Equivocal (HER2 IHC 2+) breast carcinomas: gene-protein assay testing reveals association between genetic heterogeneity, individual cell amplification status and potential treatment benefits.

AIMS: Genetic heterogeneity can pose a challenge to identifying eligible cases for targeted therapy in the human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) 2+ breast carcinoma group. In this study, we characterised this subset of tumours according to clinicopathological parameters. METHODS AND RESULTS: We assessed 1000 tumour cells per case and recorded the number of HER2 and chromosome enumeration probe 17 (CEP17) copies using gene-protein assay slides. HER2 status was determined based on ASCO/CAP 2013 guidelines. Tumours with 5-50% of cancer cells with amplification were considered to be heterogeneous, whereas those with >50% were considered to be non-heterogeneous. In a study cohort of 110 HER2 IHC 2+ carcinomas, 93 (84.5%) were non-amplified, 12 (10.9%) were amplified and five (4.5%) were ISH-equivocal. All the HER2-amplified and two of ISH-equivocal cases (12.7%) corresponded to non-heterogeneous tumours, with highly significant differences evident in the average HER2/CEP17 ratio (P = 0.0002) and the proportion of cells with HER2 >6 copies (P < 0.0001) compared with heterogeneous lesions. NST grade 3 and HER2-amplified carcinomas average HER2/CEP17 ratio correlated with an increased number of cells with HER2/CEP17 ≥2.0 (P < 0.014). Triple-negative CEP17 polysomic carcinomas showed increased metastatic capacity (P = 0.003) compared with other tumour types. CONCLUSION: Non-heterogeneous HER2 IHC 2+ tumours tend to be HER2-amplified. Adding the percentage of cells with HER2 >6 copies to the average HER2/CEP17 ratio may facilitate assessment of amplification status in ISH-equivocal cases. The proportion of cells with HER2/CEP17 ≥2.0 contributes information concerning the actual average HER2/CEP17 ratio, depending on tumour type.

Pediatric reference intervals for general clinical chemistry components - merging of studies from Denmark and Sweden.

BACKGROUND: Reference intervals are crucial tools aiding clinicians when making medical decisions. However, for children such values often are lacking or incomplete. The present study combines data from separate pediatric reference interval studies of Denmark and Sweden in order to increase sample size and to include also pre-school children who were lacking in the Danish study. METHODS: Results from two separate studies including 1988 healthy children and adolescents aged 6 months to 18 years of age were merged and recalculated. Eighteen general clinical chemistry components were measured on Abbott and Roche platforms. To facilitate commutability, the NFKK Reference Serum X was used. RESULTS: Age- and gender-specific pediatric reference intervals were defined by calculating 2.5 and 97.5 percentiles. CONCLUSION: The data generated are primarily applicable to a Nordic population, but could be used by any laboratory if validated for the local patient population.

Survival and incidence of cardiovascular diseases in participants in a long-distance ski race (Vasaloppet, Sweden) compared with the background population.

Aims: We studied the relationship between taking part in a long-distance ski race and incidence of cardiovascular diseases (CVDs) to address the hypothesis that lifestyle lowers the incidence. Methods and results: A cohort of 399 630 subjects in Sweden, half were skiers in the world's largest ski race, and half were non-skiers. Non-skiers were frequency matched for sex, age, and year of race. Individuals with severe diseases were excluded. The endpoints were death, myocardial infarction, or stroke. The subjects were followed up for a maximum of 21.8 years and median of 9.8 years. We identified 9399 death, myocardial infarction, or stroke events among non-skiers and 4784 among the Vasaloppet skiers. The adjusted hazard ratios (HRs) comparing skiers and non-skiers were 0.52 [95% confidence interval (CI) 0.49-0.54] for all-cause mortality, 0.56 (95% CI 0.52-0.60) for myocardial infarction and 0.63 (95% CI 0.58-0.67) for stroke and for all three outcomes 0.56 (95% CI 0.54-0.58). The results were consistent across subgroups: age, sex, family status, education, and race year. For skiers, a doubling of race time was associated with a higher age-adjusted risk of 19%, and male skiers had a doubled risk than female skiers, with a HR 2.06 (95% CI 1.89-2.41). The outcome analyses revealed no differences in risk of atrial fibrillation between skiers and non-skiers. Conclusion: This large cohort study provides additional support for the hypothesis that individuals with high level of physical activity representing a healthy lifestyle, as evident by their participation in a long-distance ski race, have a lower risk of CVD or death.

