Extended genetic analysis and tumor characteristics in over 4600 women with suspected hereditary breast and ovarian cancer.

BACKGROUND: Genetic screening for pathogenic variants (PVs) in cancer predisposition genes can affect treatment strategies, risk prediction and preventive measures for patients and families. For decades, hereditary breast and ovarian cancer (HBOC) has been attributed to PVs in the genes BRCA1 and BRCA2, and more recently other rare alleles have been firmly established as associated with a high or moderate increased risk of developing breast and/or ovarian cancer. Here, we assess the genetic variation and tumor characteristics in a large cohort of women with suspected HBOC in a clinical oncogenetic setting. METHODS: Women with suspected HBOC referred from all oncogenetic clinics in Sweden over a six-year inclusion period were screened for PVs in 13 clinically relevant genes. The genetic outcome was compared with tumor characteristics and other clinical data collected from national cancer registries and hospital records. RESULTS: In 4622 women with breast and/or ovarian cancer the overall diagnostic yield (the proportion of women carrying at least one PV) was 16.6%. BRCA1/2 PVs were found in 8.9% of women (BRCA1 5.95% and BRCA2 2.94%) and PVs in the other breast and ovarian cancer predisposition genes in 8.2%: ATM (1.58%), BARD1 (0.45%), BRIP1 (0.43%), CDH1 (0.11%), CHEK2 (3.46%), PALB2 (0.84%), PTEN (0.02%), RAD51C (0.54%), RAD51D (0.15%), STK11 (0) and TP53 (0.56%). Thus, inclusion of the 11 genes in addition to BRCA1/2 increased diagnostic yield by 7.7%. The yield was, as expected, significantly higher in certain subgroups such as younger patients, medullary breast cancer, higher Nottingham Histologic Grade, ER-negative breast cancer, triple-negative breast cancer and high grade serous ovarian cancer. Age and tumor subtype distributions differed substantially depending on genetic finding. CONCLUSIONS: This study contributes to understanding the clinical and genetic landscape of breast and ovarian cancer susceptibility. Extending clinical genetic screening from BRCA1 and BRCA2 to 13 established cancer predisposition genes almost doubles the diagnostic yield, which has implications for genetic counseling and clinical guidelines. The very low yield in the syndrome genes CDH1, PTEN and STK11 questions the usefulness of including these genes on routine gene panels.

Familial platelet disorder due to germline exonic deletions in RUNX1: a diagnostic challenge with distinct alterations of the transcript isoform equilibrium.

Germline pathogenic variants in RUNX1 are associated with familial platelet disorder with predisposition to myeloid malignancies (FPD/MM) with intragenic deletions in RUNX1 accounting for almost 7% of all reported variants. We present two new pedigrees with FPD/MM carrying two different germline RUNX1 intragenic deletions. The aforementioned deletions encompass exons 1-2 and 9-10 respectively, with the exon 9-10 deletion being previously unreported. RNA sequencing of patients carrying the exon 9-10 deletion revealed a fusion with LINC00160 resulting in a change in the 3' sequence of RUNX1. Expression analysis of the transcript isoform demonstrated altered RUNX1a/b/c ratios in carriers from both families compared to controls. Our data provide evidence on the impact of intragenic RUNX1 deletions on transcript isoform expression and highlight the importance of routinely performing copy number variant analysis in patients with suspected MM with germline predisposition.

A retrospective two centre study of Birt-Hogg-Dubé syndrome reveals a pathogenic founder mutation in FLCN in the Swedish population.

Birt-Hogg-Dube syndrome (BHDS) (MIM: 135150) is a rare autosomal dominant disorder with variable penetrance, caused by pathogenic variants in the FLCN gene. Only a few hundreds of families have so far been described in the literature. Patients with BHDS present with three distinct symptoms: fibrofolliculomas, pneumothorax due to lung cyst formation, and increased lifetime risk of kidney tumours. The aim of the current study was to estimate the incidence of BHDS in the Swedish population and further describe the clinical manifestations and their frequency. Splice variant c.779+1G>T was the most common pathogenic variant, found in 57% of the families, suggesting this may be a founder mutation in the Swedish population. This was further investigated using haplotype analysis in 50 families that shared a common haplotype. Moreover, according to gnomAD the carrier frequency of the c.779+1G>T variant has been estimated to be 1/3265 in the Swedish population, however our data suggest that the carrier frequency in the Swedish population may be significantly higher. These findings should raise awareness among physicians of different specialties to patients presenting with fibrofolliculomas, pneumothorax and/or kidney tumours. We also stress the importance of consensus recommendations regarding diagnosis and clinical management of this, not that uncommon, syndrome.

