Next-generation sequencing of 100 candidate genes in young victims of suspected sudden cardiac death with structural abnormalities of the heart.

BACKGROUND: In sudden, unexpected, non-traumatic death in young individuals, structural abnormalities of the heart are frequently identified at autopsy. However, the findings may be unspecific and cause of death may remain unclear. A significant proportion of these cases are most likely caused by inherited cardiac diseases, and the cases are categorized as sudden cardiac death (SCD). The purpose of this study was to explore the added diagnostic value of genetic testing by next-generation sequencing (NGS) of a broad gene panel, as a supplement to the traditional forensic investigation in cases with non-diagnostic structural abnormalities of the heart. METHODS AND RESULTS: We screened 72 suspected SCD cases (<50 years) using the HaloPlex Target Enrichment System (Agilent) and NGS (Illumina MiSeq) for 100 genes previously associated with inherited cardiomyopathies and channelopathies. Fifty-two cases had non-diagnostic structural cardiac abnormalities and 20 cases, diagnosed with a cardiomyopathy post-mortem (ARVC = 14, HCM = 6), served as comparators. Fifteen (29%) of the deceased individuals with non-diagnostic findings had variants with likely functional effects based on conservation, computational prediction, allele-frequency and supportive literature. The corresponding frequency in deceased individuals with cardiomyopathies was 35% (p = 0.8). CONCLUSION: The broad genetic screening revealed variants with likely functional effects at similar high rates, i.e. in 29 and 35% of the suspected SCD cases with non-diagnostic and diagnostic cardiac abnormalities, respectively. Although the interpretation of broad NGS screening is challenging, it can support the forensic investigation and help the cardiologist's decision to offer counselling and clinical evaluation to relatives of young SCD victims.

Next-generation sequencing of 34 genes in sudden unexplained death victims in forensics and in patients with channelopathic cardiac diseases.

Sudden cardiac death (SCD) is responsible for a large proportion of sudden deaths in young individuals. In forensic medicine, many cases remain unexplained after routine postmortem autopsy and conventional investigations. These cases are called sudden unexplained deaths (SUD). Genetic testing has been suggested useful in forensic medicine, although in general with a significantly lower success rate compared to the clinical setting. The purpose of the study was to estimate the frequency of pathogenic variants in the genes most frequently associated with SCD in SUD cases and compare the frequency to that in patients with inherited cardiac channelopathies. Fifteen forensic SUD cases and 29 patients with channelopathies were investigated. DNA from 34 of the genes most frequently associated with SCD were captured using NimbleGen SeqCap EZ library build and were sequenced with next-generation sequencing (NGS) on an Illumina MiSeq. Likely pathogenic variants were identified in three out of 15 (20%) forensic SUD cases compared to 12 out of 29 (41%) patients with channelopathies. The difference was not statistically significant (p = 0.1). Additionally, two larger deletions of entire exons were identified in two of the patients (7%). The frequency of likely pathogenic variants was >2-fold higher in the clinical setting as compared to SUD cases. However, the demonstration of likely pathogenic variants in three out of 15 forensic SUD cases indicates that NGS investigations will contribute to the clinical investigations. Hence, this has the potential to increase the diagnostic rate significantly in the forensic as well as in the clinical setting.

Carcinoma in situ of the female breast. 10 year follow-up results of a prospective nationwide study.

