Introduction/Background Aberrant Hedgehog (Hh) pathway signaling has been implicated in pathogenesis of several human cancers. Recent studies have indicated its active role in serous ovarian carcinomas. Smoothened protein (SMO), a transmembrane co-receptor in Hh pathway signal transduction, is inhibited in non-dividing cells, thus its disinhibition might be a trigger for uncontrolled cell proliferation and growth. Very few studies have explored the role of SMO in serous ovarian cancers. The aim of the study was to assess the expression of SMO protein and to explore the Smoothened gene promoter methylation in a cohort of serous ovarian carcinomas. Methodology SMO protein expression was immunohistochemically quantified in 40 high-grade serous carcinomas (HGSC), 12 low-grade serous carcinomas (LGSC), 20 normal ovarian and 9 normal fallopian tube samples (controls). SMO gene promoter methylation status was analyzed using methylation-specific polymerase chain reaction (MSP) in randomly selected HGSCs (n=10), LGSCs (n=10), and normal fallopian tube (n=9) samples. Kaplan-Meier survival plots were used to estimate the impact of SMO expression on patients‘ overall survival (OS). Results SMO nuclear expression was significantly higher in HGSCs and LGSCs compared with the fallopian tube samples (p=0.010 and p=0.003, respectively). LGSCs, compared with normal ovarian tissue, exhibited higher total, cytoplasmic/membrane and nuclear expression (p=0.000, p=0.001 and p=0.000, respectively). Comparing HGSCs and LGSCs, significantly higher total and cytoplasmic/membrane expression was found in HGSC (p=0.026 and p=0.030, respectively). SMO gene promoter was unmethylated in both LGSCs and HGSCs as well as in fallopian tube. In addition, the SMO protein expression had no significant impact on patients‘ OS (p=0.07). Conclusion Our data indicate the lack of SMO gene promoter methylation while a significant overexpression (particularly nuclear) of SMO protein characterized a substantial proportion of serous ovarian carcinomas. Further functional studies should elucidate the clinical relevance of these findings. Disclosure Nothing to disclose.

As the majority of cancers and gestational diseases are prognostically stage- and grade-dependent, the ultimate goal of ongoing studies in precision medicine is to provide early and timely diagnosis of such disorders. These studies have enabled the development of various new diagnostic biomarkers, such as free circulating nucleic acids, and detection of their epigenetic changes. Recently, extracellular vesicles including exosomes, microvesicles, oncosomes, and apoptotic bodies have been recognized as powerful diagnostic tools. Extracellular vesicles carry specific proteins, lipids, DNAs, mRNAs, and miRNAs of the cells that produced them, thus reflecting the function of these cells. It is believed that exosomes, in particular, may be the optimal biomarkers of pathological pregnancies and cancers, especially those that are frequently diagnosed at an advanced stage, such as ovarian cancer. In the present review, we survey and critically appraise novel epigenetic biomarkers related to free circulating nucleic acids and extracellular vesicles, focusing especially on their status in trophoblasts (pregnancy) and neoplastic cells (cancers).

Salivary duct carcinoma (SDC) is a rare, aggressive salivary gland malignancy, which often presents at an advanced stage. A proportion of SDC are characterized by HER2 amplification and/or overexpression of androgen receptor (AR), which could be targeted in a subset of patients, but the presence of AR splice variant‐7 (AR‐V7) in some SDC cases could result in resistance to anti‐androgen therapy. We evaluated a cohort of 28 cases of SDC for potentially targetable biomarkers and pathways using immunohistochemistry (IHC) and next‐generation sequencing (DNA and RNA) assays. Pathogenic genetic aberrations were found in all but 1 case and affected TP53 (n = 19), HRAS (n = 7), PIK3CA, ERBB2 (HER2), and NF1 (n = 5 each); KMT2C (MLL3) and PTEN (n = 3 each); BRAF (p.V600E), KDM5C and NOTCH1 (n = 2 each). Androgen receptor was expressed in all cases and 13 of 27 harbored the AR‐V7 splice variant (including a case without any other detectable genetic alteration). HER2 IHC was expressed in 11 of 28 cases. The majority of SDC cases had no biomarkers predictive of immunotherapy response: 5 cases exhibited low (1%‐8%) programmed death ligand 1 (PD‐L1) expression in tumor cells, 2 cases exhibited elevated TMB, and no samples exhibited microsatellite instability. Notably, the pre‐treatment biopsies from 2 patients with metastatic disease, who demonstrated clinical responses to anti‐androgen therapy, showed AR expression and no AR splice variants. We conclude that comprehensive molecular profiling of SDCs can guide the selection of patients for targeted therapies involving AR, HER2, PD‐L1, mitogen‐activated protein kinase, and PIK3CA pathways.