Whole-Body MRI Surveillance-Baseline Findings in the Swedish Multicentre Hereditary TP53-Related Cancer Syndrome Study (SWEP53).

A surveillance strategy of the heritable TP53-related cancer syndrome (hTP53rc), commonly referred to as the Li-Fraumeni syndrome (LFS), is studied in a prospective observational nationwide multi-centre study in Sweden (SWEP53). The aim of this sub-study is to evaluate whole-body MRI (WB-MRI) regarding the rate of malignant, indeterminate, and benign imaging findings and the associated further workup generated by the baseline examination. Individuals with hTP53rc were enrolled in a surveillance program including annual whole-body MRI (WB-MRI), brain-MRI, and in female carriers, dedicated breast MRI. A total of 68 adults ≥18 years old have been enrolled to date. Of these, 61 fulfilled the inclusion criteria for the baseline MRI scan. In total, 42 showed a normal scan, while 19 (31%) needed further workup, of whom three individuals (3/19 = 16%) were diagnosed with asymptomatic malignant tumours (thyroid cancer, disseminated upper GI cancer, and liver metastasis from a previous breast cancer). Forty-three participants were women, of whom 21 had performed risk-reducing mastectomy prior to inclusion. The remaining were monitored with breast MRI, and no breast tumours were detected on baseline MRI. WB-MRI has the potential to identify asymptomatic tumours in individuals with hTP53rc syndrome. The challenge is to adequately and efficiently investigate all indeterminate findings. Thus, a multidisciplinary team should be considered in surveillance programs for individuals with hTP53rc syndrome.

A search for modifying genetic factors in CHEK2:c.1100delC breast cancer patients.

The risk of breast cancer associated with CHEK2:c.1100delC is 2-threefold but higher in carriers with a family history of breast cancer than without, suggesting that other genetic loci in combination with CHEK2:c.1100delC confer an increased risk in a polygenic model. Part of the excess familial risk has been associated with common low-penetrance variants. This study aimed to identify genetic loci that modify CHEK2:c.1100delC-associated breast cancer risk by searching for candidate risk alleles that are overrepresented in CHEK2:c.1100delC carriers with breast cancer compared with controls. We performed whole-exome sequencing in 28 breast cancer cases with germline CHEK2:c.1100delC, 28 familial breast cancer cases and 70 controls. Candidate alleles were selected for validation in larger cohorts. One recessive synonymous variant, rs16897117, was suggested, but no overrepresentation of homozygous CHEK2:c.1100delC carriers was found in the following validation. Furthermore, 11 non-synonymous candidate alleles were suggested for further testing, but no significant difference in allele frequency could be detected in the validation in CHEK2:c.1100delC cases compared with familial breast cancer, sporadic breast cancer and controls. With this method, we found no support for a CHEK2:c.1100delC-specific genetic modifier. Further studies of CHEK2:c.1100delC genetic modifiers are warranted to improve risk assessment in clinical practice.

Risk Factors and Predictors of Mortality in Streptococcal Necrotizing Soft-tissue Infections: A Multicenter Prospective Study.

BACKGROUND: Necrotizing soft-tissue infections (NSTI) are life-threatening conditions often caused by ß-hemolytic streptococci, group A Streptococcus (GAS) in particular. Optimal treatment is contentious. The INFECT cohort includes the largest set of prospectively enrolled streptococcal NSTI cases to date. METHODS: From the INFECT cohort of 409 adults admitted with NSTI to 5 clinical centers in Scandinavia, patients culture-positive for GAS or Streptococcus dysgalactiae (SD) were selected. Risk factors were identified by comparison with a cohort of nonnecrotizing streptococcal cellulitis. The impact of baseline factors and treatment on 90-day mortality was explored using Lasso regression. Whole-genome sequencing of bacterial isolates was used for emm typing and virulence gene profiling. RESULTS: The 126 GAS NSTI cases and 27 cases caused by SD constituted 31% and 7% of the whole NSTI cohort, respectively. When comparing to nonnecrotizing streptococcal cellulitis, streptococcal NSTI was associated to blunt trauma, absence of preexisting skin lesions, and a lower body mass index. Septic shock was significantly more frequent in GAS (65%) compared to SD (41%) and polymicrobial, nonstreptococcal NSTI (46%). Age, male sex, septic shock, and no administration of intravenous immunoglobulin (IVIG) were among factors associated with 90-day mortality. Predominant emm types were emm1, emm3, and emm28 in GAS and stG62647 in SD. CONCLUSIONS: Streptococcal NSTI was associated with several risk factors, including blunt trauma. Septic shock was more frequent in NSTI caused by GAS than in cases due to SD. Factors associated with mortality in GAS NSTI included age, septic shock, and no administration of IVIG.

