RET Fluorescence In Situ Hybridization Analysis Is a Sensitive but Highly Unspecific Screening Method for RET Fusions in Lung Cancer.

INTRODUCTION: RET gene fusions are established oncogenic drivers in 1% of NSCLC. Accurate detection of advanced patients with RET fusions is essential to ensure optimal therapy choice. We investigated the performance of fluorescence in situ hybridization (FISH) as a diagnostic test for detecting functional RET fusions. METHODS: Between January 2016 and November 2019, a total of 4873 patients with NSCLC were routinely screened for RET fusions using either FISH (n = 2858) or targeted RNA next-generation sequencing (NGS) (n = 2015). If sufficient material was available, positive cases were analyzed by both methods (n = 39) and multiple FISH assays (n = 17). In an independent cohort of 520 patients with NSCLC, whole-genome sequencing data were investigated for disruptive structural variations and functional fusions in the RET and compared with ALK and ROS1 loci. RESULTS: FISH analysis revealed RET rearrangement in 48 of 2858 cases; of 30 rearranged cases double tested with NGS, only nine had a functional RET fusion. RNA NGS yielded RET fusions in 14 of 2015 cases; all nine cases double tested by FISH had RET locus rearrangement. Of these 18 verified RET fusion cases, 16 had a split signal and two a complex rearrangement by FISH. By whole-genome sequencing, the prevalence of functional fusions compared with all disruptive events was lower in the RET (4 of 9, 44%) than the ALK (27 of 34, 79%) and ROS1 (9 of 12, 75%) loci. CONCLUSIONS: FISH is a sensitive but unspecific technique for RET screening, always requiring a confirmation using an orthogonal technique, owing to frequently occurring RET rearrangements not resulting in functional fusions in NSCLC.

Anisometric Cell and Dysplastic Lipomas in a Retinoblastoma Patient.

Anisometric cell lipoma (ACL) and dysplastic lipoma (DL) are underrecognized subtypes of benign lipomatous tumors, with wide variation in cell size, microscopic fat necrosis, and no or mild nuclear changes (DL). ACL/DL appear more commonly in retinoblastoma patients, in whom an increased incidence of lipomas has been established. The occurrence of ACL/DL in retinoblastoma patients suggests that RB1 aberrations play a role in its pathogenesis, similar to spindle cell/pleomorphic lipoma. In this article, we present a patient with a history of retinoblastoma with multiple lipomas histologically consistent with ACL/DL. Analysis of the lipomas supports involvement of RB1 in the development of ACL/DL. Dysplastic changes were only seen in a single lipoma, which harbored an additional TP53 mutation. While providing further support for the occurrence of ACL/DL in retinoblastoma patients, we also suggest that DL is an ACL with TP53 mutation.

Loss of Y-Chromosome during Male Breast Carcinogenesis.

Loss of Y-chromosome (LOY) is associated with increased cancer mortality in males. The prevalence of LOY in male breast cancer (BC) is unknown. The aim of this study is to assess the presence and prognostic effect of LOY during male BC progression. We included male BC patients diagnosed between 1989 and 2009 (n = 796). A tissue microarray (TMA) was constructed to perform immunohistochemistry and fluorescent in situ hybridization (FISH), using an X and Y probe. We also performed this FISH on a selected number of patients using whole tissue slides to study LOY during progression from ductal carcinoma in situ (DCIS) to invasive BC. In total, LOY was present in 12.7% (n = 92) of cases, whereby LOY was associated with ER and PR negative tumors (p = 0.017 and p = 0.01). LOY was not associated with the outcome. Using whole slides including invasive BC and adjacent DCIS (n = 22), we detected a concordant LOY status between both components in 17 patients. In conclusion, LOY is an early event in male breast carcinogenesis, which generally starts at the DCIS stage and is associated with ER and PR negative tumors.

Somatic mutations and copy number variations in breast cancers with heterogeneous HER2 amplification.