Abstract Background Neuroendocrine carcinoma of the uterine cervix (NEC) is a rare cervical malignancy, accounting for 0.9% of all cervical carcinomas. Cervical NEC is a high-grade cancer with an aggressive clinical course and poor outcome. Given that no target therapy has been approved yet for NEC, we explored novel targetable biomarkers in a large cohort of NEC of the cervix. Methods Sixty-two NEC of the cervix were profiled for biomarkers of targeted therapy including antibody-drug conjugates (DLL3, TROP-2, and FOLR1), tropomyosin receptor kinases (NTRK1/2/3 gene fusions), and immune checkpoint inhibitors (PD-L1, TMB, and MSI) using immunohistochemistry and DNA/RNA next-generation sequencing assays. Results The study included 36 primary and 26 metastatic cervical NEC. The mean patient’s age was 43.6 years (range, 24-82 years). DLL3 expression was observed in 81% of the cases with 49% of cases expressing diffusely (≥50% of positive cancer cells) DLL3 protein. DLL3 expression was inversely correlated with commonly observed mutations: PIK3CA (7/47) (p = 0.018) and PTEN mutations (5/40) (p = 0.006). Other frequently seen mutations (TP53 17%, KRAS 11% and CTTNB1 5%) were not associated with DLL3 expression. PD-L1 expression, high TMB and MSI-H were seen in single cases. Although NTRK protein expression was observed in 21% of the cases, none of these had confirmatory NTRK gene fusions. TROP-2 and FOLR1 were negative in all tested cases. Conclusions DLL3 protein and PIK3CA/PTEN pathway genes may be potential therapeutic targets for a substantial proportion of the patients NEC of the cervix. Based on I-O biomarkers status, the patients with cervical NEC are less likely to benefit from immune checkpoint inhibitors. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure D. Arguello: Full / Part-time employment: Caris Life Sciences. E. Contreras: Full / Part-time employment: Caris Life Sciences. Z. Gatalica: Full / Part-time employment: Caris Life Sciences. All other authors have declared no conflicts of interest.

Supplemental Digital Content is available in the text. The breast is a rare site for metastases, and their molecular characteristics have not been studied yet. Intrinsic molecular genetics, cancer characteristics, and breast tissue immune responses in diverse metastases to the breast have not been previously studied. We identified 64 patients with cancers metastatic to the breast: 51 carcinomas and 13 melanomas. Programmed death ligand 1 (PD-L1), steroid receptors, and HER2/neu expressions were evaluated using immunohistochemistry. Gene sequencing, copy number alterations, microsatellite instability, and tumor mutational burden were performed using next-generation sequencing platforms. The 3 most common primary sites for metastatic carcinomas were lung (37%), ovary (29%), and fallopian tubes/peritoneum (14%). TP53 mutations were commonly (50%) observed among the carcinoma cases, while other mutations were characteristic for the primary cancers (VHL in renal, BRCA1 in the fallopian tube, and BRAF in melanomas). High tumor mutational burden was detected in 5/14 carcinomas and 3/7 melanomas. Tumor cell PD-L1 expression was detected in 6 carcinomas, but not in any of the melanomas, whereas immune cells’ expression of PD-L1 was seen in 17 carcinomas and 6 melanomas. Estrogen receptor status was positive in 13/49 carcinomas including 12 adenocarcinomas originating from the ovary and fallopian tube or peritoneum and 1 duodenal neuroendocrine carcinoma. No carcinoma was HER2/neu positive. Intrinsic genetic characteristics of the metastases to the breast followed the pattern commonly seen in primary tumors. Biomarkers of potential benefit to immune checkpoint inhibition therapy were limited to PD-L1-positive non–small cell lung cancer. No common characteristics of the heterogeneous group of tumor metastases to this organ were identified.

