List of all analyzed CHEK2 variants with results of KAP1/CHK2 kinase and localization assays and the results from recent previously published functional analyses of the CHEK2 VUS.

Detail description of the functional categorization of analyzed CHEK2 missense variants.

Characteristics of 12 case-control datasets from the ENIGMA consortium partners.

AbstractPurpose:Germline pathogenic variants in CHEK2 confer moderately elevated breast cancer risk (odds ratio, OR ∼ 2.5), qualifying carriers for enhanced breast cancer screening. Besides pathogenic variants, dozens of missense CHEK2 variants of uncertain significance (VUS) have been identified, hampering the clinical utility of germline genetic testing (GGT).Experimental Design:We collected 460 CHEK2 missense VUS identified by the ENIGMA consortium in 15 countries. Their functional characterization was performed using CHEK2-complementation assays quantifying KAP1 phosphorylation and CHK2 autophosphorylation in human RPE1–CHEK2-knockout cells. Concordant results in both functional assays were used to categorize CHEK2 VUS from 12 ENIGMA case–control datasets, including 73,048 female patients with breast cancer and 88,658 ethnicity-matched controls.Results:A total of 430/460 VUS were successfully analyzed, of which 340 (79.1%) were concordant in both functional assays and categorized as functionally impaired (N = 102), functionally intermediate (N = 12), or functionally wild-type (WT)–like (N = 226). We then examined their association with breast cancer risk in the case–control analysis. The OR and 95% CI (confidence intervals) for carriers of functionally impaired, intermediate, and WT-like variants were 2.83 (95% CI, 2.35–3.41), 1.57 (95% CI, 1.41–1.75), and 1.19 (95% CI, 1.08–1.31), respectively. The meta-analysis of population-specific datasets showed similar results.Conclusions:We determined the functional consequences for the majority of CHEK2 missense VUS found in patients with breast cancer (3,660/4,436; 82.5%). Carriers of functionally impaired missense variants accounted for 0.5% of patients with breast cancer and were associated with a moderate risk similar to that of truncating CHEK2 variants. In contrast, 2.2% of all patients with breast cancer carried functionally wild-type/intermediate missense variants with no clinically relevant breast cancer risk in heterozygous carriers.

Kinase KAP1 and CHK2 assays (A). The bar graphs show results of kinase assays for 430 CHEK2 missense variants. In both assays, variants with normalized relative CHK2 activity (mean WT-activity = 1) exceeding that of the weakest signal of WT replicas (not shown) were categorized functionally WT-like, variants with normalized signal intensity lower than the strongest signal for any of kinase-dead/empty EGFP vector controls (in-frame exon 7 deletion–p.D265_H282del; not shown) were categorized as functionally impaired. Variants with normalized CHK2 activities between these ranges were categorized functionally intermediate (0.428–0.705 and 0.479–0.710 for KAP1 and CHK2 assay, respectively; indicated by red and yellow dashed lines). Scatterplot combines results from both assays showing 340 concordant (circles) and 90 discordant (crosses) variants. The nuclear-to-cytoplasmic ratio (B) bar graph (left) displays all missense variants and a set of protein-truncating CHEK2 variants (dark red bars at left, zoomed part of the graph). The missense variants, p.R521W and p.R521Q, with an aberrant localization are highlighted as bright-red bars; the arrows denote WT (green bar) and catalytically-dead in-frame p.D265_H282del variant (white bar). The highest and lowest mean nuclear/cytoplasmic ratio values from all WT replicates are indicated by green dashed lines. Of all missense variants analyzed by ScanR microscopy, only codon 521 alterations revealed aberrant intracellular localization with intense cytoplasmic positivity (right), reminiscent of mislocalization of the c.1100delC (p.T367fsX; size bar, 10 μm) variant. In comparison, the in-frame deletion p.D265_H282del revealed normal intranuclear accumulation, similar to WT. C, Scatter plots depicting correlations between assays performed in this study and previous analyses of CHEK2 VUS. Studies of Kleiblova et al. (17) and Boonen et al. (18) used phosphorylation of KAP1 as a functional readout whereas the study of Delimitsou et al. (25) used a yeast growth retardation assay. The dots are colored according to the results of the KAP1 assay in this study (red, impaired; yellow, intermediate; green, wild-type–like). Blue line represents linear regression, R, correlation coefficient; P, P value. The scatter plot does not show the p.Arg512Trp variant classified by Boonen et al. as intermediate with impaired nuclear localization in our localization assay.

