Recommendations for somatic and germline genetic testing of single pheochromocytoma and paraganglioma based on findings from a series of 329 patients.

BACKGROUND: Nowadays, 65-80% of pheochromocytoma and paraganglioma (PPGL) cases are explained by germline or somatic mutations in one of 22 genes. Several genetic testing algorithms have been proposed, but they usually exclude sporadic-PPGLs (S-PPGLs) and none include somatic testing. We aimed to genetically characterise S-PPGL cases and propose an evidence-based algorithm for genetic testing, prioritising DNA source. METHODS: The study included 329 probands fitting three criteria: single PPGL, no syndromic and no PPGL family history. Germline DNA was tested for point mutations in RET and for both point mutation and gross deletions in VHL, the SDH genes, TMEM127, MAX and FH. 99 tumours from patients negative for germline screening were available and tested for RET, VHL, HRAS, EPAS1, MAX and SDHB. RESULTS: Germline mutations were found in 46 (14.0%) patients, being more prevalent in paragangliomas (PGLs) (28.7%) than in pheochromocytomas (PCCs) (4.5%) (p=6.62×10(-10)). Somatic mutations were found in 43% of those tested, being more prevalent in PCCs (48.5%) than in PGLs (32.3%) (p=0.13). A quarter of S-PPGLs had a somatic mutation, regardless of age at presentation. Head and neck PGLs (HN-PGLs) and thoracic-PGLs (T-PGLs) more commonly had germline mutations (p=2.0×10(-4) and p=0.027, respectively). Five of the 29 metastatic cases harboured a somatic mutation, one in HRAS. CONCLUSIONS: We recommend prioritising testing for germline mutations in patients with HN-PGLs and T-PGLs, and for somatic mutations in those with PCC. Biochemical secretion and SDHB-immunohistochemistry should guide genetic screening in abdominal-PGLs. Paediatric and metastatic cases should not be excluded from somatic screening.

CXCR4: From Signaling to Clinical Applications in Neuroendocrine Neoplasms.

There are several well-described molecular mechanisms that influence cell growth and are related to the development of cancer. Chemokines constitute a fundamental element that is not only involved in local growth but also affects angiogenesis, tumor spread, and metastatic disease. Among them, the C-X-C motif chemokine ligand 12 (CXCL12) and its specific receptor the chemokine C-X-C motif receptor 4 (CXCR4) have been widely studied. The overexpression in cell membranes of CXCR4 has been shown to be associated with the development of different kinds of histological malignancies, such as adenocarcinomas, epidermoid carcinomas, mesenchymal tumors, or neuroendocrine neoplasms (NENs). The molecular synapsis between CXCL12 and CXCR4 leads to the interaction of G proteins and the activation of different intracellular signaling pathways in both gastroenteropancreatic (GEP) and bronchopulmonary (BP) NENs, conferring greater capacity for locoregional aggressiveness, the epithelial-mesenchymal transition (EMT), and the appearance of metastases. Therefore, it has been hypothesized as to how to design tools that target this receptor. The aim of this review is to focus on current knowledge of the relationship between CXCR4 and NENs, with a special emphasis on diagnostic and therapeutic molecular targets.

MicroRNA signatures in hereditary breast cancer.

This study aims to identify signatures of miR associated with hereditary, BRCA1 or BRCA2 mutation positive breast cancer (BC), and non-hereditary BC, either sporadic (SBC) or non-informative (BRCAX). Moreover, we search for signatures associated with tumor stage, immunohistochemistry and tumor molecular profile. Twenty formalin fixed paraffin embedded (FFPE) BCs, BRCA1, BRCA2, BRCAX and SBC, five per group were studied. Affymetrix platform miRNA v.3.0 was used to perform miR expression analysis. ER, PR, HER2 and Ki67 protein expression was analyzed by immunohistochemistry. BRCA1, BRCA2 and RASSF1 methylation analysis, AURKA copy number variations, and BRCA1 and BRCA2 deletions, were studied by MLPA. We validated eight of the miR selected by the arrays in 77 BCs by qRT-PCR. The miR profiles associated with tumor features were studied applying the Sparse Partial Least Squares Discriminant Analysis. MiR discrimination capability to distinguish hereditary and non-hereditary BC was analyzed by the discriminant function. With 15 out of 1,733 hsa-miRs, it was possible to differentiate the four groups. BRCA1, BRCA2 and SBC were associated with clusters of hyper-expressed miRs, and BRCAX with hypo-expressed miRs. Hsa-miR-4417 and hsa-miR-423-3p expressions (included among the eight validated miRs) differentiated 70.1 % of hereditary and non-hereditary BCs. We found miR profiles associated with tumor features like node involvement, histological grade, ER, PR and HER2 expression. Regarding molecular parameters, we only found a weak association of miRs in BC harboring losses in AURKA. We conclude that array miR expression profiles can differentiate the four study groups using FFPE BC. However, miRs expression estimated by qRT-PCR differentiates only hereditary and non-inherited BCs. The miR expression array is a simple and rapid approach that could be useful to facilitate the identification of those SBC carrying genetic or epigenetic changes in BRCA genes responsible of BRCA-like phenotype. These patients could benefit from the treatment with PARP inhibitors.