Risk of recurrent ischaemic events after myocardial infarction in long-distance ski race participants.

AIMS: To study whether a high level of physical activity prior to myocardial infarction (MI) also protects against recurrent MI (re-MI) or death. METHODS AND RESULTS: A longitudinal study of a primary cohort consisting of 204,038 skiers with a proved substantially high level of physical activity in the world's largest long-distance ski race, Vasaloppet, and 499,543 non-skiers selected from the Swedish population. Individuals with severe diseases at baseline were excluded. In the nationwide clinical register, Swedeheart, we identified 7092 individuals with a first MI incident between 1989 and 2010. Of these, 1039 (0.5%) were skiers and 6053 (1.2%) were non-skiers. One hundred and sixty-three (15.7%) skiers and 1352 (22.3%) non-skiers suffered a re-MI or died during follow-up (median 4.44 years), corresponding to an incidence rate of 38.9 (95% confidence interval (CI) 33.2-45.4)/1000 person-years and 55.6 (95% CI 52.7-58.7)/1000 person-years, respectively. Severity of MI in both groups was the same. For skiers compared to non-skiers the unadjusted hazard ratio (HR) for re-MI was 0.66 (95% CI 0.52-0.82). For death or re-MI, HR was 0.70 (95% CI 0.59-0.82) with consistent results in subgroups based on race year, age, gender, education level, marital status. After adjustment for also smoking, diabetes, hypertension and cardiovascular medication, HR was 0.80 (95% CI 0.67-0.97). CONCLUSIONS: This large cohort study supports the hypothesis that patients with MI and with prior physical activity and healthy lifestyle, as evidenced by their participation in a long-distance ski race, have a lower risk of subsequent re-MI or death.

Biobanking multifocal breast carcinomas: sample adequacy with regard to histology and DNA content.

AIMS: To determine the volume of tumoral and normal breast tissue containing sufficient DNA (>2 µg/sample) for genetic platforms and biobanking, with a focus on multifocality, tumoral heterogeneity, and factors that critically influence sample acceptability. METHODS AND RESULTS: We examined 57 breast surgical specimens with multifocal (46/57) and unifocal (11/57) cancers. Punch biopsies were obtained from tissue slices under multimodal radiological guidance, and the colour-coded sampling sites were identified in large-format histology slides. The study comprised 415 DNA isolations from tumour (n = 105) and normal (n = 283) tissue, including skin (n = 27) samples. A single 2-mm core from invasive tumour contained sufficient DNA in 91.4% (96/105) of cases, depending on tumour type (3.8-108.2 µg/sample), number and size of additional foci in multifocal cases (P = 0.001), tumour consistency, and degree of necrosis. Three biopsies obtained with a 4-mm device were required from normal breast tissue, at least 10 mm from the tumour. Cold ischaemia for up to 82 min did not influence the yield of DNA. CONCLUSIONS: Radiological disease mapping is useful for guiding optimal specimen slicing and for targeting breast lesions. A single 2-mm core from tumour and multiple 4-mm cores from normal breast tissue yield adequate DNA in the majority of samples.

Risk of Recurrent Stroke and Death After First Stroke in Long-Distance Ski Race Participants.