Intratumour heterogeneity fuels carcinogenesis and allows circumventing specific targeted therapies. HER2 gene amplification is associated with poor outcome in invasive breast cancer. Heterogeneous HER2 amplification has been described in 5-41% of breast cancers. Here, we investigated the genetic differences between HER2-positive and HER2-negative admixed breast cancer components. We performed an in-depth analysis to explore the potential heterogeneity in the somatic mutational landscape of each individual tumour component. Formalin-fixed, paraffin-embedded breast cancer tissue of ten patients with at least one HER2-negative and at least one HER2-positive component was microdissected. Targeted next-generation sequencing was performed using a customized 53-gene panel. Somatic mutations and copy number variations were analysed. Overall, the tumours showed a heterogeneous distribution of 12 deletions, 9 insertions, 32 missense variants and 7 nonsense variants in 26 different genes, which are (likely) pathogenic. Three splice site alterations were identified. One patient had an EGFR copy number gain restricted to a HER2-negative in situ component, resulting in EGFR protein overexpression. Two patients had FGFR1 copy number gains in at least one tumour component. Two patients had an 8q24 gain in at least one tumour component, resulting in a copy number increase in MYC and PVT1. One patient had a CCND1 copy number gain restricted to a HER2-negative tumour component. No common alternative drivers were identified in the HER2-negative tumour components. This series of 10 breast cancers with heterogeneous HER2 gene amplification illustrates that HER2 positivity is not an unconditional prerequisite for the maintenance of tumour growth. Many other molecular aberrations are likely to act as alternative or collaborative drivers. This study demonstrates that breast carcinogenesis is a dynamically evolving process characterized by a versatile somatic mutational profile, of which some genetic aberrations will be crucial for cancer progression, and others will be mere 'passenger' molecular anomalies.

PIK3CA mutations in ductal carcinoma in situ and adjacent invasive breast cancer.

PIK3CA is one of the most frequently mutated genes in invasive breast cancer (IBC). These mutations are generally associated with hyper-activation of the phosphatidylinositol 3-kinase signaling pathway, which involves increased phosphorylation of AKT (p-AKT). This pathway is negatively regulated by the tumor suppressor PTEN. Data are limited regarding the variant allele frequency (VAF) of PIK3CA, PTEN and p-AKT expression during various stages of breast carcinogenesis. Therefore, the aim of this study was to gain insight into PIK3CA VAF and associated PTEN and p-AKT expression during the progression from ductal carcinoma in situ (DCIS) to IBC. We isolated DNA from DCIS tissue, synchronous IBC and metastasis when present. These samples were pre-screened for PIK3CA hotspot mutations using the SNaPshot assay and, if positive, validated and quantified by digital PCR. PTEN and p-AKT expression was evaluated by immunohistochemistry using the Histo-score (H-score). Differences in PIK3CA VAF, PTEN and p-AKT H-scores between DCIS and IBC were analyzed. PIK3CA mutations were detected in 17 out of 73 DCIS samples, 16 out of 73 IBC samples and 3 out of 23 lymph node metastasis. We detected a significantly higher VAF of PIK3CA in the DCIS component compared to the adjacent IBC component (P = 0.007). The expression of PTEN was significantly higher in DCIS compared to the IBC component in cases with a wild-type (WT) PIK3CA status (P = 0.007), while it remained similar in both components when PIK3CA was mutated. There was no difference in p-AKT expression between DCIS and the IBC component. In conclusion, our data suggest that PIK3CA mutations could be essential specifically in early stages of breast carcinogenesis. In addition, these mutations do not co-occur with PTEN expression during DCIS progression to IBC in the majority of patients. These results may contribute to further unraveling the process of breast carcinogenesis, and this could aid in the development of patient-specific treatment.

Direct Ex Vivo Observation of Homologous Recombination Defect Reversal After DNA-Damaging Chemotherapy in Patients With Metastatic Breast Cancer.

PURPOSE: Biomarkers that predict response to poly (ADP-ribose) polymerase inhibitors (PARPis) are required to detect PARPi sensitivity beyond germline BRCA-mutated (gBRCAm) cancers and PARPi resistance among reverted gBRCAm cancers. Therefore, we previously developed the Repair Capacity (RECAP) test, a functional homologous recombination (HR) assay that exploits the formation of RAD51 foci in proliferating cells after ex vivo irradiation of fresh primary breast cancer tissue. The aim of the current study was to validate the feasibility of this test on histologic biopsy specimens from metastatic breast cancer and to explore the utility of the RECAP test as a predictive tool for treatment with DNA-damaging agents, such as PARPis. METHODS: Fresh tissue biopsies from easily accessible metastatic lesions from patients with locally advanced or metastatic breast cancer were irradiated with 5 Gy and cultured for 2 hours followed by detection of RAD51 foci presence (HR proficient) or absence (HR deficient [HRD]). HRD biopsy specimens as well as platinum/PARP-resistant specimens were subjected to BRCA1/2 sequencing. RESULTS: RECAP had a success rate of 93% on biopsy specimens from metastatic breast cancer lesions (n = 44). Although HRD was detected in 13 (32%) of 41 specimens, only five showed a gBRCAm. In three patients with gBRCAm, post-treatment RECAP tests showed HR phenotype reversion after in vivo progressive disease on platinum and PARPi treatment, which was explained in one patient by a secondary BRCA1 mutation. CONCLUSION: The RECAP test, which reflects real-time HR status regardless of BRCA mutations, is feasible in metastatic breast cancer biopsy specimens. Compared with gBRCA analysis, it may identify twice as many candidates for PARPi treatment.