Cancer worry at higher-risk sample of hereditary cancer in Spain.

BACKGROUND: Few studies have examined the prevalence of cancer worry in the general and at-risk population. The objective of this study was to describe the prevalence of cancer worry in a sample of individuals at increased risk of developing hereditary cancer, determine differences in cancer worry by socio-demographic characteristics and assess the relationship between cancer worry and psychological distress. METHODS: A cross-sectional study was designed with 895 patients. The Cancer Worry Scale (CWS), Hospital Anxiety and Depression Scale (HADS) for psychological distress and sociodemographic characteristics were examined. The multiple linear regression model was developed to explore what variables were predicted for cancer worry. To identify variables associated with higher cancer worry scores, a logistic model was fitted. RESULTS: In the at-higher-risk sample of hereditary cancer, the mean of CWS was 10.20 (SD: 3.70). The significant predictors for cancer worry were gender, age, previous psychiatric treatment, patients affected by cancer and having children. In the sample, 38% of patients had higher scores on cancer worry, the variables associated were patients affected by cancer compared, women, widow/divorced participants, less than secondary school, patients with previous psychiatric treatment and patients less than 55 years old. Using the HADS cutoff score 29% of the sample showed significant psychological distress, more anxiety (35%) than depressive (22%) symptomatology. Psychological distress showed a higher variability (36%) on cancer worry. CONCLUSION: Findings highlighted distinctive profiles in socio-demographic characteristics according to the degree of cancer worry; therefore, genetic counseling should continue to be provided to address cancer worry and relieve psychological distress.

Psychiatric symptoms in a Spanish sample with hereditary cancer risk.

OBJECTIVE: An integral part of the genetic counselling process is the assessment of psychiatric morbidity. The objectives of this study were first to assess psychometric properties of the General Health Questionnaire (GHQ-28items) in a Spanish sample at increased risk of hereditary cancer, and second evaluated the prevalence of psychiatric morbidity and the contribution of socio-demographic and clinical characteristics to predict distress. METHODS: A cross-sectional study was designed with 766 patients. Psychometric analysis with exploratory factor analysis was performed. The influences of socio-demographic characteristics were investigated by multiple linear regression analyses. RESULTS: Factor analysis supported the four-factor solution of the original GHQ-28; Depression and Social dysfunction scales were more stable than Anxiety and Somatic symptom scales. Psychiatric morbidity was detected in 212 (27.9%) patients. The variables predicting psychiatric morbidity were gender, age, patient affected by cancer, previous psychiatric treatment, and patients with relatives affected by cancer. The higher prevalence of psychiatric symptoms was in the age group from 41 to 59 years (16.73%), women (24.37%), patients affected by cancer (19.89%), patients without previous psychiatric treatment (20.82%), and patients with relatives affected by cancer (21.74%). CONCLUSION: Screening psychological distress should consider socio-demographic and clinical characteristics with reference to improve the quality of care. TRIAL REGISTRATION: Clinical trials identifier: NCT04428710.

Comparison of psychological distress for breast, ovarian and colorectal cancer predisposition in a Spanish sample at high risk of hereditary cancer.

