Novel morphological and genetic features of fumarate hydratase deficient renal cell carcinoma in HLRCC syndrome patients with a tailored therapeutic approach.

The hereditary leiomyomatosis and renal cell carcinoma syndrome (HLRCC) is defined by germline mutations in the fumarate hydratase (FH) gene and associated with leiomyomas and aggressive renal cell carcinomas with FH deficiency. Here, we comprehensively characterize two new patients with HLRCC syndrome on a morphological, immunohistochemical and genetic level. The patients developed aggressive HLRCC syndrome-associated RCCs, uterine leiomyomas and dermal leiomyomas. One HLRCC syndrome-associated RCC exhibited an unusual morphology with accumulation of "colloid-like" cytoplasmic inclusions, which might serve as a novel sentinel feature to trigger further testing. This case showed partially retained FH expression, initially hampering correct diagnosis. Comprehensive next-generation sequencing analyses of HLRCC syndrome-associated RCC and leiomyomas in our patients revealed divergent genetic changes in the FH gene in different tumors from the same patient. While all leiomyomas (uterine and cutaneous) showed a FH loss of heterozygosity (LOH) as a wildtype allele inactivating event, one HLRCC-RCC showed a second, undescribed NM_000143.3; c.947C>T; p.Ala316Val FH mutation accompanying the preexisting splice site mutation c.378+2T>C. In the other HLRCC syndrome-associated RCC, the FH mutation (NM_000143.3; c.462T>G; p.Asn154Lys with a somatic LOH) represents another variant of unknown significance that we link to HLRCC - and thus classify as likely pathogenic. Due to the specific diagnosis of metastatic HLRCC syndrome-associated RCC, both cases were treated in first line with bevacizumab/erlotinib and showed remarkable and long lasting responses. These findings allow new morphological and molecular insights into the biology of the HLRCC syndrome, corroborate the "second hit" hypothesis of tumor formation in HLRCC patients and may promote a distinct therapeutic approach.

SOX2 Gene Amplification and Overexpression is Linked to HPV-positive Vulvar Carcinomas.

SOX2 (SRY-related HMG-box 2) belongs to the SOX gene family of high-mobility transcription factors indispensably involved in gene regulation in pluripotent stem cells and neural differentiation. SOX2 copy number increases have been frequently reported in various types of squamous cell cancer. To better understand the effect of SOX2 aberrations on vulvar cancer phenotype and patient prognosis, we analyzed SOX2 copy number changes using fluorescence in situ hybridization and SOX2 expression by immunohistochemistry in 55 squamous cell carcinomas of the vulva. SOX2 amplification was found in 20.8% of tumors; 27.3% of vulvar carcinomas showed SOX2 protein overexpression. SOX2 amplification was correlated with SOX2 overexpression in our data set (P<0.01). Amplification of the SOX2 locus was associated with high tumor grade (P<0.05) and human papillomavirus (HPV) positivity (P<0.01). SOX2-amplified tumors showed more frequently a basaloid phenotype than nonamplified carcinomas. SOX2 protein overexpression was also correlated with basaloid phenotype and positive HPV status of vulvar carcinomas (P<0.05, each). SOX2 amplification and expression were not associated with patient overall survival. In conclusion, SOX2 copy number increases are detectable in a substantial proportion of high-grade HPV-positive vulvar carcinomas with basaloid differentiation. Our study provides further evidence for different molecular alterations in HPV-positive and HPV-negative vulvar carcinomas.

Impact of reassessment of colonic hyperplastic polyps by expert GI pathologists.