In a Danish nationwide prospective study of in situ carcinoma of the breast, a total of 275 women, treated with excision alone, were registered from 1982 to 1989. The series included 142 cases of ductal carcinoma in situ (DCIS), 100 cases of lobular carcinoma in situ (LCIS), 26 cases of DCIS+LCIS, and seven cases of atypical hyperplasia (AH). Within a median follow-up of 120 months, a crude recurrence rate of 28% (76 cases) was found, of which 53% (40 cases) recurred as invasive carcinomas (IC) and 47% (36 cases) as CIS. CIS recurrences appeared after median 18 months, compared to median 42 months for IC recurrences. No statistical difference was found with respect to development of IC between the three groups of DCIS, DCIS+LCIS, and LCIS. The majority of recurrences were ipsilateral, also for LCIS. Forty four of 49 recurrences following DCIS, and seven of nine recurrences following DCIS+LCIS occurred as local recurrences. Histopathologically, in DCIS a strong association was found between large nuclear size and comedonecrosis. Univariate analysis showed a significant association to recurrence for nuclear size, comedonecrosis, and size of the original lesion. Multivariate analysis showed that only comedonecrosis and size of lesion were independent predictors of recurrence, however, specimen margins were not included in the analysis, as this parameter could not be adequately evaluated in the present series. Nuclear size of original DCIS lesion was related to histologic grade of the IC recurrence. The recurrence rate for DCIS of small nuclear size increased from 6% at five years of follow-up to 16% at 10 years, possibly due to a slower growth rate and a continued but delayed risk. Similarities were found between LCIS and DCIS of small nuclear size, both showing a continued risk and comparable rate of recurrence. Further, progression to IC of similar, highly differentiated type was seen, indicating a linkage between biological behavior of the two histological types.

Carcinoma in situ of the breast: correlation of histopathology to immunohistochemical markers and DNA ploidy.

In a consecutive and unselected series of 178 cases of carcinoma in situ of the breast (CIS), comprising both ductal (DCIS) and lobular type (LCIS), and a series of 48 cases of invasive carcinoma (IC) with predominance of DCIS, the association between histopathology, immunohistochemical markers (ER, PgR, MIB-1, c-erbB-2, and p53), and DNA ploidy was investigated, in order to discriminate biologically different groups. In DCIS, significant correlation was shown between large nuclear size and comedonecrosis, both of which showed also strong association to DNA aneuploidy, high proliferation activity, low steroid receptor content, and overexpression of c-erbB-2 and p53 factors that may indicate an aggressive behavior. Small nuclear CIS, whether LCIS or DCIS, on the contrary, were DNA diploid with low proliferation, and no cases showed overexpression of c-erbB-2 and p53. Heterogeneity with respect to the investigated parameters was also a frequent finding that may reflect a development complexity. In IC, comparison of the DCIS and the invasive component showed similar patterns. No significant differences were shown between DCIS without and with invasion. This may indicate that none of the investigated parameters on its own are essential for the event of invasion.

DNA ploidy analysis in breast carcinoma. Comparison of unfixed and fixed tissue analyzed by image and flow cytometry.

OBJECTIVE: To form a methodologic basis for DNA analysis of ductal carcinoma in situ (DCIS) and invasive carcinoma (IC) of the breast, including very small lesions, by comparison of flow cytometric (FCM) and image cytometric (ICM) methods for DNA quantitation. STUDY DESIGN: The material consisted of 41 DCIS lesions and 26 ICs. FCM DNA analysis of unfixed, frozen samples were compared to (1) FCM of formalin-fixed, paraffin-embedded tissue; (2) ICM of imprints; and (3) ICM of paraffin-embedded tissue sections. RESULTS: FCM of unfixed tissue showed higher DNA measurement precision and a higher number of DNA nondiploid clones as compared to the other three methods. For the classification of DNA diploid/nondiploid cases, high concordance rates were found between the methods. Discordant cases were predominantly DNA neardiploid by FCM of unfixed tissue but DNA diploid by the other methods. The reproducibility of the DNA index (DI) was best in the interval 1.2 < DI < or = 2.2; it was 74% for FCM of fixed tissue and 79% for ICM of imprints. Clones with DI > 3 were found almost exclusively by ICM of imprints. For ICM of tissue sections, DI could not be reliably estimated. By ICM, contrary to FCM, a combined DNA diploid and nondiploid pattern was found frequently. CONCLUSION: Each of the methods has its own advantages and limitations. If possible, FCM should be combined with ICM. FCM of unfixed tissue is superior to the other methods with respect to precise DI estimation. Alternatively, FCM of fixed tissue and ICM of imprints may both give a reliable estimate of DI. ICM of tissue sections can discriminate DNA diploid from nondiploid clones, except for neardiploid subpopulations, and permits the analysis of very small lesions.