OBJECTIVES: Although future treatments may speciically target the tumour phenotype, other factors should be included to confirm the efficacy of treatment and prevention strategies. The objective of this study was to compare sociodemographic characteristics and psychological distress for breast, ovarian and colorectal cancer predisposition syndrome in a sample at high risk of hereditary cancer. METHODS: A cross-sectional study was designed with 799 patients. The nonparametric test, with Kruskal-Wallis test, was used to compare three genetic cancer syndromes, with significant differences in sample size. RESULTS: There were no differences in cancer hereditary syndromes related to sociodemographic characteristics except sex, as breast/ovarian cancer mainly affects women. No group differences were observed for cancer worry (P = 0.17). Breast/ovarian cancer syndrome showed significantly higher scores in cognitive distress compared to colorectal cancer (P = 0.01). CONCLUSION: The differences in the distribution of sociodemographic characteristics in these hereditary cancer syndromes can help to better plan resources for patient care in genetic counselling units.

CASP8 D302H polymorphism delays the age of onset of breast cancer in BRCA1 and BRCA2 carriers.

The polymorphic genetic differences among individuals may modify the high risk for breast cancer (BC) and/or ovarian cancer (OC) susceptibility conferred by BRCA1 and BRCA2 mutations. In the present study we investigate the relevance of RAD51 -135C > G, TP53 R72P, NQO1*2 and CASP8 D302H polymorphisms as potential modifiers of BC and/or OC susceptibility conferred by these mutations. The study group encompasses 390 BRCA1/BRCA2 mutation carriers (182 affected with BC and/or OC and 208 unaffected) of 131 unrelated families studied in the Program of Genetic Counselling on Cancer of Valencia Community. The polymorphisms were detected in genomic DNA by ASRA method or real time PCR using fluorescently labeled probes. We found similar incidence of RAD51 -135C > G, TP53 R72P and NQO1*2 polymorphisms among affected and unaffected individuals considering BRCA1/BRCA2 mutations together and separately. However, the CASP8 D302H polymorphism was strongly associated with the absence of BC [OR = 3.41 (95% CI 1.33-8.78, P = 0.01)]. In fact, in the females with CASP8 D302H polymorphism the BC appeared at a median age of 58 in opposition to the 47 years observed for the wild type subjects (P = 0.03). Furthermore, the CASP8 D302H positive females showed a 50% probability of being free of BC by the age of 78 versus the 2% of the CASP8 negative ones. Our results support that the presence of the CASP8 D302H polymorphism diminishes the high risk of BC conferred by BRCA1 and BRCA2 mutations, making possible that some of the carriers could escape from suffering BC along their life span.

[BRCA1 and BRCA2 mutations in families studied in the program of genetic counselling in cancer of the Valencian community (Spain)]. / Mutaciones de BRCA1 y BRCA2 en familias estudiadas en el Programa de Consejo Genético en el Cáncer de la Comunidad Valenciana.

BACKGROUND AND OBJECTIVE: The objective of the present study was to investigate the mutational spectrum of BRCA1 and BRCA2 in the Valencian Community, comparing this spectrum with that reported in Spain. We also analyze the association of the mutations with the family history of the selected families. PATIENTS AND METHOD: We analyzed the mutations in the BRCA1 and BRCA2 in 147 families with history of breast and/or ovarian cancer. The detection was based on the amplification of in frame and flanking regions of BRCA1 and BRCA2 genes by polymerase chain reaction, detection of the heteroduplex formed by conformation-sensitive gel electrophoresis and their characterization by sequencing. RESULTS: We identified 24 different pathogenic mutations in 50 out of the 147 families (34.0%; 23 in BRCA1 and 27 in BRCA2). The higher incidence of pathogenic mutations was observed in families with breast and ovarian cancer or with more than 3 cases of breast cancer. The most frequent mutations in BRCA1 were the c.187_188delAG, c.2080delA and the c.3889_3890delAG, whereas for BRCA2 the mutations with higher prevalence was observed for c.9254_9258delATCAT and the c.9204delCATCAGATTTATAT. We detected 5 pathogenic mutations (p.Y1429X in BRCA1 and c.1835insT, c.5025delT, c.6722delT and p.Q3156X in BRCA2) not reported in the Breast Cancer Information Core Database. Among them, the BRCA2 mutations c.1835insT and c.5025delT were recurrent and seemed to be characteristic of the population the Valencian Community. CONCLUSIONS: We detected pathogenic mutations in BRCA1 and BRCA2 genes in 34.0% of the families studied. The mutations c.1835insT and c.5025delT were 2 new recurrent pathogenic mutations in BRCA2 that seemed to be characteristic of the population of the Valencian Community. The study reports 5 new pathogenic mutations to the world spectrum of BRCA1 and BRCA2 mutations and other 5 mutations to the Spanish spectrum.