BACKGROUND: Recommended follow-up intervals after endoscopic removal of hyperplastic polyps (HP) and sessile serrated adenomas (SSA) differ because of assumed differences in biological behaviour. However, histopathologic differentiation is difficult, with higher SSA rates reported from specialist GI histopathologists. OBJECTIVE: The objective of this study was to clarify the relevance of histologic reassessment of HP. DESIGN AND SETTING: From a prospective screening colonoscopy study relevant serrated lesions (excluding distal small HP ≤5 mm) diagnosed by private practice pathologists were reassessed by four specialized GI pathologists PATIENTS: One thousand sixty-nine screening colonoscopies were performed in patients. MAIN OUTCOME MEASUREMENTS: In terms of main outcome measurements, there is a likelihood of changes of the HP diagnosis on reassessment, as well as interrater variability. RESULTS: SSA were initially diagnosed in 7 cases (0.7%) and relevant HP in 83 (7.8%; 101 lesions). Of the latter, the chance of a change in diagnosis from HP to SSA by any of the four specialist histopathologists was higher for larger (>5 mm) and right-sided lesions (19.1 vs 1.3%, OR 18.4, p = 0.04) including a higher likelihood to change recommended follow-up intervals (32.1 vs 3.3%, p < 0.01). However, follow-up intervals were determined by concomitant adenomas in 41%. Interrater variability was also higher for these lesions (p = 0.04), with an overall kappa value of 0.48. However, this issue related to only 1.2% of the 1069 study cases. LIMITATION: The limitations this study are the limited case number as well as limited retrospective assessment. CONCLUSIONS: Right-sided HP >5 mm had a higher chance of change in diagnosis to SSA; therefore, they should probably be treated like adenomas and be removed. However, reliable data for recommendations on follow-up intervals of HP or SSA will require follow-up studies.

Secondary sentinel node biopsy after previous excision of the primary tumor in squamous cell carcinoma of the vulva.

BACKGROUND: To reduce morbidity of radical groin dissection, the sentinel-node (SLN) procedure was implemented for the treatment of vulvar cancer. It has been proven to be a safe alternative in early-stage disease. Feasibility and safety of the procedure after previous vulvar surgery remain unclear. METHODS: A total of 106 patients with primary vulvar cancer undergoing the SLN procedure were analyzed. Seventy-four patients received the SLN procedure concomitant to vulvar surgery [primary-sentinel group (PSG)], whereas 32 patients had vulvar surgery before secondary SLN [secondary-sentinel group (SSG)]. RESULTS: SLN detection was possible in all patients. Three (9.4 %) patients in the SSG and 30 (40.5 %) in the PSG had metastatic spread to the SLN and underwent radical groin dissection. Median interval between vulva surgery and secondary sentinel was 34 days (range, 7-98). In the SSG tumor, stages were earlier with smaller tumor size (median 19 mm in the PSG vs. 9 mm in the SSG) and lesser invasion depth (4 vs. 2 mm; p < 0.001). There were no groin recurrences in the SSG and 5.4 % in the PSG. No significant difference regarding disease-free survival (DFS) could be detected (3-year DFS of 72.5 % in the PSG compared with 92.5 % in the SSG (median DFS not reached, p = 0.114)). Adjusting for potential confounders (tumor stage, nodal status, tumor size, invasion depth) did not alter the results with regards to DFS. CONCLUSIONS: Our results suggest that a secondary SLN procedure after previous vulvar surgery is feasible and can accurately reflect the groin status of selected patients. Ideally, prospective trials should be conducted to verify accuracy and oncologic safety of the procedure.

Rare oncogenic mutations of predictive markers for targeted therapy in triple-negative breast cancer.

Women with triple-negative breast cancer (TNBC) do not benefit from endocrine therapy or trastuzumab. Chemotherapy is the only systemic therapy currently available. To reduce the elevated risk of disease progression in these patients, better treatment options are needed, which are less toxic and more targeted to this patient population. We performed a comprehensive analysis of potential targetable genetic aberrations affecting the receptor tyrosine kinase/RAS/MAPK pathway, which are observed at higher frequencies in adenocarcinomas of other organs. Sixty-five individual TNBCs were studied by sequence analysis for HER2 (exon 18-23), EGFR (exon 18-21), KRAS (exon 2), and BRAF (exon 15) mutations. In addition, a tissue microarray was constructed to screen for EGFR gene copy gain and EML4-ALK fusion by FISH. Triple-negative status was confirmed by immunohistochemistry and FISH on tissue microarray sections. EGFR and CK5/6 immunohistochemical analyses were performed for identification of the basal-like phenotype. In addition, mutation analysis of TP53 (exon 5-8) was included. Sequence analysis revealed HER2 gene mutation in only one patient (heterozygous missense mutation in exon 19: p.L755S). No mutations were found in EGFR, KRAS, and BRAF. High polysomy of EGFR was detected in 5 of the 62 informative cases by FISH. True EGFR gene amplification accompanied by strong membranous EGFR protein expression was observed in only one case. No rearrangement of the ALK gene was detected. Basal-like phenotype was identified in 38 of the 65 TNBCs (58.5 %). TP53 gene mutation was found in 36/63 (57.1 %) tumors. We conclude that targetable genetic aberrations in the receptor tyrosine kinase/RAS/MAPK pathway occur rarely in TNBC.