Functional Ex Vivo Assay Reveals Homologous Recombination Deficiency in Breast Cancer Beyond BRCA Gene Defects.

PURPOSE: Tumors of germline BRCA1/2 mutated carriers show homologous recombination (HR) deficiency (HRD), resulting in impaired DNA double-strand break (DSB) repair and high sensitivity to PARP inhibitors. Although this therapy is expected to be effective beyond germline BRCA1/2 mutated carriers, a robust validated test to detect HRD tumors is lacking. In this study, we therefore evaluated a functional HR assay exploiting the formation of RAD51 foci in proliferating cells after ex vivo irradiation of fresh breast cancer tissue: the recombination REpair CAPacity (RECAP) test. EXPERIMENTAL DESIGN: Fresh samples of 170 primary breast cancer were analyzed using the RECAP test. The molecular explanation for the HRD phenotype was investigated by exploring BRCA deficiencies, mutational signatures, tumor-infiltrating lymphocytes (TIL), and microsatellite instability (MSI). RESULTS: RECAP was completed successfully in 148 of 170 samples (87%). Twenty-four tumors showed HRD (16%), whereas six tumors were HR intermediate (HRi; 4%). HRD was explained by BRCA deficiencies (mutations, promoter hypermethylation, deletions) in 16 cases, whereas seven HRD tumors were non-BRCA related. HRD tumors showed an increased incidence of high TIL counts (P = 0.023) compared with HR proficient (HRP) tumors and MSI was more frequently observed in the HRD group (2/20, 10%) than expected in breast cancer (1%; P = 0.017). CONCLUSIONS: RECAP is a robust functional HR assay detecting both BRCA1/2-deficient and BRCA1/2-proficient HRD tumors. Functional assessment of HR in a pseudo-diagnostic setting is achievable and produces robust and interpretable results.

Guanylin and uroguanylin are produced by mouse intestinal epithelial cells of columnar and secretory lineage.

Guanylin (GN) and uroguanylin (UGN), through activation of guanylyl cyclase C (GCC), serve to control intestinal fluid homeostasis. Both peptides are produced in the intestinal epithelium, but their cellular origin has not been fully charted. Using quantitative PCR and an improved in situ hybridization technique (RNAscope), we have assessed the expression of GN (Guca2a), UGN (Guca2b), and GCC (Gucy2c) in mouse intestine. In the crypts of Lieberkühn, expression of Guca2a and Guca2b was restricted to cells of secretory lineage, at the crypt's base, and to a region above, previously identified as a common origin of cellular differentiation. In this compartment, comparatively uniform levels of Guca2a and Guca2b expression were observed throughout the length of the gut. In contrast, Guca2a and Guca2b expression in the villus-surface region was more variable, and reflected the distinct, but overlapping expression pattern observed previously. Accordingly, in jejunum and ileum, Guca2a and Guca2b were abundantly expressed by enterocytes, whereas in colon only Guca2a transcript was found in the surface region. In duodenum, only low levels of Guca2b transcript were observed in columnar cells, and Guca2a expression was restricted entirely to cells of the secretory lineage. Gucy2c was shown to be expressed relatively uniformly along the rostrocaudal and crypt-villus axes and was also found in the duodenal glands. Our study reveals novel aspects of the cellular localization of the GCC signaling axis that, apart from its role in the regulation of fluid balance, link it to pH regulation, cell cycle control, and host defense.

MET expression during prostate cancer progression.