Relationship of immunohistochemistry, copy number aberrations and epigenetic disorders with BRCAness pattern in hereditary and sporadic breast cancer.

The study aims to identify the relevance of immunohistochemistry (IHC), copy number aberrations (CNA) and epigenetic disorders in BRCAness breast cancers (BCs). We studied 95 paraffin included BCs, of which 41 carried BRCA1/BRCA2 germline mutations and 54 were non hereditary (BRCAX/Sporadic). Samples were assessed for BRCA1ness and CNAs by Multiplex Ligation-dependent Probe Amplification (MLPA); promoter methylation (PM) was assessed by methylation-specific-MLPA and the expression of miR-4417, miR-423-3p, miR-590-5p and miR-187-3p by quantitative RT-PCR. IHC markers Ki67, ER, PR, HER2, CK5/6, EGFR and CK18 were detected with specific primary antibodies (DAKO, Denmark). BRCAness association with covariates was performed using multivariate binary logistic regression (stepwise backwards Wald option). BRCA1/2 mutational status (p = 0.027), large tumor size (p = 0.041) and advanced histological grade (p = 0.017) among clinic-pathological variables; ER (p < 0.001) among IHC markers; MYC (p < 0.001) among CNA; APC (p = 0.065), ATM (p = 0.014) and RASSF1 (p = 0.044) among PM; and miR-590-5p (p = 0.001), miR-4417 (p = 0.019) and miR-423 (p = 0.013) among microRNA expression, were the selected parameters significantly related with the BRCAness status. The logistic regression performed with all these parameters selected ER+ as linked with the lack of BRCAness (p = 0.001) and MYC CNA, APC PM and miR-590-5p expression with BRCAness (p = 0.014, 0.045 and 0.007, respectively). In conclusion, the parameters ER expression, APC PM, MYC CNA and miR-590-5p expression, allowed detection of most BRCAness BCs. The identification of BRCAness can help establish a personalized medicine addressed to predict the response to specific treatments.

Carboplatin and tegafur-uracil concomitant with standard radiotherapy in the management of locally advanced head and neck cancer.

INTRODUCTION: We undertook a prospective study to determine the feasibility, toxicity, response and survival rate of simultaneous chemotherapy (CT) and radiotherapy (RT) for locally-advanced head and neck cancer. MATERIAL AND METHODS: Fifty eight patients were treated with carboplatin (i.e. 100 mg/m(2)) weekly, tegafur-uracil (UFT) (oral 400 mg/m(2)) daily and simultaneous treatment with a cobalt-60 source of radiation (total dose 65-70 Gy). RESULTS: Forty six patients (79%) received the total dose of RT while CT was delayed or reduced in 31 patients (53%). Grade 3-4 toxicity observed was mucositis in 27 (47%), leukopenia in 10 (17%), anaemia in 5 (9%), and diarrhoea in 4 (7%) patients. The objective response rate was 74%; 24 complete response (41%) and 19 partial response (33%). Overall, there are 11 patients (19%) disease-free, 7 (12%) alive with disease, 35 have died of progressive disease (60%) and 3 (5%) from other causes. There were 2 toxic deaths (3%). Median time to progression was 10 months and median survival was 18.4 months. CONCLUSIONS: The use of carboplatin and UFT concomitant with radiotherapy has, in our study, a slightly lower activity than other chemo-radiotherapy protocols, especially with respect to complete responses, but with no significant differences in overall survival or disease-free survival rates.

[Extra-pulmonary small-cell cancer: is its outcome similar to small cell lung cancer?]. / Carcinoma microcítico de localización extrapulmonar: es su curso similar a los microcíticos pulmonares?