Relevance of activated leukocyte cell adhesion molecule (ALCAM) in tumor tissue and sera of cervical cancer patients.

BACKGROUND: An altered expression of the activated leukocyte cell adhesion molecule (ALCAM) is associated with cancer progression in various cancer types. In some cancers ALCAM has a prognostic value or is predictive for the benefit of therapeutic interventions. To date there are no data on the role of ALCAM in cervical cancer available. METHODS: In this study, ALCAM expression was analysed by immunohistochemistry (IHC) in tissue samples of 233 patients with cervical cancer, among them 178 with complete follow-up information. In addition, soluble (s-)ALCAM was measured in sera of a subset of the included patients (n = 55) by enzyme-linked immunosorbent assay (ELISA). RESULTS: ALCAM overexpression was detected (immunoreactive score (IRS) 2-12) in 58.4% of the cervical cancer samples. The normal ectocervical or endocervical epithelium showed no ALCAM reactivity. In untreated patients, ALCAM overexpression in tumor tissue tended to be associated with shorter cancer-specific survival (CSS) and disease-free survival (DFS). Patients, whose tumor samples showed ALCAM overexpression receiving a cytotoxic therapy like radiotherapy or chemoradiation, however, had a favourable prognosis compared to those patients, whose cancers showed no or minimal ALCAM staining. This effect was particularly apparent in patients receiving chemoradiation where the CSS was significantly longer in patients with ALCAM-positive tumors (p = 0.038; cumulative incidence rates at 96 months 8%, 95% CI 0%-23%, and 26%, CI 3%-43% in ALCAM-positive and ALCAM-negative cases, respectively).Median preoperative s-ALCAM concentration in sera from tumor patients was 27.6 ng/ml (range 17.5-55.1 ng/ml, mean 28.9 ng/ml), serum levels did not correlate with intratumoral ALCAM expression. CONCLUSIONS: The data of our retrospective study suggest that the prognostic value of ALCAM expression in cervical carcinoma might be therapy-dependent, and that ALCAM might function as a predictive marker for the response to chemoradiation. This should be confirmed in further, prospective studies.

Prognostic role of lymph node metastases in vulvar cancer and implications for adjuvant treatment.

OBJECTIVE: Lymph node metastases are the most important prognostic factor for recurrence and survival in vulvar cancer. However, information regarding the impact of the number of positive nodes in vulvar cancer is inconsistent, and so are recommendations when to apply adjuvant radiotherapy. METHODS: One hundred fifty-seven consecutive patients with primary squamous cell cancer of the vulva treated at our center were analyzed. All patients underwent primary surgery by triple incision resulting in complete tumor resection. RESULTS: Median age was 61 years; 49 patients (31%) had lymph node metastases; 21 patients had 1, 13 had 2, and 15 had more than 2 positive lymph nodes. Thirty-two percent of the patients received adjuvant radiotherapy. The risk of lymph node metastases increased with age, greater tumor size, deeper invasion, and higher tumor grade. Median follow-up was 36 months; 23 patients (14.6%) developed disease recurrence (61% vulva, 35% groins, and 4% both). Compared with node-negative patients, survival in all node-positive patients was significantly impaired (P < 0.001; disease-free patients after 2 years: 88% in node-negative patients; 60%, 43%, and 29% in patients with 1, 2, and >2 affected nodes, respectively), whereas no significant difference between the node-positive subgroups could be demonstrated regarding disease-free survival. In multivariate analysis, lymph node status remained the most important prognostic factor regarding disease-free survival, but the effect of positive nodes differed significantly dependent on adjuvant treatment (P = 0.001). In patients without adjuvant radiotherapy to the groins/pelvis, the number of metastatic nodes was highly relevant for prognosis (hazard ratio, 1.752; P < 0.001), whereas this effect disappeared in patients who were treated with adjuvant radiotherapy (hazard ratio, 0.972; P = 0.828). CONCLUSIONS: The negative impact of lymph node metastases is already evident in patients with only 1 affected lymph node. In patients receiving adjuvant radiotherapy, the negative effect of additional lymph node metastases is reduced; adjuvant treatment might therefore be beneficial in patients with only 1 positive node.