Tissue disaggregation for flow cytometric DNA analysis: comparison of fine-needle aspiration and an automated mechanical procedure.

Flow cytometric DNA analysis was performed on unfixed frozen samples from 56 breast cancer patients. From each patient, two samples were analyzed. The only difference in the handling of the paired samples was the mechanical disaggregation of one sample by fine-needle aspiration compared to an automated mechanical disaggregation method (Medimachine) of the other sample. With the two methods for tissue disaggregation, the same resolution of the DNA histograms was obtained, indicated by median coefficients of variation (CV) of 1.5% for the DNA diploid G1 peaks. Also, the frequencies of DNA diploid and aneuploid cases as well as the fractions of DNA aneuploid cells were comparable. This indicates that the two methods did not differ in ability to detect DNA aneuploid tumor clones. Automated mechanical disaggregation resulted in DNA histograms with significantly less debris and with lower S-phase fractions. In practice, the procedure of automated mechanical disaggregation was rapid, easy, and safer because of minimal handling of the unfixed tissue compared to the fine-needle aspiration.

DNA aneuploidy in early breast cancer.

High-resolution flow cytometric (FCM) DNA analysis was performed on 148 unfixed, frozen tissue samples from four groups of early breast cancers: invasive carcinomas (ICs) with predominance of carcinoma in situ (DCIS) (group I), small clinical cancers < or = 15 mm (group II), node-negative, clinical cancers (group III) and small screening-detected cancers < or = 15 mm (group IV). The median tumour size was 12 mm. The aim of the study was to support, with a larger sample, our recent findings with respect to DNA ploidy pattern in the selected group of ICs with predominance of DCIS (group I). Similar results to this group were found for both the small clinical cancers and the node-negative cancers, with respect to frequency of DNA aneuploidy (79% and 90%), DNA index (DI) distribution, intratumoral DNA heterogeneity and S-phase fraction. A high frequency of DNA hyperdiploid clones was found, in particular related to highly differentiated tumours. A significant difference was found compared with the screening-detected cancers, which were characterised by a much lower frequency of DNA aneuploid samples (49%) and may represent a biologically specific group of low-malignant, slowly growing tumours. Associations were shown between histological grade and DI subclasses, and between lymph node status and DNA diploidy/aneuploidy, whereas DI was not correlated with tumour size. The DNA ploidy findings in this series of early cancers are concordant to our own results from preinvasive lesions as well as those reported from series of more advanced cancers.

DNA analysis of in situ ductal carcinoma of the breast via flow cytometry.

Flow cytometric (FCM) DNA analysis of 41 clinical cases of ductal carcinoma in situ (DCIS) of the breast was performed on fine-needle aspirates of unfixed, frozen tissue. Based on analysis of a single tissue sample, abnormal DNA content was found in 35 (85%) of the cases, and 15 (37%) disclosed heterogeneity. If five or more samples were analyzed, then heterogeneity was found in eight of ten cases. No conclusive correlation could be found between DNA index (DI) and histopathology. Our results on DCIS demonstrate a very high concordance with invasive breast cancer with respect to the frequency of DNA nondiploid cases, distribution of DI, occurrence of multiple clones, and S-phase fraction. This indicates that major genetic alterations and DNA heterogeneity are early events in carcinogenesis that are already established at the preinvasive stage.

Flow cytometric DNA analysis of breast cancers with predominance of carcinoma in situ: a comparison of the premalignant and malignant components.