INTRODUCTION: Extra-pulmonary small cell carcinoma (ESCC) is as a pathologic entity distinct from small cell lung carcinoma (SCLC). ESCC is considered a systemic disease in its origin, so the therapeutic approach is similar to SCLC with chemotherapy being considered in case of extensive and local disease. We present a retrospective comparison of ESCC and SCLC in our institution. MATERIAL AND METHODS: Using the tumour registry database of Hospital Universitario La Fe we reviewed 24 ESCC cases receiving attention between 1987 and 2003, and these were compared with a series of 341 patients with SCLC in the same institution. RESULTS: Of the 24 patients with ESCC 19 were men and 5 were women with an average age of 58 years (range 23 to 85). The most frequent site was the mediastinum with 58% having extensive disease. All patients but one received treatment. The therapeutic approaches were local and systemic in 13 patients, systemic alone in 6 and local alone in 4. Schedules based on platinum and etoposide were used. The median follow-up was 53 months (range 4 to 211). Median survival was 18.9 months; 30 in patients with local disease and 8 in those with extensive disease. In the SCLC series of patients, there were 336 men and 5 women; 62% having extensive disease. The median survival was 10 months; 12 months in those patients with local disease and 8 in those with extensive disease. CONCLUSIONS: The overall survival of patients with ESCC was slightly better than patients with SCLC. ESCC with local disease had a better survival outcome than SCLC with local disease. Chemotherapy is the cornerstone of the treatment, but sometimes local treatment could be sufficient.

[Extra-cranial anaplastic oligoastrocytoma development from a low-grade glioma]. / Afectación extracraneal de oligoastrocitoma anaplásico desarrollado sobre un glioma de bajo grado.

Oligodendrogliomas are primary brain tumours derived from oligodendroglial cells, or precursors, and represent 2%-5% of brain tumours. This type of glioma has a favourable prognosis compared to other brain tumours. The treatment is multidisciplinary and is based on three therapeutic arms: surgery, radiotherapy and chemotherapy. We present a patient who had received treatment previously for a lowgrade glioma and who subsequently developed an anaplastic oligoastrocytoma in the same zone together with skull and extra-cranial involvement in the disease progression.

Novel and recurrent BRCA1/BRCA2 mutations in early onset and familial breast and ovarian cancer detected in the Program of Genetic Counseling in Cancer of Valencian Community (eastern Spain). Relationship of family phenotypes with mutation prevalence.

During the first 6 years of the Program of Genetic Counselling in Cancer of Valencia (eastern Spain), 310 mutations (155 in BRCA1 and 155 in BRCA2) in 1,763 hereditary breast (BC) and ovarian cancer (OC) families were identified. Of the mutations found 105 were distinct (53 in BRCA1 and 52 in BRCA2), eight new and 37 recurrent. Two of the novel mutations were frame-shift placed in exons 2 and 11 of BRCA1 and the remaining six were placed in BRCA2; four frame-shift (three in exon 11 and one in exon 23), one deletion of the entire exon 19 and one in the intervening sequence of exon 22. The BRCA1 mutations with higher recurrence were c.66_68delAG, c.5123C > A, c.1961delA, c.3770_3771delAG and c.5152+5G > A that covered 45.2% of mutations of this gene. The age of onset of BCs of c.68_69delAG mutation carriers occurs later than for the other recurrent mutations of this gene (45 vs. 37 years; p = 0.008). The BRCA2 mutations with higher recurrence were c.9026_9030delATCAT, c.3264insT and c.8978_8991del14 which represented 43.2% of all mutations in this gene, being the most recurrent mutation by far c.9026_9030delATCAT that represents 21.3% of BRCA2 mutations and 10.6% of all mutations. Probands with family histories of BC and OC, or OC and/or BC in at least two first degree relatives, were the more likely to have BRCA1/BRCA2 mutations (35.2% of the total mutations). And that most BRCA1mutations (73.19% mutations) occurred in probands with early-onset BC or with family history of OC.

Low penetrance alleles as risk modifiers in familial and sporadic breast cancer.