BACKGROUND: Physical activity is of benefit for primary prevention of cardiovascular diseases, but it appears to increase the risk for atrial fibrillation. We aimed to study a cohort of patients following a first stroke in individuals with previous high physical activity, compare them to the general population with respect to recurrent stroke and death, and relate these to atrial fibrillation. METHODS AND RESULTS: From the participants of the Vasaloppet, the world's largest ski-race, and matched individuals from the general population (n=708,604), we identified 5964 patients hospitalized with a first-time stroke between 1994 and 2010. Individuals with severe diseases were excluded. One half percent of skiers and 1% of nonskiers were hospitalized due to stroke. The incidence rate was 8.3 per 100 person-years among skiers and 11.1 among nonskiers. The hazard ratio (HR) for recurrent stroke or death between the 2 groups was 0.76 (95% CI 0.67 to 0.86). The result was consistent in subgroups. The HR for death was 0.66 (95% CI 0.56 to 0.78) and for recurrent stroke 0.82 (95% CI 0.70 to 0.96). After adjustment for smoking and socioeconomic factors, the HR for death was consistent at 0.70 (95% CI 0.56 to 0.87) while the HR for recurrent stroke was not statistically significant. Outcomes for skiers with atrial fibrillation tended to show a lower risk than for nonskiers. CONCLUSIONS: This large cohort study supports the hypothesis that patients with a stroke and with prior regular physical activity have a lower risk of death, while their risk for recurrent stroke is similar to that of nonskiers. The skiers had a higher incidence of atrial fibrillation, but still no increased risk of recurring stroke.

Alkaline phosphatase in healthy children: reference intervals and prevalence of elevated levels.

BACKGROUND: Transient hyperphosphatasemia (TH) is an often unnoticed benign entity, primarily affecting children below 5 years of age. However, the prevalence among healthy children is unknown. We used data from a Swedish pediatric reference interval project to estimate the prevalence of high alkaline phosphatase (ALP) among healthy children and to calculate pediatric reference intervals. METHODS: Blood was collected from 699 subjectively healthy children aged 6 months to 18 years. After exclusion of subjects with high ALP, age- and gender-specific reference intervals were calculated. RESULTS: Six children had ALP levels >16.7 µkat/l (>1,000 U/l), including 4 females and 2 males aged 7-22 months. The prevalence in the age group from 6 months to 2 years was 6.2% (6/97). None of the older children had levels of ALP >16.7 µkat/l. The study did not include the follow-up of these apparently healthy children. Consequently, conditions others than TH explaining the elevated ALP could not be excluded. However, general chemistry analyses, such as liver enzymes, calcium, intact PTH and vitamin D, were essentially normal in these children. CONCLUSIONS: The prevalence of high ALP among subjectively healthy children was approximately 2.4% below 5 years of age and 6.2% below 2 years. Reference intervals vary with age and gender.

Tumor marker score for prognostication of early-stage squamous cell cervical cancer.

BACKGROUND/AIM: Histopathological and clinical scores to predict prognosis in cervical cancer have been of limited value. In the present study a tumor marker expression score was evaluated for prognostication in early-stage cervical cancer. MATERIALS AND METHODS: The entire study population included 128 women with invasive squamous cell cervical cancer followed-up for at least 10 years. RESULTS: Expression of 12 tumor markers (epidermal growth factor receptor (EGFR), Ki-67, c-MYC, p53, p27, E-cadherin, CD44, vascular endothelial growth factor receptor (VEGF), cyclooxygenase-2 (COX2), CD4, and leucine-rich immunoglobulin-like repeats-1 (LRIG1) and LRIG2, considered relevant for cervical cancer prognostication was evaluated by immunohistochemistry. Expression of five markers, LRIG1, LRIG2, p53, COX2 and c-MYC were useful to make a prognostication score, ranging from 0 to 5. Score 0-1 correlated to less than 5% 10-year mortality, while the mortality rate of those with score 4-5 approached 70%; those with score 2 formed an intermediate group. Using different models, a high sensitivity, specificity, positive predictive value and negative predictive value was attained. CONCLUSION: Tumor marker scoring could be an adjunct to histopathological and clinical parameters in prognostication of early-stage cervical cancer.

A systematic analysis of commonly used antibodies in cancer diagnostics.

