AGAPP: efficacy of first-line cisplatin, 5-fluorouracil with afatinib in inoperable gastric and gastroesophageal junction carcinomas. A Hellenic Cooperative Oncology Group study.

PURPOSE: Gastric cancer is the fifth most common neoplasm worldwide with high rates of mortality. Afatinib, a low molecular, irreversible potent inhibitor of ErbB trans-membrane receptor family, has shown promising results according to preclinical and phase I clinical trial data when combined with chemotherapy. We aimed at evaluating the safety and efficacy of the combination of cisplatin, 5FU with afatinib in molecularly unselected patients with advanced gastric cancer. METHODS: Patients with locally advanced or metastatic gastric/gastroesophageal junction adenocarcinoma received first line combination therapy of cisplatin, 5FU and afatinib every 21 days, followed by afatinib maintenance monotherapy. The primary endpoint was the Objective Response Rate (ORR); secondary endpoints included Overall Survival (OS), Progression Free Survival (PFS) and the safety profile. Unplanned exploratory analysis of HER2 and tumor mutational profile was performed. RESULTS: Among 55 patients (ITT population) enrolled, 19 (34.5%) achieved an objective tumor response; stable disease was observed in 16 patients (29.1%) and progressive disease in 10 patients (18.2%). The ORR in the per protocol population (PP) was 42.9%. Within a median follow-up of 56 months, the median PFS and OS in the ITT population was 5.0 and 8.7 months, respectively. Seven of the 47 HER2 informative cases carried HER2 positive tumors while TP53, BRCA2 and SMAD4 were the most frequently mutated genes. The most common toxicities were neutrophil count and white blood cell decrease occurring in 56.4% of patients, followed by anemia (50.9%), hyperglycemia (40%), and diarrhea (38.2%). CONCLUSIONS: The combination of cisplatin/5FU with afatinib did not surpass the benchmarks of efficacy of the contemporary therapeutic regimens that are being applied for the treatment of patients with advanced gastric cancer. However, the observed efficacy and the improved safety profile support that our administration schedule may be further investigated to overcome toxicity problems when integrating afatinib to cytotoxic chemotherapy. CLINICAL TRIAL REGISTRATION: NCT01743365.

Evaluation of the Role of p95 HER2 Isoform in Trastuzumab Efficacy in Metastatic Breast Cancer.

BACKGROUND/AIM: Human epidermal growth factor receptor 2 (HER2) P95-isoform could be involved in trastuzumab resistance in HER2 metastatic breast cancer. MATERIALS AND METHODS: A total of 114 metastatic breast cancer patients treated with trastuzumab were evaluated retrospectively. HER2 was centrally reviewed. P95 was evaluated along with other markers possibly affecting trastuzumab efficacy in regards to progression-free survival and overall survival. RESULTS: HER2 was centrally negative in 54 cases. P95 expression was significantly higher in HER2-positive tumors. High p95 was associated with gain of HER2 copy number variations (CNVs), high pHER2Tyr877, Ki67 and HER2 mRNA. P95 as a continuous variable was positively correlated with mRNA expression of HER2 and negatively correlated with HER4 and IGF1. HER2-negative p95-high patients had a marginally higher risk for death (HR=2.15, p=0.055). CONCLUSION: p95 was associated with higher HER2 CNVs and mRNA expression, pHER2Tyr877 expression and high Ki67, indicating a more aggressive phenotype.

Genotyping data of routinely processed matched primary/metastatic tumor samples.

Genotypic and phenotypic comparisons of tumors in multiple tissue samples from the same patient are important for understanding disease evolution and treatment possibilities. Panel NGS genotyping is currently widely used in this context, whereby NGS variant filtering and final evaluation constitute the basis for meaningful comparisons. Here, we present the genotype data used for genotype / phenotype comparisons between matched primary / metastatic colorectal tumors in the work by Chatzopoulos et al (doi: 10.1016/j.humpath.2020.10.009), as well as the process followed for obtaining these data. We describe key issues while processing routinely formalin-fixed paraffin-embedded (FFPE) tumors for genotyping, NGS application (Ion Torrent), a stringent variant filtering algorithm for genotype analyses in FFPE tissues and particularly in matched tumor samples, and provide the respective datasets. Apart from research, tumor NGS genotyping is currently applied for clinical diagnostic purposes in Oncology. The datasets and method description provided herein (a) are important for comprehending the peculiarities of FFPE tumor genotyping, which is still mostly based on principles of germline DNA genotyping; (b) can be used in pooled analyses, e.g., of primary / metastatic tumors for the investigation of tumor evolution.