The aim of the study is to investigate the relevance of rs1056663 and rs2708861 HUS1 polymorphisms, and rs104548, rs2981582 and rs2910164 polymorphisms of CASP8, FGFR2 and micro RNA 146A genes, respectively, as risk modifiers in hereditary breast or ovarian cancer (BC/OC) and risk factors in sporadic BC. We performed a case-control study in 189 healthy controls (CG) and 538 BC/OC cases, 340 with familial history of BC/OC (130 carriers of BRCA1/2 mutations and 210 non-carriers) and 198 sporadic BC/OC. The polymorphisms were assessed by real-time PCR using primers and fluorescent-labelled hybridization probes. We found statistically significant differences between familial BC/OC and CG for rs1056663 and rs2708861 HSU1 polymorphisms and rs2981582 FGFR2 polymorphism, particularly in non-carriers of BRCA1/2 mutations. In this group we found statistical differences for rs1056663 HSU1 and rs2981582 FGFR2 polymorphisms (p-trend < 0.006). The logistic regression confirmed that rs2981582 FGFR2 polymorphism (OR = 2.09; 95 % CI 1.35, 3.20) and the interaction between rs1056663 and rs2708861 HUS1 polymorphisms increased the risk of cancer (OR = 1.87; 95 % CI 1.19, 2.92). Furthermore, we found that the presence of rs1056663 and rs2708861 HUS1 polymorphisms is associated with early age of presentation of BC (p = 0.015) in the group of non-carriers of BRCA1/2 mutations. In addition, no association of the polymorphisms studied in sporadic BC was observed. In conclusion, the HUS1 and FGFR2 polymorphisms act as risk BC modifiers in familial BC/OC, particularly in the group of non-carriers of BRCA1/2 mutations.

Relationship of BRCA1 and BRCA2 mutations with cancer burden in the family and tumor incidence.

The aim of the present study is to analyze the relationship of the incidence of mutations in the two major genes BRCA1 and BRCA2 conferring risk of breast cancer (BC) and ovarian cancer (OC) with the cancer burden in families and with the presence and age of onset of BC/OC. We included 704 index patients (IP) and 668 family members of the IP who tested positive for BRCA1/BRCA2 who were studied in the Program of Genetic Counselling in Cancer of the Valencia Community (Spain). We found 129 IPs with deleterious mutations (18.3%), 59 in BRCA1 and 70 in BRCA2, detecting 396 mutations in this kindred. The incidence of mutations and their distribution between BRCA1 and BRCA2 showed a significantly uneven incidence among the family groups (P < 0.001). We found 179 tumors in the 396 mutation carriers (45%) and detected only 11 cancers among the 272 non-mutation carriers (P < 0.001). No differences in the tumor prevalence or the age of onset of cancer between the genes among the mutation carriers were found. The mutation carriers showed a 50% probability of having BC/OC at a median age of 49 years (95% CI 46-52 years) and 78% at the age of 70 years (95% CI: 71-85%). In conclusion the family burden of BC and OC is strongly associated with the incidence of BRCAs mutations and could foretell which of the two BRCAs genes is more likely to have mutations. Mutation carriers have a 50% risk of having BC/OC by the age of 50 years.

Identification of a novel BRCA1 large genomic rearrangement in a Spanish breast/ovarian cancer family.

BACKGROUND: Alterations in BRCA1 gene are responsible for the majority of hereditary breast and/or ovarian cancers. However, the frequency of detected germline mutations is lower than expected by linkage analysis. Standard PCR-based screening methods are mainly used for detecting mutations, but the large genomic rearrangements are commonly overlooked. The purpose of this study was to confirm and characterize a novel deletion identified in BRCA1 gene which has not yet been reported to date. METHODS: Multiplex ligation-dependent probe amplification was used to analyze BRCA1 rearrangements in 255 unrelated index patients with familial breast and/or ovarian cancer negative for BRCA1/BRCA2 mutations studied in Program of Genetic Counselling on Cancer of Valencia Community (Spain). The breakpoints of detected novel rearrangement were characterized by sequencing. RESULTS AND DISCUSSION: Five different rearrangements in the BRCA1 gene were identified in five unrelated index patients out of the 225 (2%). We found four large genomic rearrangements already described consisting in a 1A/1B and 2 deletion; deletion of exons 5-7; deletion of exons 8-13; exon 20 deletion. Additionally, we found the novel g.8097_22733del14637 deletion that encompasses exons 3-5. This deletion affects the RING domain of the BRCA1 protein and it is suggestive of having a negative impact on its function. CONCLUSION: The new mutation here reported broadens the mutational spectrum of large rearrangements. Furthermore, the five large rearrangements found in patients non-carriers of BRCA1/BRCA2 mutations reinforce the need of studying BRCA1 large genomic rearrangements in genetic counselling programs.