: Neuroendocrine carcinomas (NECs) are aggressive malignant diseases. Platinum-etoposide (PE) combination is the standard first-line treatment, whatever the primary location. The NEC score and also retinoblastoma protein (Rb) status have been suggested to be predictive/prognostic factors in NEC. The primary objective of our multicentric retrospective study was to evaluate the prognostic relevance of the NEC score and Rb status, assessed by immunohistochemistry in PE-treated patients with metastatic NEC. METHODS: Seven centres participated. The inclusion criteria were NEC, whatever the primary site, metastatic stage, first-line treatment with PE and tissue samples available. Rb status was determined centrally. RESULTS: We report multicentric data from 185 metastatic patients (37% women, median age 63). There were 108 small-cell NECs (SCNECs, 58.4%), 50 large-cell NECs (LCNECs, 27%) and 27 not otherwise specified NECs (nosNECs, 14.6%). The primary sites were the thorax (37%), gastroenteropancreatic sites (38%), unknown (15%) and other (9%). The mean Ki-67 index was 76% (range 20-100). Rb status was interpretable in 122 cases. Rb expression was lost in 74% of the cases: 84% of SCNEC vs. 60% and 63% of LCNEC and nosNEC, respectively (p = 0.016). Objective response was seen in 70% of SCNEC, 45% of LCNEC and 48% of nosNEC (p < 0.001) and in 62% of Rb-negative tumours vs. 46% of Rb-positive tumours (p = 0.3). There was no difference in median progression-free survival or overall survival (OS) as per Rb status. Age, NEC score and response to chemotherapy were the main factors associated with OS in our cohort. CONCLUSION: In our series, Rb status had no prognostic impact in PE-treated metastatic patients with NEC, whereas age, NEC score and response to chemotherapy were the main factors associated with OS.

RB1 gene mutations are a distinct predictive factor in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a rare cutaneous neuroendocrine carcinoma that tends to show local recurrence and metastasis. Typically, MCC is polyomavirus (MCPyV)-associated and cytokeratin 20 (CK20) positive. However, little is known about this tumor and its origins. Here, we aimed to determine the developmental origins of MCC and to identify prognostic clinicopathologic factors. Initial examinations revealed that CK20 and MCPyV expression (CK20+, MCPyV+ (60%); CK20+, MCPyV- (10%); CK20-, and MCPyV- (30%)) did not affect overall survival. With RB1 gene sequencing of FFPE specimens, which covered an entire exon, all RB1 mutation-positive cases showed positive regional lymph node and/or distant metastases (8/8 cases, 100%), whereas the frequency of the metastasis was statistically significantly lower in RB1 mutation-negative cases, (10/16 cases, 62%, P = 0.033). The results were also confirmed with immunohistochemistry, and either RB1 alterations, entire exon sequencing, or immunohistochemistry was associated with the metastasis (P = 0.007). RB1 alterations may be used to access the aggressive clinical course of MCC.

p16 in highly malignant esophageal carcinomas: the correlation with clinicopathological factors and human papillomavirus infection.

p16 is generally considered to be a surrogate maker of human papillomavirus (HPV) infection and also a predictive marker of favorable clinical outcome of patients with squamous cell carcinoma of the oropharynx. p16 overexpression is also known to be induced by deregulation of RB1 in neuroendocrine carcinomas. In highly malignant esophageal neoplasms, however, the status of p16 has remained largely unknown. We immunolocalized p16 and Rb1 in 82 surgically resected esophageal high-grade squamous cell carcinomas (46 poorly differentiated and 36 basaloid squamous cell carcinomas) and 15 esophageal small-cell carcinomas in order to clarify the clinical and biological significance of p16. p16 immunoreactivity was detected in 7/82 (9%) high-grade squamous cell carcinomas and 15 (100%) small-cell carcinomas. p16 immunoreactivity was significantly associated with Rb1 protein loss in both groups (P < 0.001). HPV was detected in none of the p16-positive cases examined. Clinical outcome of the p16-positive high-grade squamous cell carcinomas was not different from that of the p16-negative counterparts (P = 0.687) but significantly better than those with the small-cell carcinomas (P = 0.023). p16 was therefore considered to be induced through an inactivation of the RB1 signaling pathway and not through HPV infection in highly malignant esophageal neoplasms. Nevertheless, patients' clinical outcome of these neoplasms significantly differs; therefore, small-cell carcinomas have to be carefully differentiated from other neoplasms. In addition, p16 overexpression is not predictive of favorable clinical outcome in high-grade squamous cell carcinomas of the esophagus.