Genotype-phenotype associations in colorectal adenocarcinomas and their matched metastases.

Although primary colorectal carcinomas (CRCs) frequently share genetic alterations with their metastases, morphologic surrogates reflecting the genotype contexture of metastases remain largely unknown. We investigated phenotype/genotype associations in paired primary and metastatic colorectal adenocarcinomas from 75 patients. Thirty-three (44%) metastatic lesions were synchronous and 42 (56%) were metachronous. Tumor budding, micronecrosis, and tumor-infiltrating lymphocyte (TIL) density were compared with matched next-generation sequencing genotypes. Micronecrosis in the primary were significantly associated with nodal status (P = 0.0054) and with micronecrosis in metastatic sites (P = 0.0216), particularly in metachronous metastases (P = 0.0033). With a 57-gene panel, one or more mutations were identified in 64 (85.3%) cases. In metastases, high (brisk) TILs were associated with overall mutational burden (P = 0.0058) and with mutations in EGF (P = 0.0325), RAS genes (P = 0.0043), and MMR genes (P = 0.0069), whereas high-level micronecrosis correlated with mutations in APC (P = 0.0004) and MSH6 (P = 0.0385) genes. Genomic alterations were shared in 90.1% of primary/metastatic pairs, but clonality of the same mutation was shared in only 57.1% of paired lesions. Compared with synchronous, metachronous metastases had more private clonal alterations (P = 0.0291); in this group, clonal alterations coincided with brisk TILs (P = 0.0334) and high micronecrosis (P = 0.0133). High TILs in metastatic lesions were predictive of favorable overall survival (log-rank P = 0.044). The observed phenotype/genotype associations favor the clonal evolution model in CRC metastases that seems accompanied by intense host immune response. If the role of micronecrosis and brisk TILs in metachronous metastases is validated in larger studies, these histologic parameters will be worth adding in the armamentarium for the evaluation of metastatic CRC.

Testis-Sparing Surgery for Non-Palpable Leydig Cell Tumors in Prepubertal Children.

Leydig cell tumor (LCT) is an infrequent stromal neoplasm of the testis with an incidence of less than 3% of all gonadal tumors in the general male population. Only 25% is found in prepubertal children, where Leydig cell tumors are always reported benign. The hospital records of two prepubertal male children, who underwent organ sparing surgery for testicular LCT the last five years, were retrospectively reviewed. In both of them, the lesion was incidentally found during a scrotal ultrasonography for testicular pain. The diagnosis of a benign LCT was based on the pre-operative physical examination and imaging (Ultrasound-US, Magnetic Resonance Imaging-MRI) as well as the negative tumor markers. A testicle-sparing procedure was decided and the pathologic examination of the surgical specimen confirmed the diagnosis. No tumor recurrence was noted on follow-up. Testis-sparing surgery provides the possibility of complete excision of such lesions and should be considered as the treatment of choice.

Prognostic impact of stromal and intratumoral CD3, CD8 and FOXP3 in adjuvantly treated breast cancer: do they add information over stromal tumor-infiltrating lymphocyte density?

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) and their subsets contribute to breast cancer prognosis. We investigated the prognostic impact of CD3+, CD8+ and FOXP3+ TILs in patients with early intermediate/high-risk breast cancer treated with adjuvant anthracycline-based chemotherapy within two randomized trials conducted by our Group. METHODS: We examined 1011 patients (median follow-up 130.9 months) and their tumors for total, stromal (s) and intratumoral (i) CD3, CD8 and FOXP3 lymphocyte density (counts/mm2) on tissue-microarray cores by immunohistochemistry. Morphological sTIL density on whole H&E-stained sections was also evaluated. RESULTS: The majority of TILs were CD3+. Total CD3 and CD8, sCD3 and sCD8, iCD3 and iCD8, sFOXP3 and iFOXP3 were strongly correlated (Spearman's rho values > 0.6). High individual lymphocytic subsets and sTIL density were strongly associated with high tumor grade, higher proliferation and HER2-positive and triple-negative tumors (all p values < 0.001). Higher sTIL density (10% increments), high density of almost each individual marker and all-high profiles conferred favorable prognosis. However, when adjusted for sTIL density, stromal and intratumoral lymphocytic subsets lost their prognostic significance, while higher sTIL density conferred up to 15% lower risk for relapse. Independently of sTIL density, higher total CD3+ and CD8+ TILs conferred 35% and 28% lower risk for relapse, respectively. CONCLUSIONS: Stromal and intratumoral CD3+, CD8+ and FOXP3+ TIL density do not seem to add prognostic information over the morphologically assessed sTIL density, which is worth introducing in routine histology reports.