Abstract Rationale: Appendiceal mucocele is a rare entity of mucinous cystic dilatation of the appendix. It has no typical clinical presentation and is considered a potentially premalignant condition. Patient concerns: We present a case of accidental intraoperative finding of an appendiceal mucocele in a 54-year old woman that clinically presented with an exacerbated chronic tubo-ovarian abscess. Diagnoses: Trans-vaginal ultrasonography showed an encapsulated, oval, unilocular mass above the uterus with a heteroechogenic structure, homogeneous fluid content, and smooth regular walls without inner proliferation. The histopathologic diagnosis was consistent with an appendiceal cystadenoma. Interventions: The patient underwent a simple appendectomy. Outcomes: There were no clinical, biochemical or imaging signs of the disease recurrence at 6 months follow up. Lessons: To our knowledge, this is the only well-documented case of appendiceal mucocele mimicking exacerbated chronic tubo-ovarian abscess reported in the literature. Awareness of a rare entity such as an appendiceal mucocele, which is frequently misdiagnosed as a potential cause of acute abdomen, is necessary for the appropriate management strategy in order to prevent complications.

The treatment of several solid and hematologic malignancies with immune checkpoint inhibitors (against PD-1/PD-L1) has dramatically changed the cancer treatment paradigm. However, no checkpoint inhibitors were previously approved for the treatment of triple-negative breast cancer (TNBC), a difficult-to-treat disease with a high unmet therapeutic need. Based on IMpassion130 clinical trial (NCT02425891), FDA has recently granted an accelerated approval for atezolizumab (TECENTRIQ®), a monoclonal antibody drug targeting PD-L1, plus chemotherapy (Abraxane; nab®-Paclitaxel) for the treatment of adults with PD-L1-positive, unresectable, locally advanced or metastatic TNBC. FDA has also approved the Ventana diagnostic antibody SP142 as a companion test for selecting TNBC patients for treatment with atezolizumab. In the present review, we briefly discuss the importance of this breakthrough as the first cancer immunotherapy regimen to be approved for the management of breast cancer.

I read with a great interest a recently published clinical study of Takala et al 1 on metaplastic carcinoma of the breast. The authors explored 78 patients with the diagnosis of metaplastic carcinoma in the period 2002‐2016. In line with previous studies, the authors confirmed predominantly triple‐negative phenotype of metaplastic carcinomas (85%) exhibiting a poor therapeutic response with an ag‐ gressive clinical course and poor outcome. Two things are worthy of further discussion: First, 12% of the pa‐ tients were estrogen receptor (ER)‐positive, eight of which received adjuvant endocrine therapy. I would appreciate more information from the authors regarding the percentage of ER positivity in these metaplastic carcinomas and a potential response to endocrine treat‐ ment. It is well known that most metaplastic carcinomas are ER‐neg‐ ative and if ER‐positive, the percentage of positive cancer cells is usually low (range, 1%‐10%). This fact may substantially affect the response to endocrine therapy, as the patients with lower ER posi‐ tivity in their breast cancers are less responsive to anti‐ER treatment modalities. Another important issue is a targeted therapy of metaplastic carcinomas that has recently come into focus. Multiple molecular studies have revealed potentially targetable biomarkers in met‐ aplastic carcinomas of the breast. These include PI3K (PIK3CA, PTEN, PIK3R1), MAPK (NF1, KRAS, NRAS), RB1, and Wnt path‐ ways.2‐9 Of these, PI3K pathway alterations are of particular im‐ portance in metaplastic carcinoma given that several studies have shown their relevance in predicting the response to mTOR/PIK3CA inhibitors.6,10‐13 In addition, programmed death‐ligand 1 (PD‐L1) expression in cancer and/or inflammatory cells, as a predictor of response to immune checkpoint inhibitors, has also been described in a substantial proportion of metaplastic carcinomas.3,14‐16 A pecu‐ liar case report of Adams et al revealed an impressive therapeutic response of the PD‐L1‐positive metaplastic carcinoma to the com‐ bined treatment with pembrolizumab (anti‐PD‐1 drug) and nab‐ paclitaxel therapy.16 Of note, the Food and Drug Administration (FDA) has recently approved atezolizumab, an anti‐PD‐L1 drug, for the treatment of PD‐L1‐positive triple‐negative breast cancers.17,18 Given the period of your cohort, I was wondering if any of the pa‐ tients were profiled for the aforementioned targetable biomarkers and treated accordingly? Semir Vranic MD, PhD