AIMS: Immunohistochemistry plays a pivotal role in cancer differential diagnostics. To identify the primary tumour from a metastasis specimen remains a significant challenge, despite the availability of an increasing number of antibodies. The aim of the present study was to provide evidence-based data on the diagnostic power of antibodies used frequently for clinical differential diagnostics. METHODS AND RESULTS: A tissue microarray cohort comprising 940 tumour samples, of which 502 were metastatic lesions, representing tumours from 18 different organs and four non-localized cancer types, was analysed using immunohistochemistry with 27 well-established antibodies used in clinical differential diagnostics. Few antibodies, e.g. prostate-specific antigen and thyroglobulin, showed a cancer type-related sensitivity and specificity of more than 95%. A majority of the antibodies showed a low degree of sensitivity and specificity for defined cancer types. Combinations of antibodies provided limited added value for differential diagnostics of cancer types. CONCLUSIONS: The results from analysing 27 diagnostic antibodies on consecutive sections of 940 defined tumours provide a unique repository of data that can empower a more optimal use of clinical immunohistochemistry. Our results highlight the benefit of immunohistochemistry and the unmet need for novel markers to improve differential diagnostics of cancer.

Molecular phenotype of the foci in multifocal invasive breast carcinomas: intertumoral heterogeneity is related to shorter survival and may influence the choice of therapy.

BACKGROUND: Multiple synchronous, ipsilateral, invasive foci of breast carcinomas are frequent and are associated with a poorer prognosis. Few studies have investigated the prognostic and therapeutic implications of heterogeneity of such foci. METHODS: The authors reviewed the tumor type, grade, and size of all invasive foci in a series of 110 multifocal breast carcinomas documented on large-format slides. Molecular phenotype was determined by immunohistochemistry in tissue microarray blocks using 3 classification systems. The survival of patients who had tumors with microscopic (tumor type and/or grade) heterogeneity and of those who had tumors with phenotypic heterogeneity was compared with the survival of patients who had multifocal homogeneous tumors using Kaplan-Meier curves. The hazard ratio of dying from breast cancer was also calculated. RESULTS: Intertumoral heterogeneity in tumor type and grade was detected in 16 of 110 tumors (14.6%) and in 6 of 110 tumors (5.5%), respectively. The molecular phenotype of invasive tumor foci within the same breast differed in 10% to 12.7% of patients (11-14 of 110 tumors), depending on the classification system used. Patients who had phenotypically heterogeneous, multifocal cancers had a greater risk of dying from disease (HR=2.879; 95%CI=1.084-7.649; P = .034) and had significantly shorter survival (P = .016). Phenotypic differences were most common in patients who had tumors that were homogeneous in terms of tumor type (11 of 18 tumors) and histology grade (14 of 18 tumors). Phenotyping additional tumor foci had the potential to influence the therapeutic decisions in up to 8 patients. CONCLUSIONS: Phenotyping more than 1 invasive focus of multifocal breast carcinomas only if the individual foci deviate microscopically appears to be insufficient, because phenotypic intertumoral heterogeneity may be observed in microscopically identical foci and has potential prognostic and therapeutic consequences.

Estimating reliable paediatric reference intervals in clinical chemistry and haematology.

UNLABELLED: Very few high-quality studies on paediatric reference intervals for general clinical chemistry and haematology analytes have been performed. Three recent prospective community-based projects utilising blood samples from healthy children in Sweden, Denmark and Canada have substantially improved the situation. CONCLUSION: The present review summarises current reference interval studies for common clinical chemistry and haematology analyses.

Markers of Celiac Disease and Gluten Sensitivity in Children with Autism.