Assessment of cervical intraepithelial neoplasia (CIN) with colposcopic biopsy and efficacy of loop electrosurgical excision procedure (LEEP).

PURPOSE: Conization for suspected high grade cervical intraepithelial neoplasia (CIN) is often performed based on abnormal cytology only. Loop electrosurgical excision procedure (LEEP) is a very common technique in this context. The present study analyses the accuracy of preoperative assessment of CIN with cytology plus colposcopic biopsy and assesses the efficacy of LEEP for the treatment of CIN. METHODS: Two-hundred and sixty-six consecutive patients treated with LEEP for suspected CIN at our center were retrospectively analyzed. Cytology, HPV-DNA testing, colposcopically directed cervical biopsy and/or endocervical curettage were performed to assess cervical lesions before and 3-6 months after surgery. RESULTS: Median age of the patients was 34 years. Median follow-up was 50 months. Preoperative HPV testing was positive for high risk types in 77.9%. All patients underwent LEEP without further ablative procedures. Complete excision of the lesion could be achieved in 84.3%; in 13.5% margins were not securely cleared and in 2.2% the lesion was not excised entirely. Overall complication rate was 5.4% (mainly postoperative bleeding and pain). Overall concordance of colposcopic biopsy and cone histology was 85.8%. The concordance rate was higher for CIN 2/3 (95.1%) compared with CIN 1 (63.2%). Nine patients (3.4%) had persistent disease after 3 months, 4 (1.5%) developed disease recurrence and underwent re-conization. HPV testing at 3-6 months after surgery was negative in 78.5%; 2 of the patients developing disease recurrence had a persistent HPV infection after surgery. CONCLUSIONS: Assessment of cervical lesions with colposcopic biopsy is an accurate method (concordance with cone histology 85.8%). Surgical treatment of high grade CIN with LEEP is a safe procedure with low recurrence rates, resulting in a clearance of cervical HPV infection in the majority of cases.

Prognostic value of pathological resection margin distance in squamous cell cancer of the vulva.

BACKGROUND: A tumor-free resection margin of at least 8 mm is considered state of the art in vulvar cancer. This standard is based on small and heterogeneous patient cohorts, and its implementation can result in mutilation. METHODS: One hundred two consecutive patients with primary squamous cell vulvar cancer were analyzed. All patients received resection of the primary tumor and the inguinofemoral lymph nodes via three separate incisions, resulting in complete tumor resection. Median follow-up was 31 months. Minimal margin distances were pathologically determined in all dimensions after fixation. RESULTS: Median age of the patients was 62 years; 38.2% had International Federation of Gynecology and Obstetrics (FIGO) stage I, 17.6% stage II, 24.4% stage III, and 8.8% stage IV disease. The median minimal resection margin was 5 mm (range 0.5-25 mm). Sixteen patients (15.6%) developed disease recurrence, of whom 10 (62.5%) at the vulva. Margin distance had no significant impact on disease-free survival when analyzed continuously (p = 0.388). When cases were divided into three subgroups of <3 mm (28.4%), ≥3 to <8 mm (42.2%), and ≥8 mm (29.4%) resection margin, neither univariate nor multivariate analysis revealed a difference in disease-free survival. There was no significant difference between any of the subgroups regarding tumor stages and adjuvant radiotherapy of the vulva. These results were independent of the direction of the minimal margin distance and consistent when only vulvar recurrences were analyzed. CONCLUSIONS: A tumor-free resection margin is essential for locoregional control in vulvar cancer. However, in this large, single-center study, we could not demonstrate any prognostic impact of pathological margin distance.

Carbonic anhydrase IX in tumor tissue and sera of patients with primary cervical cancer.