Patient's characteristics and outcomes in necrotising soft-tissue infections: results from a Scandinavian, multicentre, prospective cohort study.

PURPOSE: Necrotising soft-tissue infections (NSTI) are characterised by necrosis, fast progression, and high rates of morbidity and mortality, but our knowledge is primarily derived from small prospective studies and retrospective studies. METHODS: We performed an international, multicentre, prospective cohort study of adults with NSTI describing patient's characteristics and associations between baseline variables and microbiological findings, amputation, and 90-day mortality. RESULTS: We included 409 patients with NSTI; 402 were admitted to the ICU. Cardiovascular disease [169 patients (41%)] and diabetes [98 (24%)] were the most common comorbidities; 122 patients (30%) had no comorbidity. Before surgery, bruising of the skin [210 patients (51%)] and pain requiring opioids [172 (42%)] were common. The sites most commonly affected were the abdomen/ano-genital area [140 patients (34%)] and lower extremities [126 (31%)]. Monomicrobial infection was seen in 179 patients (44%). NSTI of the upper or lower extremities was associated with monomicrobial group A streptococcus (GAS) infection, and NSTI located to the abdomen/ano-genital area was associated with polymicrobial infection. Septic shock [202 patients (50%)] and acute kidney injury [82 (20%)] were common. Amputation occurred in 22% of patients with NSTI of an extremity and was associated with higher lactate level. All-cause 90-day mortality was 18% (95% CI 14-22); age and higher lactate levels were associated with increased mortality and GAS aetiology with decreased mortality. CONCLUSIONS: Patients with NSTI were heterogeneous regarding co-morbidities, initial symptoms, infectious localisation, and microbiological findings. Higher age and lactate levels were associated with increased mortality, and GAS infection with decreased mortality.

Novel variants in Nordic patients referred for genetic testing of telomere-related disorders.

Telomere-related disorders are a clinically and genetically heterogeneous group of disorders characterized by premature telomere shortening and proliferative failure of a variety of tissues. This study reports the spectrum of telomere-related gene variants and telomere length in Nordic patients referred for genetic testing due to suspected telomere-related disorder. We performed Sanger sequencing of the genes TERT, TERC, DKC1, and TINF2 on 135 unrelated index patients and measured telomere length by qPCR on DNA from peripheral blood leukocytes. We identified pathogenic or likely pathogenic variants in 10 index patients, all of which had short telomeres compared to age-matched healthy controls. Six of the 10 variants were novel; three in TERC (n.69_74dupAGGCGC, n.122_125delGCGG, and n.407_408delinsAA) and three in TERT (p.(D684G), p.(R774*), and p.(*1133Wext*39)). The high proportion of novel variants identified in our study highlights the need for solid interpretation of new variants that may be detected. Measurement of telomere length is a useful approach for evaluating pathogenicity of genetic variants associated with telomere-related disorders.

The BRCA1 c. 5096G>A p.Arg1699Gln (R1699Q) intermediate risk variant: breast and ovarian cancer risk estimation and recommendations for clinical management from the ENIGMA consortium.

BACKGROUND: We previously showed that the BRCA1 variant c.5096G>A p.Arg1699Gln (R1699Q) was associated with an intermediate risk of breast cancer (BC) and ovarian cancer (OC). This study aimed to assess these cancer risks for R1699Q carriers in a larger cohort, including follow-up of previously studied families, to further define cancer risks and to propose adjusted clinical management of female BRCA1*R1699Q carriers. METHODS: Data were collected from 129 BRCA1*R1699Q families ascertained internationally by ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) consortium members. A modified segregation analysis was used to calculate BC and OC risks. Relative risks were calculated under both monogenic model and major gene plus polygenic model assumptions. RESULTS: In this cohort the cumulative risk of BC and OC by age 70 years was 20% and 6%, respectively. The relative risk for developing cancer was higher when using a model that included the effects of both the R1699Q variant and a residual polygenic component compared with monogenic model (for BC 3.67 vs 2.83, and for OC 6.41 vs 5.83). CONCLUSION: Our results confirm that BRCA1*R1699Q confers an intermediate risk for BC and OC. Breast surveillance for female carriers based on mammogram annually from age 40 is advised. Bilateral salpingo-oophorectomy should be considered based on family history.