Validation of KAP1-pS473 and CHK2-pS516 antibodies. A, Parental RPE, RPE1–CHEK2-KO cells or RPE1–CHEK2-KO cells transfected with the wild-type or mutant pEGFP–CHEK2 were left untreated or were exposed to ionizing radiation (5 Gy, 3 hours). After fixation, cells were probed with KAP1-pS473 antibody. Representative images are shown. B, Quantification of A. The mean nuclear intensity of the KAP1-pS473 signal is plotted. Each dot represents one cell; more than 300 cells were analyzed. Red line, error bars and numbers indicate mean ± SDs. Statistical significance was evaluated by the Mann–Whitney test (****, P < 0.0001). A representative experiment is shown from two independent replicates. C, Cells were grown and treated as in A and were probed with CHK2-pS516 antibody. Representative images are shown. D, Quantification of C. The mean nuclear intensity of the CHK2-pS516 signal is plotted. Each dot represents one cell; more than 300 cells were analyzed. Red line, error bars and numbers indicate mean ± SDs. Statistical significance was evaluated by the Mann–Whitney test (****, P < 0.0001). A representative experiment is shown from two independent replicates. E, Cells were grown and treated as in A. Whole-cell lysates were analyzed by immunoblotting with indicated antibodies.

Funnel plot (left) and forest plots (right) for individual datasets of breast cancer cases and controls from 12 datasets (10 countries) stratified according to the functional categorization.

Results of KAP1 and CHK2 kinase assays for 430 successfully analyzed missense CHEK2 variants (shown as an average relative CHK2 kinase activity). Bars are colored as functionally WT-like (green), intermediate (IM; yellow), and impaired (ID; red), respectively, with thresholds for IM variants (0.428 and 0.479) and ID variants (0.705 and 0.710) for KAP1 and CHK2 assays, respectively (dashed lines). Error bars represent standard errors of mean. Color/gray letters for protein variants indicate concordant/discordant functional assays result, respectively. Blue boxes denote conserved CHK2 domains. DNL, variants that do not localize into the nucleus.

Purpose To describe a snapshot of international genetic testing practices, specifically regarding the use of multigene panels, for hereditary breast/ovarian cancers. We conducted a survey through the Evidence-Based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium, covering questions about 16 non- BRCA1/ 2 genes. Methods Data were collected via in-person and paper/electronic surveys. ENIGMA members from around the world were invited to participate. Additional information was collected via country networks in the United Kingdom and in Italy. Results Responses from 61 cancer genetics practices across 20 countries showed that 16 genes were tested by > 50% of the centers, but only six ( PALB2, TP53, PTEN, CHEK2, ATM, and BRIP1) were tested regularly. US centers tested the genes most often, whereas United Kingdom and Italian centers with no direct ENIGMA affiliation at the time of the survey were the least likely to regularly test them. Most centers tested the 16 genes through multigene panels; some centers tested TP53, PTEN, and other cancer syndrome–associated genes individually. Most centers reported (likely) pathogenic variants to patients and would test family members for such variants. Gene-specific guidelines for breast and ovarian cancer risk management were limited and differed among countries, especially with regard to starting age and type of imaging and risk-reducing surgery recommendations. Conclusion Currently, a small number of genes beyond BRCA1/ 2 are routinely analyzed worldwide, and management guidelines are limited and largely based on expert opinion. To attain clinical implementation of multigene panel testing through evidence-based management practices, it is paramount that clinicians (and patients) participate in international initiatives that share panel testing data, interpret sequence variants, and collect prospective data to underpin risk estimates and evaluate the outcome of risk intervention strategies.

BACKGROUND:Breast cancer (BC) in males is a rare disease and comprises 0.5-1% of all BC cases. Due to its rarity, there are limited data regarding risk factors, biology and relevant treatment.AIM:A prospective observational study of demographic, clinical and histological characteristics of serially-admitted men with breast cancer was carried out from 1999 to 2009.PATIENTS AND METHODS:Data were recorded and analyzed from a database including 1,315 cases of BC. Registered data concerned age, initial presentation, family and lifestyle history (risk factors), histological features, phenotypic subtypes and TNM staging.RESULTS:Twenty two men with BC were identified, with a median age of 63 years. The most common initial presentation was a palpable lump in 12 patients, nipple contraction in three and ulceration in three. According to their medical history, nine men were overweight, 10 suffered from hypertension and 12 were smokers. The most prevalent phenotype was luminal-A followed by triple-negative type. BC in none of the cases was HER 2-amplified. The majority of cases were grade II or III and stage II or III.CONCLUSION:In the present small study, we confirm that BC in males is rare. It is a disease of middle-age and presents at advanced stages. Most of patients had 1-3 risk factors for BC. Expression of hormonal receptors occurs in the majority of BC tumors in males and with rarity in HER 2 amplification.

Collecting duct carcinoma is a rare renal malignant neoplasm, arising from the medullary collecting duct and accounting for less than 1 % of renal cell carcinomas. It is more common in middle-aged men and is usually presented with hematuria, abdominal mass and back or flank pain. Its immunohistochemical analysis detects the expression of various markers, such as low and high molecular weight keratins, Ulex europaeus agglutinin-1, epithelial membrane antigen and peanut lectin. Here, we present a case of a 29-year-old woman with CDC presented with back pain and supraclavicular lymphadenopathy that produced carcinoembryonic antigen and CA-125.