Tyrosine-kinase inhibitors of the hepatocyte growth factor receptor MET are under investigation for the treatment of hormone-refractory prostate cancer (HRPC) metastasis. Analysis of MET protein expression and genetic alterations might contribute to therapeutic stratification of prostate cancer patients. Our objective was to investigate MET on protein, DNA and RNA level in clinical prostate cancer at various stages of progression. Expression of MET was analyzed in hormone-naive primary prostate cancers (N=481), lymph node (N=40) and bone (N=8) metastases, as well as HRPC (N=54) and bone metastases (N=15). MET protein expression was analyzed by immunohistochemistry (D1C2 C-terminal antibody). MET mRNA levels and MET DNA copy numbers were determined by in situ hybridization. None of the hormone-naive primary prostate cancer or lymph node metastases demonstrated MET protein or mRNA expression. In contrast, MET protein was expressed in 12/52 (23%) evaluable HRPC resections. RNA in situ demonstrated cytoplasmic signals in 14/54 (26%) of the HRPC patients, and was associated with MET protein expression (p=0.025, χ2), in absence of MET amplification or polysomy. MET protein expression was present in 7/8 (88%) hormone-naive and 10/15 (67%) HRPC bone metastases, without association of HRPC (p=0.37; χ2), with MET polysomy in 8/13 (61%) evaluable cases. In conclusion, MET was almost exclusively expressed in HRPC and prostate cancer bone metastasis, but was not related to MET amplification or polysomy. Evaluation of MET status could be relevant for therapeutic stratification of late stage prostate cancer.

Somatic aberrations of mismatch repair genes as a cause of microsatellite-unstable cancers.

Lynch syndrome (LS) is caused by germline mutations in mismatch repair (MMR) genes, resulting in microsatellite-unstable tumours. Approximately 35% of suspected LS (sLS) patients test negative for germline MMR gene mutations, hampering conclusive LS diagnosis. The aim of this study was to investigate somatic MMR gene aberrations in microsatellite-unstable colorectal and endometrial cancers of sLS patients negative for germline MMR gene mutations. Suspected LS cases were selected from a retrospective Clinical Genetics Department diagnostic cohort and from a prospective multicentre population-based study on LS in The Netherlands. In total, microsatellite-unstable tumours of 40 sLS patients (male/female 20/20, median age 57 years) were screened for somatic MMR gene mutations by next-generation sequencing. In addition, loss of heterozygosity (LOH) of the affected MMR genes in these tumours as well as in 68 LS-associated tumours and 27 microsatellite-unstable tumours with MLH1 promoter hypermethylation was studied. Of the sLS cases, 5/40 (13%) tumours had two pathogenic somatic mutations and 16/40 (40%) tumours had a (likely) pathogenic mutation and LOH. Overall, LOH of the affected MMR gene locus was observed in 24/39 (62%) tumours with informative LOH markers. Of the LS cases and the tumours with MLH1 promoter hypermethylation, 39/61 (64%) and 2/21 (10%) tumours, respectively, demonstrated LOH. Half of microsatellite-unstable tumours of sLS patients without germline MMR gene mutations had two (likely) deleterious somatic MMR gene aberrations, indicating their sporadic origin. Therefore, we advocate adding somatic mutation and LOH analysis of the MMR genes to the molecular diagnostic workflow of LS.

An immunohistochemical procedure to detect patients with paraganglioma and phaeochromocytoma with germline SDHB, SDHC, or SDHD gene mutations: a retrospective and prospective analysis.