Distantly Metastatic Retinoblastoma to Soft Tissue and Bone: A Challenging Diagnosis Highlighting the Utility of CRX.

Distant metastasis of retinoblastoma to sites outside the central nervous system is rare; such cases may present years following primary treatment. Diagnosis may be difficult given the rarity of such events and considerable histologic mimics. We describe the clinicopathologic features of 6 cases of metastatic retinoblastoma to distant bone and soft tissue sites from 2 large academic centers. Patients were 3 female and 3 male children; median age was 9.5 years (range: 5 to 15 y) with a mean interval from primary disease diagnosis of 8.0 years (range: 0.75 to 14 y). Metastasis to bones of the lower extremities was most common, occurring in 4 of 6 cases. Tumors showed typical histologic features of retinoblastoma, with sheets of primitive round cells with minimal cytoplasm and indistinct nucleoli; however, characteristic Flexner-Wintersteiner rosettes were absent. A subset of cases demonstrated an alveolar growth pattern, and 2 cases showed higher grade cytology with nuclear anaplasia and prominent nucleoli. Immunohistochemistry for CRX and RB1 showed uniform positivity and loss of expression, respectively. Metastatic retinoblastoma outside the central nervous system may present following long disease-free intervals. Immunohistochemistry for CRX is helpful to confirm this challenging diagnosis.

Immunohistochemistry-based assessment of androgen receptor status and the AR-null phenotype in metastatic castrate resistant prostate cancer.

BACKGROUND: Molecular and immunohistochemistry-based profiling of prostatic adenocarcinoma has revealed frequent Androgen Receptor (AR) gene and protein alterations in metastatic disease. This includes an AR-null non-neuroendocrine phenotype of metastatic castrate resistant prostate cancer which may be less sensitive to androgen receptor signaling inhibitors. This AR-null non-neuroendocrine phenotype is thought to be associated with TP53 and RB1 alterations. Herein, we have correlated molecular profiling of metastatic castrate resistant prostate cancer with AR/P53/RB immunohistochemistry and relevant clinical correlates. DESIGN: Twenty-seven cases of metastatic castrate resistant prostate cancer were evaluated using histopathologic examination to rule out neuroendocrine differentiation. A combination of a hybridization exon-capture next-generation sequencing-based assay (n = 26), fluorescence in situ hybridization for AR copy number status (n = 16), and immunohistochemistry for AR (n = 27), P53 (n = 24) and RB (n = 25) was used to profile these cases. RESULTS: Of 27 metastatic castrate resistant prostate cancer cases, 17 had AR amplification and showed positive nuclear expression of AR by immunohistochemistry. Nine cases lacked AR copy number alterations using next-generation sequencing/fluorescence in situ hybridization. A subset of these metastatic castrate resistant prostate cancer cases demonstrated the AR-null phenotype by immunohistochemistry (five cases and one additional case where next-generation sequencing failed). Common co-alterations in these cases involved the TP53, RB1, and PTEN genes and all these patients received prior therapy with androgen receptor signaling inhibitors (abiraterone and/or enzalutamide). CONCLUSIONS: Our study suggests that AR immunohistochemistry may distinguish AR-null from AR-expressing cases in the metastatic setting. AR-null status informs clinical decision-making regarding continuation of therapy with androgen receptor signaling inhibitors and consideration of other treatment options. This might be a relevant and cost-effective diagnostic strategy when there is limited access and/or limited tumor material for molecular testing.

The Utility of ERBB4 and RB1 Immunohistochemistry in Distinguishing Chromophobe Renal Cell Carcinoma From Renal Oncocytoma.

Objectives. Differentiating renal oncocytoma (RO) from chromophobe renal cell carcinoma (ChRCC) can occasionally be challenging. We evaluated the expression of RB1 and ERBB4 in RO and ChRCC, and compared the immunohistochemistry (IHC) results to RB1 and ERBB4 gene abnormalities detected by fluorescence in situ hybridization (FISH). Materials and Methods. Fifty-three kidney resections (ChRCC, n=28; RO, n=25) were stained for RB1 and ERBB4 IHC and FISH was performed to evaluate gene copy number analysis. Results. A loss of RB1 staining was identified in 64% (18/28) of ChRCCs, which was not found in any ROs (0/25; P <.001). FISH analysis revealed 36% (10/28) of ChRCCs contained a RB1 hemizygous deletion with a concordance of 56% (10/18) between the IHC and FISH findings. No RB1 gene copy number variations were detected in any of the ROs (0/25; P <.001) and retained expression of RB1 by IHC. ERBB4 showed cytoplasmic/membranous staining in all ROs and ChRCCs. However, 75% (21/28) of ChRCCs also contained nuclear positivity for ERBB4, which was uncommonly seen in ROs (3/25, 12%; P < .001). A hemizygous ERBB4 gene deletion was detected in 46% of ChRCCs (13/28), but none of the ROs (0/25; 0%). Loss of labeling by RB1 or nuclear staining for ERBB4 IHC identified 25 of 28 (89%) of ChRCCs. Conclusion. In summary, the loss of RB1 expression is a highly specific diagnostic biomarker in distinguishing ChRCC from RO. Nuclear ERBB4 expression also appears to be a sensitive diagnostic biomarker for ChRCC, albeit the mechanism is unknown.

