When molecular outsmarts morphology: Malignant ossifying fibromyxoid tumors masquerading as osteosarcomas, including a novel CREBZF::PHF1 fusion.

We present two cases of malignant ossifying fibromyxoid tumor (OFMT) which eluded diagnosis due to compelling clinicopathologic mimicry, compounded by similarly elusive underlying molecular drivers. The first is of a clavicle mass in a 69 year-old female, which histologically showed an infiltrative nested and trabeculated proliferation of monomorphic cells giving rise to scattered spicules of immature woven bone. Excepting SATB2 positivity, the lesion showed an inconclusive immunoprofile which along with negative PHF1 FISH led to an initial diagnosis of high-grade osteosarcoma. Next generation sequencing (NGS) revealed a particularly rare CREBBP::BCORL1 fusion. The second illustrates the peculiar presentation of a dural-based mass in a 52 year-old female who presented with neurologic dyscrasias. Sections showed a sheeted monotonous proliferation of ovoid to spindle cells, but in contrast to Case #1, the tumor contained an exuberance of reticular osteoid and woven bone deposition mimicking malignant osteogenic differentiation. NGS showed a novel CREBZF::PHF1 fusion. Both tumors recurred locally less than 1 year post-operatively. As such we reiterate that careful morphologic examination is axiomatic to any diagnosis in our discipline, but this paradigm must shift to recognize that molecular diagnostics can provide closure where traditional tools have notable limitations.

Expression of high molecular weight cytokeratin-A novel feature of aggressive and innate hormone-refractory prostatic adenocarcinoma.

BACKGROUND: Castration-resistance is common in advanced prostatic adenocarcinomas (PACs) treated with androgen deprivation therapy (ADT) and usually occurs after 2 years following treatment. A minority of PACs confer innate ADT resistance without prior hormonal treatment. The expression of HMWCK in PAC cells has not been studied. This study aimed to investigate the clinicopathologic and genomic features of HMWCK-expressing PACs and the relationship to ADT resistance. METHODS: A total of 469 PACs were studied for HMWCK expression (39 postradiotherapy, 57 post-ADT, 373 treatment-naïve PACs). Clinicopathologic correlations of the HMWCK expression with tumor grade groups, specific tumor morphologies, tumor stages and disease recurrence/persistence/progression were performed. Five HMWCK+ PACs were also sequenced for genetic alterations. RESULTS: Thirty one of the 469 cases (6.6%) showed variable HMWCK+ PAC. The HMWCK+ PAC often focally presented in the tumor and vast majority were associated with high Gleason scores and unfavorable growth patterns (cribriform, comedo-necrosis, and intraductal carcinoma) as well as high tumor stages. A small percentage of the HMWCK+ PCA (2/31, 6.5%) presented with frank squamous histomorphology. Vast majority (22/31, 87%) had no history of prior ADT. The HMWCK+ PAC all displayed diminished to lost expression of AR/NKX3.1. Most of the cases progressed within 12 months of ADT or disease persisted despite ADT. Of the 5 HMWCK+ PACs subjected to gene sequencing, 4 presented with PTEN/PI3K/MAPK pathway alterations. CONCLUSION: The study demonstrated HMWCK+ PAC to be a novel type of innate ADT-resistant PAC. Overexpression of HMWCK in PAC can be potentially used as a surrogate biomarker for aggressive and innate hormone-refractory PACs. The genetic alterations imply potential therapeutic implications of PI3K/MAPK inhibitors in the treatment of these deadly diseases.

Prognostic value of cribriform size, percentage, and intraductal carcinoma in Gleason score 7 prostate cancer with cribriform Gleason pattern 4.