BACKGROUND: Carbonic anhydrase IX (CAIX) is a membranous expressed metalloenzyme involved in pH homeostasis and cell adhesion. The protein is overexpressed in a variety of tumors and potentially associated with negative outcome. This study was designed to investigate the prognostic role of CAIX in serum and tumor tissue of patients with primary cervical cancer. METHODS: Tumor samples of 221 consecutive patients with primary cervical cancer who underwent surgery between 1993 and 2008 were analyzed for CAIX expression by immunohistochemistry. Additionally, preoperative serum CAIX concentrations were determined by ELISA in a subset of patients. Correlation with intratumoral CAIX expression as well as clinicopathological factors and outcome was analyzed. RESULTS: CAIX expression was observed in 81.9% of the tumor specimens; 62.0% showed a moderate or strong staining intensity. Moderate/strong expression was associated with squamous histology (p=0.024), advanced tumor stage (p=0.001), greater invasion depth (p=0.025), undifferentiated tumor grade (p<0.001) and high preoperative SCC-Ag values (p=0.042). Furthermore patients with moderate/strong intratumoral CAIX expression had a higher number of metastatic lymph nodes compared to those with none/weak intratumoral expression levels (p=0.047) and there was a non-significant association between high intratumoral CAIX expression and shorter survival (p=0.118). Preoperative serum concentrations of CAIX ranged between 23 and 499 pg/mL and did not correlate with intratumoral expression or other clinicopathological variables. CONCLUSION: CAIX is associated with advanced tumor stages and lymph node metastases in cervical cancer, potentially representing a new target in this disease. In contrast to other epithelial cancers we could not observe a correlation between serum CAIX and its intratumoral expression.

Role of TP53 mutations in vulvar carcinomas.

Human papillomavirus (HPV)-independent development of vulvar carcinomas is common and the disruption of the TP53 pathway seems to play a key role in these tumors. Overexpression of TP53 in precursor lesions (differentiated VIN) and associated invasive carcinomas is regarded as an important diagnostic feature of this subtype of vulvar cancer. To determine the relationship of TP53 mutation status with clinicopathologic parameters, HPV status, and patient outcome, 18 squamous cell carcinomas of the vulva with TP53 overexpression along with 21 immunohistochemically TP53-negative tumors were analyzed. TP53 mutations were found in 17 (43.6%) of vulvar cancers, 18 (46.2%) tumors were HPV associated, and 8 (20.5%) carcinomas showed no relation to HPV infection or TP53 mutations. The presence of TP53 mutations was significantly linked to TP53 overexpression (P=0.002) and negative HPV status (P=0.012). The specificity of TP53 protein overexpression for the occurrence of TP53 mutations was 68.2%, with a positive predictive value of 66.7%. The most frequent mutation types were C:G →T:A transitions (57.9%). This mutation pattern strongly indicates the important role of oxidative stress in vulvar carcinogenesis. There were no relationships between TP53 mutation status and tumor stage, grading, nodal status, depth of invasion, or patient prognosis. In summary, TP53 mutations play a crucial role in a substantial proportion of vulvar carcinomas and are probably associated to cellular oxidative stress in chronically degenerative diseases of the vulva, such as lichen sclerosus. These data support the potential utility of restoring TP53 function as a therapeutic alternative in vulvar cancer. Further studies are necessary to clarify the prognostic implications of TP53 mutations in vulvar carcinomas.

Serum carbonic anhydrase IX and its prognostic relevance in vulvar cancer.

INTRODUCTION: Therapeutic options in advanced or recurrent vulvar cancer are limited. The identification of new prognostic factors and markers for therapy stratification is therefore highly desirable. Carbonic anhydrase IX (CAIX) is up-regulated in various solid tumors and a promising new target. We therefore determined CAIX serum concentration and its prognostic relevance in correlation to intratumoral CAIX expression in patients with primary vulvar cancer. METHODS: Thirty-one serum samples of patients with primary vulvar cancer were prospectively collected before surgery and analyzed for CAIX by enzyme-linked immunosorbent assay. In addition, intratumoral CAIX expression was determined by immunohistochemistry and correlation with serum CAIX and clinicopathological factors, and outcome was analyzed. RESULTS: Preoperative serum concentration of CAIX ranged between 56 and 879 pg/mL (median, 147 pg/mL; mean, 237.29) and was significantly higher in patients with high intratumoral expression (median, 269 pg/mL vs 126 pg/mL, P = 0.03). High serum CAIX was not associated with any of the analyzed clinicopathological parameters. However, disease-free survival was shorter in patients with high preoperative serum CAIX (above median; P = 0.012). By immunohistochemistry, 26% of the tumors showed a moderate or strong expression of CAIX, whereas 74% showed weak or no expression. High intratumoral expression of CAIX was also associated with unfavorable disease-free survival (P = 0.043). CONCLUSIONS: Carbonic anhydrase IX serum concentration is higher in patients with high intratumoral expression, and elevated preoperative serum values are associated with unfavorable prognosis. Serum CAIX might therefore be an easily assessable marker to stratify patients for adjuvant therapy and potentially monitor response. Carbonic anhydrase IX is differentially expressed in vulvar cancer and potentially associated with negative outcome.