"Fat-Rich" (Spindle Cell-Poor) Variant of Atypical Spindle Cell/Pleomorphic Lipomatous Tumor: Striking Mimic of "Classical" Atypical Lipomatous Tumor/Well-Differentiated Liposarcoma.

We report the case of a "fat-rich" (spindle cell-poor) variant of an atypical spindle cell/pleomorphic lipomatous tumor in a 63-year-old female patient presenting with a firm, painless soft tissue mass on the right hip. Atypical spindle cell/pleomorphic lipomatous tumor is a very recently described low-grade adipocytic neoplasm, which occurs predominantly in adults with a predilection for the limbs and limb girdles. In the present case, the diagnosis of an atypical spindle cell/pleomorphic lipomatous tumor was challenging because the tumor was almost exclusively composed of an atypical adipocytic component (resembling "classical" atypical lipomatous tumor/well-differentiated tumor) with only very focal presence of the diagnostic atypical morphologic features (atypical spindle-shaped cells, floret-like multinucleated cells, and "bizarre" pleomorphic [multinucleated] cells). The possibility of a "classical" atypical lipomatous tumor/well-differentiated liposarcoma was ruled out by immunohistochemistry (lack of MDM2 expression and loss of Rb expression) and molecular genetic testing (no amplification of MDM2 and presence of monoallelic deletion of RB1). Another interesting morphologic observation in this case was the striking perivascular location of the atypical spindle/pleomorphic cells in some areas (so-called "pericytic mimicry"). To our knowledge, pericytic mimicry has not been reported in the setting of an atypical spindle cell/pleomorphic lipomatous tumor.

Expression of MT4-MMP, EGFR, and RB in Triple-Negative Breast Cancer Strongly Sensitizes Tumors to Erlotinib and Palbociclib Combination Therapy.

PURPOSE: Here, we investigated the clinical relevance of an unprecedented combination of three biomarkers in triple-negative breast cancer (TNBC), both in human samples and in patient-derived xenografts of TNBC (PDX-TNBC): EGFR, its recently identified partner (MT4-MMP), and retinoblastoma protein (RB).Experimental Design: IHC analyses were conducted on human and PDX-TNBC samples to evaluate the production of the three biomarkers. The sensitivity of cancer cells expressing or not MT4-MMP to anti-EGFR (erlotinib) or anti-CDK4/6 inhibitor (palbociclib) was evaluated in vitro in 2D and 3D proliferation assays and in vivo using xenografts and PDX-TNBC displaying different RB, MT4-MMP, and EGFR status after single (erlotinib or palbociclib) or combined (erlotinib + palbociclib) treatments. RESULTS: EGFR and MT4-MMP were coexpressed in >70% of TNBC samples and PDX-TNBC, among which approximately 60% maintained RB expression. Notably, approximately 50% of all TNBC and PDX-TNBC expressed the three biomarkers. Single erlotinib and palbociclib treatments drastically reduced the in vitro proliferation of cells expressing EGFR and MT4-MMP when compared with control cells. Both TNBC xenografts and PDX expressing MT4-MMP, EGFR, and RB, but not PDX-TNBC with RB loss, were sensitive to erlotinib and palbociclib with an additive effect of combination therapy. Moreover, this combination was efficient in another PDX-TNBC expressing the three biomarkers and resistant to erlotinib alone. CONCLUSIONS: We defined a new association of three biomarkers (MT4-MMP/EGFR/RB) expressed together in 50% of TNBC and demonstrated its usefulness to predict the TNBC response to anti-EGFR and anti-CDK4/6 drugs used in single or combined therapy.

Benign Fibromyxoid Lesion of the Breast: A Distinct Entity From Benign Spindle Cell Tumors of the Mammary Stroma?