Cribriform Gleason pattern 4 (CGP4) is an indicator of poor prognosis in Gleason Score 7 prostate cancer; however, the significance of the size and percentage of this pattern and the presence of concomitant intraductal carcinoma (IDC) in these patients is unclear. To study the significance of these parameters in radical prostatectomy specimens, 165 cases with CGP4 were identified and reviewed (2017-2019). The size and percentage cribriform pattern and presence of IDC were noted and correlated with adverse pathological features and biochemical recurrence (BCR)-free survival. On review, 156 cases had CGP4 (Grade Group 2: 87 and Grade Group 3: 69). Large cribriform pattern and cribriform percentage of >20% showed significant association with extraprostatic extension, surgical margin positivity, and presence of IDC, whereas the presence of IDC was associated with all the analyzed adverse pathological features. BCR was seen in 22 of 111 (20%) patients after a median follow-up of 11 months, and of these, 21 had large cribriform pattern. On univariate analysis, all parameters had significant predictive values for BCR-free survival except for tertiary Gleason pattern 5. On multivariate analysis, while >20% cribriform pattern was trending to be an independent predictor, only lymphovascular invasion was statistically significant. Large cribriform pattern, >20% cribriform, and presence of IDC are additional pathologic parameters of potential value in identifying patients with high risk for early BCR.

Comparative expression of immunohistochemical biomarkers in cribriform and pattern 4 non-cribriform prostatic adenocarcinoma.

The morphology of Gleason 4 prostate cancer (PCa) can be subdivided into cribriform and non-cribriform patterns. A large body of evidence has shown that pattern 4 cribriform PCa (especially non-glomeruloid type) is associated with adverse pathologic features and clinical outcomes compared with non-cribriform pattern 4 PCa. The underlying mechanisms for the aggressiveness of cribriform PCa are not fully understood. The aim of this study is to compare the immunohistochemical expression of various biomarkers and to determine the potential proteins that may account for their biologic and clinical differences. A total of 14 biomarkers were studied. The number of non-glomeruloid cribriform PCa cases studied for each biomarker ranged from 18 to 74 and the number of non-cribriform pattern 4 PCa studied for each biomarker ranged from 29 to 112. We demonstrated that, compared with non-cribriform Gleason pattern 4 PCa, EGFR was significantly upregulated and standard CD44 (CD44s) was significantly downregulated in cribriform PCa; no significant differences were found in the expression of AR, NKX3.1, ERG, EZH2, p53, Rb, C-Myc, BCL2, p16, CyclinD1, Her2/Neu, and Synaptophysin between these two groups of pattern 4 PCa. The study also showed, compared to non-cribriform PCa, cribriform PCa presented with significantly higher serum PSA and more advanced tumor stage. The significant overexpression of EGFR and downregulation of CD44s in non-glomeruloid cribriform PCa may, at least, partly explain the unfavorable pathology and clinical results for this growth pattern. Given that EGFR targeted inhibitors are now available, the findings may also have significant therapeutic implications.

Stratification of Atypical Intraepithelial Prostatic Lesions Based on Basal Cell and Architectural Patterns.

OBJECTIVES: High-grade prostatic intraepithelial neoplasia (HPIN) and atypical cribriform lesion of the prostate are considered the precursors or associators of invasive prostate cancer (iPCa). Given loss of basal cells being the hallmark of iPCa, we hypothesized that a subset of these atypical intraepithelial lesions (AILs) with sparse basal cells can be classified as prostatic intraepithelial carcinoma (PIC) with frequent iPCa association and that different morphologic patterns of PIC are associated with specific Gleason (G) patterns and scores for iPCa. METHODS: We stratified 153 foci of AILs from 110 patients based on the integrity of the basal cell layer and architectural patterns and their association with iPCa. RESULTS: We demonstrated that AILs could be stratified into usual HPIN (intact basal cell layer and simple patterns) with low-risk of iPCa association and PIC (sparse basal cell layer) with high risk of iPCa association. Furthermore, PIC could be divided into low-grade (simple patterns and associated with G3 and G3/4 iPCa) and high-grade PIC (complex patterns and associated with G4 and G3/4/5 iPCa). CONCLUSIONS: Such stratification is of great clinical significance and instrumental to clinical patient management. It not only increases the predictability of AILs for iPCa but also accommodates a clinical scenario for lesions with features of intraductal carcinoma when iPCa is not found, particularly in biopsies.

ZBTB16: A new biomarker for primitive neuroectodermal tumor element / Ewing sarcoma.