OBJECTIVE: Gastrointestinal symptoms are a common feature in children with autism, drawing attention to a potential association with celiac disease or gluten sensitivity. However, studies to date regarding the immune response to gluten in autism and its association with celiac disease have been inconsistent. The aim of this study was to assess immune reactivity to gluten in pediatric patients diagnosed with autism according to strict criteria and to evaluate the potential link between autism and celiac disease. METHODS: Study participants included children (with or without gastrointestinal symptoms) diagnosed with autism according to both the Autism Diagnostic Observation Schedule (ADOS) and the Autism Diagnostic Interview, Revised (ADI-R) (n = 37), their unaffected siblings (n = 27), and age-matched healthy controls (n = 76). Serum specimens were tested for antibodies to native gliadin, deamidated gliadin, and transglutaminase 2 (TG2). Affected children were genotyped for celiac disease associated HLA-DQ2 and -DQ8 alleles. RESULTS: Children with autism had significantly higher levels of IgG antibody to gliadin compared with unrelated healthy controls (p<0.01). The IgG levels were also higher compared to the unaffected siblings, but did not reach statistical significance. The IgG anti-gliadin antibody response was significantly greater in the autistic children with gastrointestinal symptoms in comparison to those without them (p<0.01). There was no difference in IgA response to gliadin across groups. The levels of celiac disease-specific serologic markers, i.e., antibodies to deamidated gliadin and TG2, did not differ between patients and controls. An association between increased anti-gliadin antibody and presence of HLA-DQ2 and/or -DQ8 was not observed. CONCLUSIONS: A subset of children with autism displays increased immune reactivity to gluten, the mechanism of which appears to be distinct from that in celiac disease. The increased anti-gliadin antibody response and its association with GI symptoms points to a potential mechanism involving immunologic and/or intestinal permeability abnormalities in affected children.

Population-based pediatric reference intervals for HbA1c, bilirubin, albumin, CRP, myoglobin and serum enzymes.

BACKGROUND: Many previous studies on reference intervals are hampered by the inclusion of only hospital-based populations of children and adolescents. METHODS: This study included 694 children, evenly distributed from 6 months to 18 years of age. They were recruited as volunteers at child care units and schools. All subjects were apparently healthy. A questionnaire on diseases and medications was filled out by parents and by the older children. RESULTS: Alanine aminotransferase (ALT), albumin, aspartate aminotransferase (AST), bilirubin, conjugated bilirubin, C-reactive protein (CRP), creatine kinase (CK), Gamma-glutamyltransferase (GGT), HbA1c (mono S and IFCC calibrations), lactate dehydrogenase (LD), myoglobin and panceratic amylase were analyzed on Abbott Architect ci8200, and for HbA1c on Tosoh G7 and a mono S-system. Age- and gender-related 2.5th and 97.5th percentiles were estimated. For some analytes the differences to comparable studies were substantial. CONCLUSION: The study gives age- and gender-specific pediatric reference intervals, measured with modern methods for a number of important analytes. The results emphasize the importance to evaluate pediatric reference intervals in different populations and ethnic groups including only healthy subjects.

Socioeconomic characteristics, housing conditions and criminal offences among women with cervical neoplasia.

OBJECTIVE: To investigate the association between cervical neoplasia and socioeconomic factors, housing conditions and criminal offences. DESIGN: Longitudinal observational study. SETTING: Falun county hospital, Sweden. POPULATION: A total of 1331 women diagnosed with cervical intraepithelial neoplasia I-III or cervical cancer between 1967 and 1978 were compared with 2604 age-matched controls from the same geographical area in Sweden. METHODS: The Population and Housing Censuses were used for information about civil status, education, housing conditions, employment and socioeconomic status. The Swedish Register of Conviction Decisions was used to access information on criminal offences. MAIN OUTCOME MEASURES: Socioeconomic status, housing conditions, criminal offences. RESULTS: Women with cervical neoplasia had a lower socioeconomic status and a lower educational level than their age-matched controls. They were more often divorced and did not own their home as often as controls. A significant association with criminal offences was observed, and it persisted after adjustment for socioeconomic status. Differences in socioeconomic factors between women with cervical neoplasia and their controls had not diminished in the younger, compared with the older, part of the study population. CONCLUSIONS: The results indicate that women with cervical neoplasia belong to a socioeconomically disadvantaged group. Furthermore, the study provides information about an association with criminal offences.