PPFIA1 and CCND1 are frequently coamplified in breast cancer.

Recently, amplification of PPFIA1, encoding a member of the liprin family located about 600 kb telomeric to CCND1 on chromosome band 11q13, was described in squamous cell carcinoma of head and neck. Because 11q13 amplification is frequent in breast cancer, and PPFIA1 has been suggested to contribute to mammary gland development, we hypothesized that PPFIA1 might also be involved in the 11q13 amplicon in breast cancer and contribute to breast cancer development. A tissue microarray containing more than 2000 human breast cancers was analyzed for gene copy numbers of PPFIA1 and CCND1 by means of fluorescence in situ hybridization. PPFIA1 amplification was found in 248/1583 (15.4%) of breast cancers. Coamplification with CCND1 was found in all (248/248, 100%) PPFIA1-amplified cancers. CCND1 amplification without PPFIA1 coamplification was found in additional 117 (4.7%) tumors. Amplification of both PPFIA1 and CCND1 were significantly associated with high-grade phenotype (P = 0.0002) but were unrelated to tumor stage (P = 0.7066) or nodal stage (P = 0.5807). No difference in patient prognosis was found between 248 CCND1/PPFIA1 coamplified tumors and 117 tumors with CCND1 amplification alone (P = 0.6419). These data show that PPFIA1 amplification occurs frequently in breast cancer. The higher incidence of CCND1 amplification when compared with PPFIA1, the lack of prognostic relevance of coamplifications, and the fact that PPFIA1 amplification was found exclusively in CCND1-amplified cancers suggest that PPFIA1 gene copy number changes represent concurrent events of CCND1 amplification rather than specific biological incidents.

Deep learning for the standardized classification of Ki-67 in vulva carcinoma: A feasibility study.

BACKGROUND: The aim of this study is to demonstrate the feasibility of automatic classification of Ki-67 histological immunostainings in patients with squamous cell carcinoma of the vulva using a deep convolutional neural network (dCNN). MATERIAL AND METHODS: For evaluation of the dCNN, we used 55 well characterized squamous cell carcinomas of the vulva in a tissue microarray (TMA) format in this retrospective study. The tumor specimens were classified in 3 different categories C1 (0-2%), C2 (2-20%) and C3 (>20%), representing the relation of the number of KI-67 positive tumor cells to all cancer cells on the TMA spot. Representative areas of the spots were manually labeled by extracting images of 351 × 280 pixels. A dCNN with 13 convolutional layers was used for the evaluation. Two independent pathologists classified 45 labeled images in order to compare the dCNN's results to human readouts. RESULTS: Using a small labeled dataset with 1020 images with equal distribution among classes, the dCNN reached an accuracy of 90.9% (93%) for the training (validation) data. Applying a larger dataset with additional 1017 labeled images resulted in an accuracy of 96.1% (91.4%) for the training (validation) dataset. For the human readout, there were no significant differences between the pathologists and the dCNN in Ki-67 classification results. CONCLUSION: The dCNN is capable of a standardized classification of Ki-67 staining in vulva carcinoma; therefore, it may be suitable for quality control and standardization in the assessment of tumor grading.

Discrepancies between radiological and histological findings in preoperative core needle (CNB) and vacuum-assisted (VAB) breast biopsies.

BACKGROUND: Ultrasound (US)-guided breast biopsy is a routine diagnostic method used to correlate imaging finding to a histological diagnosis which is still the gold standard in preoperative diagnostics. The accuracy of US-guided breast biopsies relies on a precise radiologic-histopathologic correlation, which is discussed amongst an interdisciplinary team of gynecologists, radiologists and pathologists. However, false-negative or non-diagnostic biopsy results occur. Hence, a thorough and honest discussion to clarify the reason for discrepancies and to decide the next diagnostic step between specialists of the different disciplines is warranted. In this retrospective study, we analyzed discrepant findings between imaging and pathology results on preoperative breast biopsies. METHODS: Core and vacuum-assisted breast biopsies from 232 patients were included in this study. Inclusion criteria were (1) non-diagnostic (B1) category on histology independent from imaging category and (2) histological benign (B2) category with a BIRADS 5 (Breast Imaging Reporting and Data System) rating on imaging. Histological diagnoses were retrieved from all cases. Follow-up data were available in most cases. RESULTS: 138 biopsies were classified as B1, 94 biopsies as B2 category. 51 of 138 B1 cases (37%) underwent re-biopsy. Re-biopsy found malignancy (B5) in 19 of 51 cases, and B3/4 (premalignant) lesions in 3 of 51 cases. All B2 cases underwent second-look imaging-diagnosis, in 57 of 94 cases (66%) consecutive direct surgery or re-biopsy. Of these, malignancy was diagnosed histologically in 26 of 57 cases (45.6%). CONCLUSION: Determining imaging-pathology concordance after US-guided breast biopsy is essential. Discrepant cases and further diagnostic steps need to be discussed with an interdisciplinary approach.