The present study describes a rare case of a mesenteric liposarcoma that resulted in a complete remission (CR) following treatment with trabectedin (Yondelis®). The patient presented with abdominal pain and fever. An abdominal mass was identified that corresponded to a mixed-type high-grade mesenteric liposarcoma with wide areas of necrosis, areas of dedifferentiation and features of a leiomyosarcoma. Three months after the removal of the first mass, the patient underwent a second laparotomy, followed by treatment with doxorubicin and ifosfamide. Subsequently, the patient was started on therapy with trabectedin and a CR was noted following only four cycles of therapy. The best responses that are reported in the literature for cases of liposarcoma treated with trabectedin are mostly for liposarcomas of the myxoid/round cell type and are mainly partial responses. In the present study, trabectedin was used for the treatment of a mesenteric liposarcoma of mixed morphological features and a CR was achieved.

Dermatomyositis represents an idiopathic inflammatory connective-tissue disease, characterized by inflammation of the muscles and the skin. There is a high incidence of malignancy in patients with dermatomyositis. The main purpose of the present paper is to describe and underline the clinical significance of dermatomyositis manifestations as a precursor and early clinical signs of small cell lung cancer. A physical examination, laboratory tests, anti-Jo-1 antibody and muscle biopsy were performed. The most important findings were SGOT 284 IU/L, CPK 11083 IU/L, aldolase 76.3 IU/L (normal values <7.6). The patient was treated with chemotherapy and a significant improvement of clinical and laboratory findings were noted. The diagnosis of lung cancer could be correlated with the clinical existence of dermatomyositis. Increased awareness is needed regarding the association of dermatomyositis with malignancies in order to achieve correct and timely diagnosis of the underling cancer.

PURPOSEWe present the case of a patient treated for intracranial germ cell tumor in which elements of craniopharyngioma were found in the residual tumor mass.FINDINGSA 17 year old patient presented with a history of secondary amenorrhea. She deteriorated with headache and left eyelid drop, paresis of the abducent nerve and convergent strabismus (Parinaud syndrome). β-HCG was 722mIU/ml and pregnancy was excluded. AFP was 6322 ng/ml. Brain CT scan showed a large endosellar tumor to the hypersellar region. There was left papillary atrophy. MRI confirmed a tumor to dorsum sellae. Primary germ cell intracranial tumor was diagnosed. Severe clinically evident pituitary failure developed with signs of increased intracranial pressure and brain edema as well as diabetes insipidus, while AFP increased to 15786,3ng/ml. Urgent treatment with combination chemotherapy including cisplatin etoposide and bleomycin (ΡEB) was administered for 4 courses. As a result her clinical condition improved and tumor markers dropped but nevertheless did not become normal. In addition CT scans revealed a remaining endocranial mass and therefore the patient was subjected to high-dose chemotherapy followed by autologous stemcell rescue which resulted in complete clinical and biochemical remission. Due to the persisting mass in the area, it was delivered radiotherapy.CONCLUSIONSThe above case is extremely rare in worldwide literature. Dysgerminoma may coexist with craniopharyngioma which in fact may be part of a germ cell tumor in the context of dysembryogenesis and benign "teratoma".

Neoplastic pericarditis represents approximately 5%-7% of the cases with acute pericarditis and is rarely the initial manifestation of malignancy. The most common cause is lung cancer, followed by breast cancer, lymphomas, leukemia, and esophageal cancer. Neoplastic pericardial disease is extremely rare in thyroid cancer, especially as the first manifestation. Here, we present a papillary thyroid carcinoma that was manifested with pericarditis and cardiac tamponade in a 49-year-old female.

Breast cancer (BC), the most common type of cancer in women of the Western world, is often associated with paraneoplastic syndromes (PNS). Autoimmune pancreatitis is a recently recognized entity belonging to the spectrum of IgG4-related systemic diseases, which are characterized by target-organ plasmacytic infiltration and fibrosis. In this report we review PNS associated with BC and we present the first case of BC-associated autoimmune pancreatitis as well as its successful management with steroids and immunosuppressive BC-tailored chemotherapy.

Background: The prognosis of patients with metastatic breast cancer who have failed to respond to at least two different chemotherapy regimens is poor. Such patients with metastatic disease to the liver have even worse prognosis. Cisplatin and 5-fluorouracil (5-FU) can be given in patients with impaired hepatic function but their combination has not been extensively studied in this setting. Patients and Methods: We retrospectively collected data from our registry on patients with advanced metastatic breast cancer who received combination of cisplatin/5-FU. We sought to determine the toxicity, the response rate, the disease control rate and the survival of this combination. Results: We identified 25 heavily pre-treated patients, out of which 19 (76%) had liver metastases. They had been treated before with a median of three lines of cytotoxic chemotherapy. The majority of patients had also received hormonal manipulation or trastuzumab. The median number of cisplatin/5-FU administered cycles, without toxic deaths or unexpected toxicities was four. The partial response (PR) rate was 32% and the disease control rate (DCR) was 68%. The time to progression was five months and the median survival after starting on cisplatin/5-FU was six months. Conclusion: The combination of cisplatin/5-FU is active and safe in heavily pre-treated patients with metastatic breast cancer even in the presence of liver metastases and jaundice.