Primitive neuroectodermal tumor (PNET) traditionally encompasses two different classes of tumors with similar morphology - PNET of the peripheral nervous system (pPNET) and PNET of the central nervous system (cPNET). The latter also includes germ cell tumor-derived PNET (gPNET). There are currently no specific markers for gPNET. This study seeks to investigate the expression of ZBTB16 in PNET and other small round blue cell tumors as well as its potential diagnostic utility. Immunohistochemical expression of the ZBTB16 was studied in a total of 27 PNETs (12 pPNETs, 8 cPNETs, 3 primary testicular gPNETs, and 4 metastatic gPNETs) and 38 small round blue cell tumors. Positive expression for ZBTB16 was seen diffusely in 9/12 (75%), moderately in 2/12 (17%) and focally in 1/12 (8%) of pPNETs, diffusely in 3/7 (43%) and moderately in 4/7 (57%) of gPNETs, and diffusely in 2/8 (25%), moderately in 2/8 (25%) and focally in 4/8 (50%) of cPNETs. Whereas, all of the 38 non-PNET small round blue cell tumors were nonreactive. The results suggest that ZBTB16 is a highly sensitive and specific biomarker for both pPNET and gPNET/cPNET. ZBTB16 effectively differentiates PNETs from other small round blue cell tumor mimics, including the two most common germ cell tumor-derived somatic malignancies - rhabdomyosarcoma and nephroblastoma. Of note, compared to the expression of ZBTB16 in pPNET/Ewing sarcoma and gPNET, the expression of ZBTB16 in cPNET was more variable, which appears consistent with the heterogeneity of cPNET. The close proximity of ZBTB16 and FLI-1 genes on chromosome 11q may explain the overexpression of ZBTB16 in PNET, especially in pPNET with t(1122) translocation.

Long term oncologic outcome in patients with bladder cancer after radical cystectomy: Impact of carcinoma in situ in the era of neoadjuvant chemotherapy.

PURPOSE: To assess the impact of carcinoma in situ (CIS) on oncologic outcomes in patients who underwent radical cystectomy, with a focus on those who received neoadjuvant chemotherapy (NAC) including patients with down-staging to ≤ pT1cancer after chemotherapy. MATERIALS AND METHODS: All patients who underwent radical cystectomy for urothelial cancer with curative intent from 1985 to 2011 were included. The impact of CIS on recurrence free and overall survival (OS) was assessed in the whole cohort and a subgroup who received NAC as well as those with response to chemotherapy and down-staging to ≤ pT1. RESULTS: A total of 2518 patients with a median follow-up period of 9 years were included. Among all, 1397 (55.5%) had concomitant CIS on final pathology. CIS was associated with high risk pathologic features including high-grade disease, multifocality, and nodal involvement as well as worse recurrence free survival (RFS) with no impact on OS. We did not find a significant association between CIS and oncologic outcomes in a subset of patients who received NAC including those with down-staging to ≤ pT1 disease. In multivariate analysis, CIS had no association with either recurrence free or OS. CONCLUSIONS: Concomitant CIS in radical cystectomy specimens is associated with decreased RFS; however, in multivariate analysis, it was not an independent predicting factor of oncologic outcomes. Moreover, the impact of CIS on oncologic outcomes in a subset of patients who received NAC was insignificant.

Novel Gene Expression Signature Predictive of Clinical Recurrence After Radical Prostatectomy in Early Stage Prostate Cancer Patients.

BACKGROUND: Current clinical tools have limited accuracy in differentiating patients with localized prostate cancer who are at risk of recurrence from patients with indolent disease. We aimed to identify a gene expression signature that jointly with clinical variables could improve upon the prediction of clinical recurrence after RP for patients with stage T2 PCa. METHODS: The study population includes consented patients who underwent a radical retropubic prostatectomy (RP) and bilateral pelvic lymph node dissection at the University of Southern California in the PSA-era (1988-2008). We used a nested case-control study of 187 organ-confined patients (pT2N0M0): 154 with no recurrence ("controls") and 33 with clinical recurrence ("cases"). RNA was obtained from laser capture microdissected malignant glands representative of the overall Gleason score of each patient. Whole genome gene expression profiles (29,000 transcripts) were obtained using the Whole Genome DASL HT platform (Illumina, Inc). A gene expression signature of PCa clinical recurrence was identified using stability selection with elastic net regularized logistic regression. Three existing datasets generated with the Affymetrix Human Exon 1.0ST array were used for validation: Mayo Clinic (MC, n = 545), Memorial Sloan Kettering Cancer Center (SKCC, n = 150), and Erasmus Medical Center (EMC, n = 48). The areas under the ROC curve (AUCs) were obtained using repeated fivefold cross-validation. RESULTS: A 28-gene expression signature was identified that jointly with key clinical variables (age, Gleason score, pre-operative PSA level, and operation year) was predictive of clinical recurrence (AUC of clinical variables only was 0.67, AUC of clinical variables, and 28-gene signature was 0.99). The AUC of this gene signature fitted in each of the external datasets jointly with clinical variables was 0.75 (0.72-0.77) (MC), 0.90 (0.86-0.94) (MSKCC), and 0.82 (0.74-0.91) (EMC), whereas the AUC for clinical variables only in each dataset was 0.72 (0.70-0.74), 0.86 (0.82-0.91), and 0.76 (0.67-0.85), respectively. CONCLUSIONS: We report a novel gene-expression based classifier identified using agnostic approaches from whole genome expression profiles that can improve upon the accuracy of clinical indicators to stratify early stage localized patients at risk of clinical recurrence after RP. Prostate 76:1239-1256, 2016. © 2016 Wiley Periodicals, Inc.