Breast cancer is the second most common cause of cancer-related deaths among women worldwide. It is a heterogeneous disease with four major molecular subtypes. One of the subtypes, human epidermal growth factor receptor 2 (HER2)-enriched (HER2-positive) is characterized by the absence of estrogen and progesterone receptors and overexpression of HER2 receptor, and accounts for 15–20% of all breast cancers. Despite the anti-HER2 and cytotoxic chemotherapy, HER2 subtype is an aggressive disease with significant mortality. Recent advances in molecular biology techniques, including gene expression profiling, proteomics, and microRNA analysis, have been extensively used to explore the underlying mechanisms behind human breast carcinogenesis and metastasis including HER2-positive breast cancer, paving the way for developing new targeted therapies. This review focuses on recent advances on gene expression and miRNA status in HER2-positive breast cancer.

Aim Barrett’s esophagus (BE) is a predisposing factor of esophageal adenocarcinoma/gastroesophageal junction adenocarcinoma (ECA/GEJ Aca). BE patients are stratified and subsequently monitored according to the risk of malignant progression by the combination of endoscopy and biopsy. This study is to evaluate the maspin expression patterns as early diagnostic markers of malignancy in BE patients. Materials and methods Immunohistochemistry (IHC) staining was performed on 62 archival core biopsies from 35 patients, including BE without dysplasia (intestinal metaplasia, IM), BE with low grade dysplasia, BE with high grade dysplasia, carcinoma in situ, and well to poorly differentiated ECA/GEJ Aca (PD-ECA/GEJ Aca). The intensity and the subcellular distribution of immunoreactivity were evaluated microscopically. Statistical analysis was performed using the χ2 and Fisher exact tests. Results The level of epithelial-specific tumor suppressor maspin protein inversely correlated with the progression from IM to PD-ECA/GEJ Aca. Lesions of each pathological grade could be divided into subtypes that exhibited distinct maspin subcellular distribution patterns, including nuclear only (Nuc), combined nuclear and cytoplasmic (Nuc+Cyt), cytoplasmic only (Cyt) and overall negligible (Neg). The Cyt subtype, which was minor in both IM and dysplasia (approximately 10%), was predominant in ECA/GEJ Aca as early as well-differentiated lesions (more than 50%: p = 0.0092). In comparison, nuclear staining of the tumor suppressor TP53 was heterogeneous in dysplasia, and did not correlate with the differentiation grades of ECA/GEJ Aca. Conclusion The Cyt subtype of maspin expression pattern in core biopsies of BE patients may serve as a molecular marker for early diagnosis of ECA/GEJ Aca.

Dear Editor, We highly appreciate Dr Altundag's feedback regarding our recently published manuscript in The Breast Journal.1 We are also thankful to the editor in chief (Dr S. Masood) for giving us the opportunity to address Dr Altundag's comments. Neo‐adjuvant chemotherapy has been widely used for breast cancer treatment due to the effective pathologic responses seen with newer therapeutic agents.2 Recently, it has also been introduced for the treatment of early breast cancer.3 Despite this, there is ongoing debate and controversies related to the use of neo‐adjuvant chemotherapy in breast cancer (critically appraised in a recent review by Vaidya et al2). We find Dr Altundag's point regarding our neo‐adjuvant cohort quite valid. In our study, ~43% of patients with invasive apocrine carcinoma (IAC) presented at the advanced stage (III or IV) with only 19% of the patients having the tumor size ≤2 cm at presentation. This is mainly due to the lack of organized screening program at the national level. In this regard, our small IAC cohort treated in neo‐adjuvant setting is somehow biased but it essentially reflected the previous and current overall breast cancer presentation in Bosnia and Herzegovina. Consequently, the response rates to neo‐adjuvant therapy in our study may be different from the previously published data. Noteworthy, IAC is a rare breast cancer subtype (~1%‐2% of all breast cancers)4,5 and future larger and multi‐institutional studies are required to validate the effectiveness of (neo)adjuvant chemotherapy in patients with IAC.