Myxoid lesions of the breast can be diagnostically challenging entities. We report 4 cases of CD34+ fibromyxoid lesion that have been previously diagnosed as "benign myxoid lesion," "nodular mucinosis," or "mammary myofibroblastoma, myxoid type" on the basis of CD34-positivity. The lesions were microscopically well circumscribed and composed of a paucicellular spindle cell proliferation in a background of myxoid stroma. No epithelial component was identified. The spindle cells showed immunohistochemical reactivity for CD34 and smooth muscle actin. Based on morphologic and immunohistochemical similarities between these cases and myxoid myofibroblastoma, we compared 4 myxoid lesions with cases of typical myofibroblastoma, utilizing retinoblastoma (Rb) antibody and fluorescent in situ hybridization for 13q14 gene rearrangement (encoding the Rb gene). The myxoid lesions showed retention of Rb protein by immunohistochemistry, whereas Rb expression was lost in cases of myofibroblastoma. We identified loss of 13q14 in 3 of 4 cases of myofibroblastoma. Notably, 13q14 gene rearrangement was not observed in any of the myxoid lesions. Our data show that there is at least a subset of CD34+ fibromyxoid lesions that, despite overlapping morphologic and immunohistochemical phenotype and proposed common histogenesis with myofibroblastomas, is genetically distinct from the latter based on Rb analysis.

Bladder cancer.

Bladder cancer is a highly prevalent disease and is associated with substantial morbidity, mortality and cost. Environmental or occupational exposures to carcinogens, especially tobacco, are the main risk factors for bladder cancer. Most bladder cancers are diagnosed after patients present with macroscopic haematuria, and cases are confirmed after transurethral resection of bladder tumour (TURBT), which also serves as the first stage of treatment. Bladder cancer develops via two distinct pathways, giving rise to non-muscle-invasive papillary tumours and non-papillary (solid) muscle-invasive tumours. The two subtypes have unique pathological features and different molecular characteristics. Indeed, The Cancer Genome Atlas project identified genetic drivers of muscle-invasive bladder cancer (MIBC) as well as subtypes of MIBC with distinct characteristics and therapeutic responses. For non-muscle-invasive bladder cancer (NMIBC), intravesical therapies (primarily Bacillus Calmette-Guérin (BCG)) with maintenance are the main treatments to prevent recurrence and progression after initial TURBT; additional therapies are needed for those who do not respond to BCG. For localized MIBC, optimizing care and reducing morbidity following cystectomy are important goals. In metastatic disease, advances in our genetic understanding of bladder cancer and in immunotherapy are being translated into new therapies.

SOX2 and Rb1 in esophageal small-cell carcinoma: their possible involvement in pathogenesis.

Clinicopathological features and pathogenesis of esophageal small-cell carcinoma remain unclear. We hypothesized common cellular origin and pathogenesis in small-cell carcinoma of esophagus and lung associated with SOX2 overexpression and loss of Rb1. Expression of squamous-basal markers (CK5/6 and p40), glandular markers (CK18 and CEA), SOX2, and Rb1 were evaluated in 15 esophageal small-cell carcinomas, 46 poorly differentiated squamous cell carcinomas, and 88 small-cell lung carcinoma, as well as 16 embryonic esophagus. Esophageal small-cell carcinoma expressed higher levels of glandular markers and lower levels of squamous-basal markers than poorly differentiated squamous cell carcinoma. No significant differences were observed in immunohistochemistry profiles between small-cell carcinoma of the esophagus and the lung. SOX2 expression was high in esophageal small-cell carcinoma (70%±33% of nuclei), small-cell lung carcinoma (70%±26%), and the embryonic esophagus (75%±4%), and it was significantly lower in poorly differentiated squamous cell carcinoma (29%±28%). Rb1 expression was significantly lower in esophageal small-cell carcinoma (0.3%±1%), small-cell lung carcinoma (2%±6%), and the embryonic esophagus (7%±5%), and it was significantly higher in poorly differentiated squamous cell carcinoma (51%±24%). The immunohistochemistry profiles of small-cell carcinoma of the esophagus and the lung are highly similar. The loss of Rb1 function is a key contributor to the pathogenesis of both neoplasms. In addition, SOX2 overexpression observed in esophageal and lung small-cell carcinoma as well as in the embryonic esophagus indicated that esophageal small-cell carcinoma may arise from embryonic-like stem cells in the esophageal epithelium. The two distinct differentiation patterns (neuroendocrine and glandular) of esophageal small-cell carcinoma further support the fact that SOX2 has a pivotal role in the differentiation of pluripotent stem cells into esophageal small-cell carcinoma cells.

Superficial acral fibromyxoma: clinicopathological, immunohistochemical, and molecular study of 11 cases highlighting frequent Rb1 loss/deletions.