Double-Blind Phase III Randomized Trial of the Antiprogestin Agent Mifepristone in the Treatment of Unresectable Meningioma: SWOG S9005.

PURPOSE: Progesterone receptors are expressed in approximately 70% of meningiomas. Mifepristone is an oral antiprogestational agent reported to have modest activity in a phase II study. This multicenter, prospective, randomized, placebo-controlled phase III trial conducted by SWOG was planned to define the role of mifepristone in the treatment of unresectable meningioma. PATIENTS AND METHODS: Eligible patients were randomly assigned to receive either mifepristone or placebo for 2 years unless disease progressed. Patients who were stable or responding to protocol therapy after 2 years had the option to continue with the same blinded therapy. Serial follow-up allowed assessment of efficacy and toxicity. Time to treatment failure and overall survival were ascertained for all randomly assigned patients. On progression, patients receiving placebo could cross over and receive active drug. RESULTS: Among 164 eligible patients, 80 were randomly assigned to mifepristone and 84 to placebo. Twenty-four patients (30%) were able to complete 2 years of mifepristone without disease progression, adverse effects, or other reasons for discontinuation. Twenty-eight patients (33%) in the placebo arm completed the 2-year study. There was no statistical difference between the arms in terms of failure-free or overall survival. CONCLUSION: Long-term administration of mifepristone was well tolerated but had no impact on patients with unresectable meningioma.

FCGR2A and FCGR3A polymorphisms associated with clinical outcome of epidermal growth factor receptor expressing metastatic colorectal cancer patients treated with single-agent cetuximab.

PURPOSE: Cetuximab, a chimeric immunoglobulin G 1 (IgG1) anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb), has shown efficacy in 10% of patients with metastatic colorectal cancer (CRC). Recent studies demonstrate antibody-dependent cell-mediated cytotoxicity (ADCC) is one of the modes of action for rituximab and trastuzumab. Fragment c (Fc) portion of IgG1 mAb has shown to induce ADCC. Fragment c gamma receptors (FcgammaR) play an important role in initiating ADCC. Studies have shown that two IgG FcgammaR polymorphisms (FCGR2A-H131R and FCGR3A-V158F) independently predict response to rituximab in patients with follicular lymphoma. We tested the hypothesis of whether these two polymorphisms are associated with clinical outcome in metastatic CRC patients treated with single-agent cetuximab. PATIENTS AND METHODS: Thirty-nine metastatic CRC patients were enrolled onto the ImClone0144 trial. Using an allele-specific polymerase chain reaction (PCR) -based method, gene polymorphisms of FCGA2A-H131R and FCGA3A-V158F were assessed from genomic DNA extracted from peripheral blood samples. RESULTS: FCGR2A-H131R and FCGR3A-V158F polymorphisms were independently associated with progression-free survival (PFS; P = .037 and .055, respectively; log-rank test). Combined analysis of these two polymorphisms showed that patients with the favorable genotypes (FCGR2A, any histidine allele, and FCGR3A, any phenylalanine allele) showed a median PFS of 3.7 months (95% CI, 2.4 to 4.4 months), whereas patients with any two unfavorable genotypes (FCGR2A arginine/arginine or valine/valine) had a PFS of 1.1 months (95% CI, 1.0 to 1.4 months; P = .004; log-rank test). CONCLUSION: Our preliminary data suggest that these two polymorphisms may be useful molecular markers to predict clinical outcome in metastatic CRC patients treated with cetuximab and that they may indicate a role of ADCC of cetuximab.