Population-based pediatric reference intervals for hematology, iron and transferrin.

Reference intervals are crucial decision-making tools aiding clinicians in differentiating between healthy and diseased populations. However, for children such values often are lacking or incomplete. Blood samples were obtained from 689 healthy children, aged 6 months to 18 years, recruited in day care centers and schools. Hematology and anemia analytes were measured on the Siemens Advia 2120 and Abbott Architect ci8200 platforms (hemoglobin, erythrocyte volume fraction [EVF], erythrocytes, mean corpuscular volume [MCV], mean corpuscular hemoglobin [MCH], mean corpuscular hemoglobin concentration [MCHC], reticulocytes, leukocytes, lymphocytes, monocytes, neutrophils, eosinophils, basophils, platelets, iron, transferrin, transferrin saturation). Age- and gender-specific pediatric reference intervals were defined by calculating 2.5th and 97.5th percentiles. The data generated is primarily applicable to a Caucasian population, but could be used by any laboratory if verified for the local patient population.

Girls who masturbate in early infancy: diagnostics, natural course and a long-term follow-up.

AIM: To evaluate the natural course, onset, diagnostics and long-term follow-up masturbation in infant girls, which up to now has only been described in case reports. METHODS: Nineteen consecutive healthy, masturbating girls, diagnosed in early infancy between three and 15 months, were followed up for an average of 8 years. All were diagnosed in the same Swedish hospital between May 1996 and June 2010. Ten girls were videotaped and/or directly observed while masturbating. RESULTS: Mean age at onset of masturbation was 10.4 months. The diagnosis was based on history taking and clinical observation. Parents reported that masturbation ranged from a few times a week to two-to-fifty episodes a day. Twelve girls had stopped masturbating when this study was written, after a mean duration of 66 months. Their symptoms and diagnostics are described in detail. CONCLUSION: This is the first follow-up study of girls who started masturbating in early infancy, with a mean duration of five-and-a-half years. Diagnosis may be difficult, but with awareness and knowledge of the condition, a normal physical and neurological examination, a detailed history from the parents and, in particular, video documentation, it can be settled without extensive investigations and the parents reassured.

Multifocal breast cancer documented in large-format histology sections: long-term follow-up results by molecular phenotypes.

BACKGROUND: The prognostic significance of molecular phenotype in breast cancer is well established in the literature. Recent studies have demonstrated that subgross lesion distribution (unifocal, multifocal, and diffuse) and disease extent also carry prognostic significance in this disease. However, the correlation of molecular phenotypes with subgross parameters has not yet been investigated in detail. METHODS: In total, 444 consecutive invasive breast cancers that were documented in large-format histology slides and worked up with detailed radiologic-pathologic correlation were sampled into tissue microarray blocks and stained immunohistochemically to delineate the molecular subtypes. RESULTS: Diffuse or multifocal distribution of the invasive component of breast carcinomas in this series was associated with a 4.14-fold respectively 2.75-fold risk of cancer-related death compared with unifocal tumors irrespective of molecular phenotype. Patients who had human epidermal growth factor receptor 2 (HER2)-positive cancers; estrogen receptor-negative, progesterone receptor-negative, and HER2-negative (triple-negative) cancers; or basal-like cancers had a 2.18-fold, 2.33-fold, and 4.07-fold risk of dying of disease, respectively, compared with patients who had luminal A carcinomas. Unifocal luminal A, HER2-positive, and basal-like cancers were associated with significantly better long-term survival outcomes than their multifocal or diffuse counterparts; luminal B and triple-negative tumors also had the same tendency. In multivariate analysis, patient age, tumor size category, lymph node status, lesion distribution, and molecular phenotypes remained significant. CONCLUSIONS: Multifocality and diffuse distribution of the invasive component were associated with significantly poorer survival in women with breast carcinomas compared with unifocal disease in patients with luminal A, HER2 type, and basal-like cancers. Molecular classification of breast cancer is a powerful tool but gains in power when combined with conventional and subgross morphologic parameters.