Flow cytometric DNA analysis was performed on unfixed frozen tissue samples from 48 cases of invasive breast cancer (IC) with a predominance of ductal carcinoma in situ (DCIS). In 15 cases the samples contained only the DCIS component, in 17 cases only the IC component, whereas in 16 cases separate samples from the DCIS as well as the IC part within the individual lesion were available. In the latter 16 cases, complete or partial accordance in DNA ploidy between DCIS and IC was found in 12 cases, whereas no correspondence could be demonstrated in the remaining 4 cases, possibly due to intratumoral DNA heterogeneity. Comparison of the DNA index distribution in samples of DCIS and IC from the 48 cases showed concordant results except for the DNA hyperdiploid subclass, in which 6 clones were found in the DCIS portion compared to 18 clones in the IC portion. S-phase fractions were also comparable in the two groups. A comparison of the DCIS component from the present series of breast cancers to our previous series of pure DCIS also showed similar results with respect to the DNA index distribution, DNA heterogeneity, and S-phase fraction. No differences could be demonstrated between DCIS with and without invasion. The results indicate that the DNA ploidy pattern of breast cancer, as detected by flow cytometric DNA analysis, is established at the preinvasive stage of carcinogenesis.

Lobular carcinoma in situ of the female breast. Short-term results of a prospective nationwide study. The Danish Breast Cancer Cooperative Group.

In a Danish nationwide prospective study of in situ carcinomas and atypical lesions of the breast, 88 women, comprising 69 patients with lobular carcinoma in situ (LCIS) and 19 patients with combined lobular and ductal carcinoma in situ (LCIS + DCIS), were accrued from 1982 through 1987. All cases were treated with excision only. Within a median follow-up time of 61 months, a recurrence rate of 17% (15 cases) was found, excluding nine cases of refinding of LCIS. No contralateral recurrences occurred. The recurrences were in eight cases invasive carcinomas (IC), in six cases LCIS + DCIS, and in one case DCIS alone. The recurrence rates among cases of LCIS and of LCIS + DCIS were not significantly different. The histopathological review included an estimate of the number of lobules with LCIS and nuclear size, both of which were significantly related to recurrence. The risk of developing IC was calculated to be increased by a factor 11 as compared with the reference population.

Ductal carcinoma in situ of the female breast. Short-term results of a prospective nationwide study. The Danish Breast Cancer Cooperative Group.

In a Danish nationwide prospective study of in situ carcinoma of the breast, 112 women with ductal carcinoma in situ, treated with excision only, were registered from 1982 to 1987. Within a median follow-up of 53 months, a crude recurrence rate of 22% (25 cases) was found, of which five cases recurred as invasive carcinomas and 20 cases as in situ carcinomas. The histopathologic review included a single-parameter analysis of histological growth pattern, size of lesion, nuclear size, presence of comedonecrosis, and subhistologic type. A strong interrelationship was found for histological growth pattern, nuclear size, and comedonecrosis. These parameters were also significantly related to recurrence. Cases that had clinical symptoms had a high recurrence rate as compared with cases that were discovered by mammography only or incidentally.

The significance of the frequency and occurrence of breast cancer for treatment. Essential aspects of new Danish studies of the natural history of breast cancer.

The natural history of breast cancer (BC) has been evaluated by two entirely different types of consecutive and unselected materials: The general female population. Females with the breast cancer disease. The frequency of in situ carcinoma in the general female population is about 25 per cent. The growth pattern of the in situ carcinomas is predominantly microfocal contrary to findings in mammography selected surgical materials where tumor forming and/or diffuse types contribute to more than 30 per cent. Evidence is established of three different stages of the development of invasive breast carcinoma: The benign epithelial hyperplastic stage, the microfocal in situ carcinoma stage and the cancer stage. Among females with the BC-disease the residual breast glandular tissue in the contralateral breast develops a new primary invasive carcinoma in 33 per cent and furthermore in situ carcinoma in another 35 per cent. Death of disseminated metastases and axillary lymphnode metastases is significantly related to these new primaries. Residual breast glandular tissue left behind after total mastectomy contributes to new primary invasive carcinoma. Thus among clinical chest-wall recurrences not less than 20 per cent were primary invasive cancers. Our investigations have considerable impact on treatment: As concern microfocal in situ lesions they support a conservative attitude as regard treatment, providing the females are included in prospective follow-up studies including mammography. As concern the contralateral breast they support a more active attitude, but more studies are needed. Meanwhile a close follow-up program is an inevitable consequence.(ABSTRACT TRUNCATED AT 250 WORDS)