Association of Notch and Hedgehog Pathway Activation With Prognosis in Early-stage Colorectal Cancer.

BACKGROUND/AIM: Early-stage colorectal cancer (CRC) carries a wide range of survival probabilities. Novel biomarkers in this setting are eagerly awaited. Cancer stem cells (CSCs) are considered one of the reasons for treatment failure. This study sought to determine whether activation of pathways governing the function of CSC's could correlate with treatment outcomes. MATERIALS AND METHODS: Tumor specimens from 325 patients were analyzed with immunohistochemistry (IHC) for Hedgehog and Notch pathway activation and results were correlated with prognosis. RESULTS: Positive Notch3 protein expression was an unfavorable prognostic factor for disease-free survival (DFS) and overall survival (OS) (HR=2.43, p=0.024 and HR=2.56, p=0.028, respectively). Activation of the Shh pathway showed univariately longer DFS (HR=0.49, p=0.032). Possible crosstalk between the two pathways was indicated. No further associations between pathway activation and outcome were evident. CONCLUSION: Apart from Notch 3, activation of the pathways, as indicated by IHC expression of their components, did not result in differences in terms of DFS or OS.

Prognostic Subcellular Notch2, Notch3 and Jagged1 Localization Patterns in Early Triple-negative Breast Cancer.

BACKGROUND: The Notch pathway has been implicated in triple-negative breast cancer (TNBC). Herein, we studied the subcellular localization of the less investigated Notch2 and Notch3 and that of the Jagged1 (Jag1) ligand in patients with operable TNBC. PATIENTS AND METHODS: We applied immunohistochemistry for Notch2, Notch3 and Jag1 in 333 tumors from TNBC patients treated with adjuvant anthracycline-based chemotherapy. We evaluated cytoplasmic (c), membranous (m) and nuclear (n) protein localization. RESULTS: c-Notch2 (35% positive tumors), c-Notch3 (63%), c-Jag1 (43%), m-Notch3 (23%) and n-Jag1 (17%) were analyzed individually and by using hierarchical clustering for prognostic evaluation. Upon multivariate analysis, compared to high m-Notch3 in the absence of n-Jag1 (cluster 4), all other marker combinations (clusters 1, 2, 3) conferred significantly higher risk for relapse (p<0.05). CONCLUSION: Specific Notch3 and Jag1 subcellular localization patterns may provide clues for the behavior of the tumors and potentially for Jag1 targeting in TNBC patients.

The Prognostic Value of the Immunohistochemical Expression of Phosphorylated RB and p16 Proteins in Association with Cyclin D1 and the p53 Pathway in a Large Cohort of Patients with Breast Cancer Treated with Taxane-based Adjuvant Chemotherapy.

BACKGROUND: The retinoblastoma (RB) gene is a tumor-suppressor gene that plays a central role in regulating the cell cycle. Inactivation of this gene is involved in breast cancer. PATIENTS AND METHODS: A total of 827 patients with breast cancer treated with taxane-based adjuvant chemotherapy were included in the study. Protein expression of RB, phosphorylated RB (pRB), p16, cyclin D1 and p53 was evaluated by immunohistochemistry. RESULTS: Neither of the retinoblastoma markers (RB and pRB) reached statistical significance in terms of their association with disease-free or overall survival. Nevertheless, when clustering analysis was performed, patients with tumors featuring low levels of p16, cyclin D1 and p53 with concomitantly high levels of pRB had reduced risk for relapse (Wald's p=0.015). CONCLUSION: The p53-mediated sensitivity of breast cancer cells to chemotherapeutic agents appears to be driven mostly by pRB. Using agents that enhance RB phosphorylation might possibly increase the chemosensitivity of breast cancer cells.

Adult type granulosa cell tumor of the testis: Radiological evaluation and review of the literature.

A testicular granulosa cell tumor of the adult type is very rare. Isolated case reports and small series have been published in the English literature. Here, we analyze an incidentally discovered testicular granulosa cell tumor in a 29-year-old man to discuss the radiological evaluation of this entity and review the literature.