Superficial acral fibromyxoma (SAF) is an uncommon benign dermal mesenchymal lesion of adults with predilection for acral sites, in particular the nail region. To date, less than 300 cases have been reported. SAFs consistently express CD34, but other diagnostic markers or specific genetic alterations have not been established yet. We describe 11 SAFs occurring in 7 men and 4 women aged 37 to 86years (median, 48 years). Mean size was 6mm (range, 4-20mm). Affected sites were fingers (n=5), toes (n=3), heel (n=1), calf (n=1), and unspecified digit (n=1). None of 10 patients with available follow-up (2-60months; median, 24months) developed recurrence. Histology showed relatively hypocellular vaguely lobulated nodules composed of bland-looking spindled or stellate fibroblast-like cells arranged into storiform or loose fascicles within a variably myxoid, fibromyxoid, or collagenous vascularized stroma. Immunohistochemistry showed expression of CD34 (9/10) and focal weak reactivity for epithelial membrane antigen (2/11). None of the lesions expressed protein S100 (0/11), MUC4 (0/11), or STAT6 (0/11). Loss of Rb1 immunoexpression was observed in 9 (90%) of 10 cases. All 7 cases with successful RB1 fluorescence in situ hybridization testing showed RB1 gene deletions, which was variably associated with co-loss of the corresponding 13q12 signal (monosomy at the 13q region). To our knowledge, this is the first study investigating the expression status of the tumor suppressor Rb1 in SAF by immunohistochemistry and fluorescence in situ hybridization. Our results showed frequent Rb1 deficiency as a possible driver molecular event in SAF (seen in 90% of cases) indicating relationship of SAF to the RB1-deleted tumor family.

Characterization of the leiomyomatous variant of myofibroblastoma: a rare subset distinct from other smooth muscle tumors of the breast.

Mammary myofibroblastoma is a benign spindle cell tumor that can show variable morphologic patterns and lines of differentiation. Myofibroblastoma belongs to a family of CD34-positive tumors with similar morphology that show a deletion of 13q14, which includes RB1 and FOXO1A genes. A subset of these tumors demonstrates distinct smooth muscle differentiation. We aimed to characterize 4 cases of the leiomyomatous variant of myofibroblastoma arising in the breast by clinicopathological, immunohistochemical, and molecular means. All 4 examples arose in women aged 41 to 62 years (median, 46.5 years). Tumors ranged in size from 1.7 to 2.5 cm (median, 2.2 cm). Morphologically, all tumors were characterized by bundles of smooth muscle cells with elongated cigar-shaped nuclei and eosinophilic cytoplasm. All 4 tumors showed diffuse positive staining with desmin, caldesmon, smooth muscle actin, estrogen receptor, and Bcl-2. CD34 staining was diffusely positive in 2 cases, was weak and patchy in 1 case, and was negative in 1 case. Two (50%) of 4 tumors showed deletion of RB1 by fluorescence in situ hybridization. Loss of Rb staining was seen in 1 tumor with RB1 deletion by fluorescence in situ hybridization, whereas intact Rb staining was observed in 1 nondeleted case studied. In conclusion, this rare variant of myofibroblastoma is a distinct subgroup of tumors among an already uncommon category of (smooth muscle) breast tumors. Some reported examples of "parenchymal leiomyoma" may represent the leiomyomatous variant of myofibroblastoma.

Genetic analysis in retinoblastoma and peripheral blood correlation.

OBJECTIVE: To determine the importance of intratumoral genetic analysis in the diagnosis of germ-line mutations in patients with retinoblastoma. To underline the importance of performing these genetic tests in every case of retinoblastoma. METHOD: Intratumoral genetic analysis of RB1 mutation was performed on 17 enucleated eyes that were non-responsive to conservative treatment. Patients had no family history of retinoblastoma, and lesions were always single. The identified mutations were then also studied in peripheral blood analysis. RESULTS: There were 12 (70.6%) cases with positive results in intratumoral analysis. In 8 cases (47.1%) mutation of both RB1 alelli were detected, and in 4 (23.5%) cases only one allele was found mutated. In 5 patients (29.4%) no mutation was identified. In the first hit, mutations comprised 7 frameshift or nonsense and 2 splice, whereas in the second hit, one splice mutation, 2 nonsense and 8 loss of heterozygosity were identified. Among 6 patients where intratumoral analysis detected a single mutation associated with a loss of heterozygosity, the peripheral blood analysis was able to detect the same mutation in 3 cases (50%). CONCLUSIONS: Intratumoral genetic analysis of sporadic retinoblastoma can detect germ-line mutations. These patients are at higher risk of bilateralization and development of second tumors or trilateral retinoblastoma. Genetic screening is recommended in every patient diagnosed with retinoblastoma.