Amplification of 8q21 in breast cancer is independent of MYC and associated with poor patient outcome.

Copy number gains involving the long arm of chromosome 8, including high-level amplifications at 8q21 and 8q24, have been frequently reported in breast cancer. Although the role of the MYC gene as the driver of the 8q24 amplicon is well established, the significance of the 8q21 amplicon is less clear. The breast cancer cell line SK-BR-3 contains three separate 8q21 amplicons, the distal two of which correspond to putative target genes TPD52 and WWP1. To understand the effect of proximal 8q21 amplification on breast cancer phenotype and patient prognosis, we analyzed 8q21 copy number changes using fluorescence in situ hybridization (FISH) in a tissue microarray containing more than 2000 breast cancers. Amplification at 8q21 was found in 3% of tumors, and was associated with medullary type (P<0.03), high tumor grade (P<0.0001), high Ki67 labeling index (P<0.05), amplification of MYC (P<0.0001), HER2, MDM2, and CCND1 (P<0.05 each), as well as the total number of gene amplifications (P<0.0001). 8q21 copy number gains were significantly related to unfavorable patient outcome in univariate analysis. However, multivariate Cox regression analysis did not reveal an independent prognostic value of 8q21 amplification. The position of our FISH probe and data of a previously performed high-resolution CGH study in the breast cancer cell line SK-BR-3 involve TCEB1 and TMEM70 as new possible candidate oncogenes at 8q21 in breast cancer.

Homogeneous EGFR amplification defines a subset of aggressive Barrett's adenocarcinomas with poor prognosis.

AIMS: The epidermal growth factor receptor (EGFR) is a tyrosine kinase (TK) involved in the tumour progression of many cancer types and may serve as an important therapeutic target (erlotinib, cetuximab). Heterogeneity of EGFR amplification and expression could represent a major drawback for anti-EGFR therapy. The aim of this study was performed to determine the potential impact of tumour heterogeneity on anti-EGFR therapy in Barrett's adenocarcinoma (BAC). METHODS AND RESULTS: Tissue microarray (TMA) sections of 112 BAC and 45 lymph node metastases were analysed for EGFR amplification and expression using fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). A subset of 20 samples was also sequenced for EGFR exons 18-21 and Kirsten rat sarcoma viral oncogene homologue (KRAS) exons 2-3 mutations. EGFR amplification was seen in seven (6.25%) of 112 interpretable BAC and typically high-level with more than 10-20 EGFR copies per tumour cell (EGFR/centromere 7 ratio >3). EGFR amplification was associated with high pT, pN and poor prognosis (P = 0.0004). Identical EGFR amplification status was found in 29 primary tumours and 29 matched lymph node metastases. Moreover, FISH analysis of three to 16 large sections from all amplified BAC and corresponding lymph node metastases did not reveal any heterogeneity of EGFR amplification. No EGFR mutation but one KRAS mutation was found. CONCLUSION: The high level and homogeneity of EGFR amplification in primary tumours and metastases suggests the potential therapeutic utility of anti-EGFR drugs in BAC.

Juvenile papillomatosis of the breast (Swiss cheese disease) has frequent associations with PIK3CA and/or AKT1 mutations.