Telomerase antagonist imetelstat inhibits esophageal cancer cell growth and increases radiation-induced DNA breaks.

Telomerase is mainly active in human tumor cells, which provides an opportunity for a therapeutic window on telomerase targeting. We sought to evaluate the potential of the thio-phosphoramidate oligonucleotide inhibitor of telomerase, imetelstat, as a drug candidate for treatment of esophageal cancer. Our results showed that imetelstat inhibited telomerase activity in a dose-dependent manner in esophageal cancer cells. After only 1 week of imetelstat treatment, a reduction of colony formation ability of esophageal cancer cells was observed. Furthermore, long-term treatment with imetelstat decreased cell growth of esophageal cancer cells with different kinetics regarding telomere lengths. Short-term imetelstat treatment also increased γ-H2AX and 53BP1 foci staining in the esophageal cancer cell lines indicating a possible induction of DNA double strand breaks (DSBs). We also found that pre-treatment with imetelstat led to increased number and size of 53BP1 foci after ionizing radiation. The increase of 53BP1 foci number was especially pronounced during the first 1h of repair whereas the increase of foci size was prominent later on. This study supports the potential of imetelstat as a therapeutic agent for the treatment of esophageal cancer.

Telomerase activity is not a key determinant of sensitivity to standard cytotoxic drugs in human esophageal carcinoma cell lines.

The aim of the present study was to investigate if basal telomerase activity levels may predict sensitivity to cytotoxic drugs in a panel of human esophageal carcinoma cell lines. The TRAPeze telomerase detection assay was used to investigate telomerase activity in the cell lines. Cytotoxic drug sensitivity for 20 standard cytotoxic agents was assessed using the fluorometric microculture cytotoxicity assay (FMCA). Telomerase activity was detected in all cell lines with a broad range of activity levels. Drug sensitivity also varied considerably between the cell lines. Except for a P value towards a correlation between mitoxantrone and telomerase activity (P=0.054), no statistically significant correlation was found between telomerase activity levels and sensitivity to investigated drugs, including key drugs such as cisplatin (P=0.9), 5-fluorouracil (P=0.8) and doxorubicin (P=0.54). We therefore conclude that basal telomerase activity level is not a key determinant of sensitivity to standard cytotoxic drugs in esophageal carcinoma cell lines.

Interferon-induced sensitization to apoptosis is associated with repressed transcriptional activity of the hTERT promoter in multiple myeloma.

The aim of the present study was to explore hTERT as a target for IFN-induced sensitization to apoptosis in multiple myeloma (MM). IFN-alpha and IFN-gamma downregulated telomerase activity in the IL-6-dependent MM cell line U-266-1970. In MM cells undergoing IFN-induced sensitization to Fas-mediated apoptosis, the repression of telomerase was increased as compared to IFN-alpha treatment alone. Similar to the sensitization effect of IFN, the use of a dominant negative IkappaBalpha vector inhibiting hTERT activity via transcriptional targeting resulted in augmentation of Fas-mediated apoptosis. The mechanism underlying the reduction of telomerase activity by IFN was shown to be transcriptional repression of the hTERT gene. The present study does not support a direct effect of IFN on NF-kappaB binding to the hTERT promoter as underlying the transcriptional repression. We conclude that one potential mechanism whereby IFNs induce apoptosis sensitization is by repressing hTERT transcription and telomerase activity, thereby constituting attractive targets for MM therapy.

Differential effects of TGF-beta1 on telomerase activity in thyroid carcinoma cell lines.

The aim of the present study was to investigate the effect of transforming growth factor-beta1 (TGF-beta1) on telomerase activity in a panel of human anaplastic thyroid carcinoma (ATC) cell lines. Addition of TGF-beta1 decreased the telomerase activity in HTh 74 and KTC-1 cells, while in C 643 and HTh 7 an increased activity was observed. The decreased telomerase activity appeared to be due to transcriptional repression of the hTERT promoter. Addition of a PI-3 kinase inhibitor (LY294002) abrogated the stimulatory effect of TGF-beta1 on the telomerase activity, indicating the possible involvement of hTERT activation via phosphorylation. Furthermore, the MEK-inhibitor U0126 had similar effects suggesting dual regulatory mechanisms. Interestingly, the cell lines differed genetically in that ATC cell lines responding with increased telomerase activity harbored a p53 mutation. In conclusion, TGF-beta1 exerts opposing effects on telomerase activity in ATC cell lines, possibly reflecting deregulation of TGF-beta1 signaling in a more malignant genotype.