Association of methylenetetrahydrofolate reductase gene polymorphisms and sex-specific survival in patients with metastatic colon cancer.

PURPOSE: Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme regulating intracellular folate levels, which affects DNA synthesis and methylation. Two MTHFR gene polymorphisms, C677T and A1298C, are linked to altered enzyme activity. Several studies have shown these two polymorphisms to be associated with response to fluorouracil (FU) -based treatment in advanced colon cancer patients, but data are inconsistent and contradictory. Meanwhile, epidemiologic studies demonstrated that these MTHFR polymorphisms were associated with cancer risk in a sex-specific manner. We tested the hypothesis of whether these two polymorphisms are associated with sex-specific clinical outcome in metastatic colon cancer patients treated with FU-based chemotherapy. PATIENTS AND METHODS: This study included 318 patients (177 men and 141 women) with metastatic colon cancer treated between 1992 and 2003 at the University of Southern California/Norris Comprehensive Cancer Center or Los Angeles County/University of Southern California Medical Center. Peripheral blood samples were collected from each patient, and genomic DNA was extracted from WBCs. Two MTHFR gene polymorphisms (C677T and A1298C) were tested by fluorogenic 5'-nuclease assay. RESULTS: The A1298C polymorphism showed statistically significant differences in overall survival (OS) in female, but not male, patients with metastatic colon cancer (log-rank test, P = .038). Among females, OS was greater for patients with the A/A genotype (n = 67; median OS, 18.4 months) compared with patients with the A/C genotype (n = 50; median OS, 13.9 months) or C/C genotype (n = 10; median OS, 15.6 months). CONCLUSION: Although preliminary, these data support the role of the A1298C polymorphism in MTHFR as prognostic marker in female patients with metastatic colon cancer. Further studies are needed to confirm these findings.

Molecular determinants of irinotecan efficacy.

Molecular markers predicting the efficacy of CPT-11 based chemotherapies in patients with colorectal cancer (CRC) are unknown. Therefore, we investigated whether mRNA levels of drug targets (Topoisomerase I, TS), enzymes involved in 5-FU metabolism (DPD), in angiogenesis (EGFR, IL-8, VEGF) and in DNA-repair/drug detoxification (ERCC1, GST-P1) are associated with the clinical outcome of patients with CRC treated with first-line CPT-11 based chemotherapy. Thirty three patients with metastatic CRC were included in the study. Intratumoral gene expression levels were assessed from paraffin-embedded tissue samples, using laser capture microdissection and quantitative Real-Time PCR. Complete response was observed in 1 patient, partial response in 12 patients, stable disease in 13 patients and progressive disease in 6 patients. Response was inevaluable for 1 patient. Patients with complete response or partial response were classified as responders, while patients with stable disease or progressive disease were classified as nonresponders. High intratumoral mRNA levels of EGFR, ERCC1 and GSPT-P1 were each significantly associated with response to CPT-11 based chemotherapy. Recursive partitioning analysis showed that mRNA levels of EGFR and ERCC1 are primarily responsible for delineating responders from nonresponders. Also, the combination of high intratumoral gene expression levels of both EGFR and ERCC1 was significantly associated with progression-free survival. The mRNA levels of EGFR had a significant correlation with expression levels of ERCC1, GST-P1 and VEGF. This small retrospective study suggests that gene expression levels of EGFR, ERCC1 and GST-P1 may be useful in predicting the clinical outcome of patients with metastatic CRC treated with first-line CPT-11 based chemotherapy.

Cyclin D1 and epidermal growth factor polymorphisms associated with survival in patients with advanced colorectal cancer treated with Cetuximab.