Juvenile papillomatosis (JP), the so-called Swiss cheese disease, is a rare benign breast disease of young adults. An association (up to 28%) with breast cancer within the family of affected patients has been reported. A multinodular cystic breast mass lesion and calcifications characterizes JP in imaging studies. The histological picture is diverse and comprises multiple intraductal papillomas, usual ductal hyperplasia, ductectasias, perifocal sclerosing adenosis, and calcification. Patients with complete excision of JP lesions have an excellent follow-up; breast cancer develops only on a very low subset of patients. Molecular background of JP has not been investigated until now. In this study, we addressed mutational analysis of JP cases and correlated these results with follow-up and family history in context with a comprehensive review of the JP literature. We identified 13 cases fulfilling the criteria of JP. All patients were women with a median age of 38 years (26-50 years). Follow-up information was available for 11 of 13 patients. Sufficient paraffin-embedded tissue and good DNA quality for next-generation sequencing (NGS) was available for 10 patients. Paraffin blocks were microdissected in the area of intraductal proliferative disease; the tissue cores underwent NGS analysis using the Oncomine Comprehensive Panel. In 5 of 10 patients, we found PIK3CA mutations; in 2 of 10 patients, we found AKT1 mutations in known hot spot regions. Further mutations in MET, FGFR3, PTEN, ATM, NF1, and GNAS genes were detected in individual patients. Some of these mutations were present at high allele frequencies suggesting germ line mutations. Two of 3 patients with positive family history had PIK3CA mutation; one patient with positive family history had an AKT1 mutation. One patient who subsequently developed invasive ductal carcinoma in the contralateral breast possibly had a germ line ATM mutation. Our results confirm hot spot mutations in PIK3CA and AKT1 genes in JP associated with positive family history for breast cancer, although these mutations are not specific for JP. The genetic link between JP, positive family history, and subsequent risk of breast cancer needs to be analyzed in further studies.

Radiation dosimetry of 18F-AzaFol: A first in-human use of a folate receptor PET tracer.

BACKGROUND: The folate receptor alpha (FRα) is an interesting target for imaging and therapy of different cancers. We present the first in-human radiation dosimetry and radiation safety results acquired within a prospective, multicentric trial (NCT03242993) evaluating the 18F-AzaFol (3'-aza-2'-[18F]fluorofolic acid) as the first clinically assessed PET tracer targeting the FRα. MATERIAL AND METHODS: Six eligible patients presented a histologically confirmed adenocarcinoma of the lung with measurable lesions (≥ 10 mm according to RECIST 1.1). TOF-PET images were acquired at 3, 11, 18, 30, 40, 50, and 60 min after the intravenous injection of 327 MBq (range 299-399 MBq) of 18F-AzaFol to establish dosimetry. Organ absorbed doses (AD), tumor AD, and patient effective doses (E) were assessed using the OLINDA/EXM v.2.0 software and compared with pre-clinical results. RESULTS: No serious related adverse events were observed. The highest AD were in the liver, the kidneys, the urinary bladder, and the spleen (51.9, 45.8, 39.1, and 35.4 µGy/MBq, respectively). Estimated patient and gender-averaged E were 18.0 ± 2.6 and 19.7 ± 1.4 µSv/MBq, respectively. E in-human exceeded the value of 14.0 µSv/MBq extrapolated from pre-clinical data. Average tumor AD was 34.8 µGy/MBq (range 13.6-60.5 µGy/MBq). CONCLUSIONS: 18F-Azafol is a PET agent with favorable dosimetric properties and a reasonable radiation dose burden for patients which merits further evaluation to assess its performance. TRIAL REGISTRATION: ClinicalTrial.gov, NCT03242993, posted on August 8, 2017.

Estrogen receptor gene amplification occurs rarely in ovarian cancer.

Amplification of the gene encoding estrogen receptor-alpha occurs in about 20% of breast cancers and is an important mechanism for estrogen receptor overexpression in this tumor type. In ovarian cancer, overexpression of estrogen receptor protein has been described in more than two thirds of cases. To study a potential role of estrogen receptor-alpha gene amplification for estrogen receptor overexpression in ovarian cancer, a tumor tissue microarray containing 428 ovarian cancers was analyzed by fluorescence in situ hybridization for estrogen receptor-alpha gene amplification and immunohistochemistry for estrogen receptor expression. The estrogen receptor-alpha gene status was successfully determined in 243 of 428 arrayed cancers. Estrogen receptor gene amplification was found in 5 of 243 (2%) of tumors. Amplification levels were usually low, with 4-8 estrogen receptor-alpha gene copies. However, one case had a high-level amplification, with more than 30 estrogen receptor-alpha gene copies. All five amplified tumors were estrogen receptor positive, with 3 of 5 tumors showing highest (Allred score, 7-8) estrogen receptor levels. The data demonstrate that estrogen receptor-alpha amplification occurs only rarely in ovarian cancer.