The study aimed to investigate whether polymorphisms in genes of the EGFR signaling pathway are associated with clinical outcome in advanced colorectal cancer (CRC) patients treated with single-agent Cetuximab. Polymorphisms of interest in the EGFR pathway include: cyclin D1 (CCND1) A870G, cyclooxygenase 2 (Cox-2) G-765C, epidermal growth factor (EGF) A61G, epidermal growth factor receptor (EGFR) codon R497 K, EGFR CA dinucleotide repeat in intron 1, interleukin (IL)-8 T-251A and vascular endothelial growth factor (VEGF) C936 T gene polymorphisms. Thirty-nine metastatic CRC patients were enrolled in the IMCL-0144 trial and treated with single-agent Cetuximab. Using the polymerase chain reaction-restriction fragment length polymorphism method, gene polymorphisms of CCND1, COX-2, EGF, EGFR, IL-8 and VEGF were assessed from genomic DNA extracted from blood samples. A significant association was found between the CCND1 A870G polymorphism and overall survival in our 39 CRC subjects. Patients with the AA homozygous genotype survived for a median of 2.3 months [95% confidence interval (CI)=2.1-5.7], whereas those with any G allele (AG, GG genotype) survived for a median of 8.7 months (95% CI=4.4-13.5) (P=0.019, log-rank test). When we analysed the cyclin D1 and EGF polymorphisms together, patients with favourable genotypes (EGF any A allele and CCND1 any G allele) showed a median survival time of 12 months (95% CI=4.8-15.2), whereas patients with any two unfavourable genotypes (EGF GG or CCND1 AA) showed a median survived time of 4.4 months (95% CI=2.1-5.7) (P=0.004, log-rank test). The findings of this pilot study suggest that the cyclin D1 A870G and the EGF A61G polymorphisms may be useful molecular markers for predicting clinical outcome in CRC patients treated with single-agent Cetuximab.

Molecular determinants of cetuximab efficacy.

PURPOSE: To investigate whether mRNA expression levels of cyclin D1 (CCND1), cyclooxygenase 2 (Cox-2), epidermal growth factor receptor (EGFR), interleukin 8 (IL-8), and vascular endothelial growth factor (VEGF), all members of the EGFR signaling pathway, are associated with clinical outcome in patients with EGFR-expressing metastatic colorectal cancer (CRC) treated with cetuximab. PATIENTS AND METHODS: Thirty-nine patients with metastatic CRC, refractory to both irinotecan and oxaliplatin, were enrolled on IMCL-0144 and treated with single-agent cetuximab. The intratumoral mRNA levels of CCND1, Cox-2, EGFR, IL-8, and VEGF were assessed from paraffin-embedded tissue samples using laser-capture microdissection and quantitative real-time polymerase chain reaction. RESULTS: There were 21 women and 18 men with a median age of 64 years (range, 35 to 83 years). Higher gene expression levels of VEGF were associated with resistance to cetuximab (P = .038; Kruskal-Wallis test). The combination of low gene expression levels of Cox-2, EGFR, and IL-8 was significantly associated with overall survival (13.5 v 2.3 months; P = .028; log-rank test). Both findings were independent of skin toxicity that was itself significantly correlated to survival. Patients with a lower mRNA amount of EGFR had a longer overall survival compared with patients that had a higher mRNA amount (7.3 v 2.2 months; P = .09; log-rank test). Patients with lower expression of Cox-2 had a significantly higher rate of grade 2 to 3 skin reactions under cetuximab treatment. CONCLUSION: This pilot study suggests that gene expression levels of Cox-2, EGFR, IL-8, and VEGF in patients with metastatic CRC may be useful markers of clinical outcome in single-agent cetuximab treatment.

Cytologic identification of reinke crystalloids in scrapings and imprints of fresh testicular tumors: a simple and rapid technique for intraoperative use.

Although not required for the diagnosis, crystalloids of Reinke are pathognomonic for Leydig cell tumor. However, conventional frozen section rarely reveals their presence. A method of rapid identification of crystalloids of Reinke could improve the intraoperative diagnosis. We tested the efficacy of touch imprints and scrape smears for the identification of crystalloids in 2 cases of Leydig cell tumor of the testis. Intraoperative smears of the tumors yielded abundant crystals. Scrape cytologic testing was the better method. We speculate that the process of scraping, and to a lesser extent touch imprinting, disrupts the cytoplasm of the Leydig cells and releases the crystalloids. We conclude that cytologic testing is an effective method of identifying crystalloids of Reinke in Leydig cell tumors of the testis.