BACKGROUND: Phaeochromocytomas and paragangliomas are neuro-endocrine tumours that occur sporadically and in several hereditary tumour syndromes, including the phaeochromocytoma-paraganglioma syndrome. This syndrome is caused by germline mutations in succinate dehydrogenase B (SDHB), C (SDHC), or D (SDHD) genes. Clinically, the phaeochromocytoma-paraganglioma syndrome is often unrecognised, although 10-30% of apparently sporadic phaeochromocytomas and paragangliomas harbour germline SDH-gene mutations. Despite these figures, the screening of phaeochromocytomas and paragangliomas for mutations in the SDH genes to detect phaeochromocytoma-paraganglioma syndrome is rarely done because of time and financial constraints. We investigated whether SDHB immunohistochemistry could effectively discriminate between SDH-related and non-SDH-related phaeochromocytomas and paragangliomas in large retrospective and prospective tumour series. METHODS: Immunohistochemistry for SDHB was done on 220 tumours. Two retrospective series of 175 phaeochromocytomas and paragangliomas with known germline mutation status for phaeochromocytoma-susceptibility or paraganglioma-susceptibility genes were investigated. Additionally, a prospective series of 45 phaeochromocytomas and paragangliomas was investigated for SDHB immunostaining followed by SDHB, SDHC, and SDHD mutation testing. FINDINGS: SDHB protein expression was absent in all 102 phaeochromocytomas and paragangliomas with an SDHB, SDHC, or SDHD mutation, but was present in all 65 paraganglionic tumours related to multiple endocrine neoplasia type 2, von Hippel-Lindau disease, and neurofibromatosis type 1. 47 (89%) of the 53 phaeochromocytomas and paragangliomas with no syndromic germline mutation showed SDHB expression. The sensitivity and specificity of the SDHB immunohistochemistry to detect the presence of an SDH mutation in the prospective series were 100% (95% CI 87-100) and 84% (60-97), respectively. INTERPRETATION: Phaeochromocytoma-paraganglioma syndrome can be diagnosed reliably by an immunohistochemical procedure. SDHB, SDHC, and SDHD germline mutation testing is indicated only in patients with SDHB-negative tumours. SDHB immunohistochemistry on phaeochromocytomas and paragangliomas could improve the diagnosis of phaeochromocytoma-paraganglioma syndrome. FUNDING: The Netherlands Organisation for Scientific Research, Dutch Cancer Society, Vanderes Foundation, Association pour la Recherche contre le Cancer, Institut National de la Santé et de la Recherche Médicale, and a PHRC grant COMETE 3 for the COMETE network.

Identification of a 7.1-mega base pairs minimal deletion at 14q31.1-32.11 in adenocarcinomas of the gastroesophageal junction.

In a recent evaluation by comparative genomic hybridization, we demonstrated chromosome 14q31-32.1 to be frequently deleted in adenocarcinomas of the gastroesophageal junction. This suggests the presence of a tumor suppressor gene in the deleted region. In the present study, we have performed a detailed loss of heterozygosity analysis in 34 gastroesophageal junction adenocarcinomas and 1 tumor-corresponding dysplastic Barrett's epithelium sample with 37 polymorphic microsatellite markers. Thirty-five markers are in the 14q24.3-32.33 region with a mean distance of 800 kilo base pairs. Of 34 tumor samples, 14 (41%) showed loss of 14q markers. We identified a minimal region of allelic loss of 7105440 base pairs between markers D14S1000 and D14S256 at cytogenetic location 14q31.1-32.11. Within this region, markers D14S1035, D14S55, D14S1037, D14S1022, D14S1052, D14S974, D14S73, D14S1033, D14S67, D14S68, and D14S1058 showed loss in all informative tumors with 14q loss. The region between markers D14S1000 and D14S256 contains 7 known genes. The identification of this minimal deletion and the data base information on the genes present in this region facilitate the search for the candidate tumor suppressor gene(s).

p53 alterations and their relationship to SDHD mutations in parasympathetic paragangliomas.

Experimental and observational evidence suggests that chronic hypoxic stimulation can induce parasympathetic paraganglioma. This is emphasized by the identification of germline mutations in genes of the mitochondrial succinate dehydrogenase enzyme complex II in hereditary paraganglioma. Because of inactivating mutations in the succinate dehydrogenase subunit B (SDHB), C (SDHC), or D (SDHD) gene, the paraganglia undergo a chronic hypoxic stimulus leading to proliferation of the paraganglionic cells. Hypoxia is a known inducer of p53 up-regulation, which triggers cell cycle arrest and apoptosis. Inactivation of the p53 pathway, by gene mutation or by MDM2 overexpression, would enable cells to escape from cell cycle arrest and apoptosis and could contribute to tumorigenesis. To determine whether p53 inactivation plays a role in paraganglioma tumorigenesis, we investigated a series of 43 paragangliomas from 41 patients (of whom 24 patients harbored a germline SDHD mutation) for mutations in p53 exons 5-8 by PCR-SSCP. In addition, these tumors were investigated for p53 and MDM2 protein expression by immunohistochemistry, and the results were compared with clinical data and the presence of SDHD mutations. No aberrations in p53 exons 5-8 were found. The immunohistochemical experiments showed nuclear p53 expression in 15 tumors. Three tumors were positive for MDM2 that were also positive for p53. There was no correlation between p53 and MDM2 expression and clinical data or SDHD status. Given the fact that hypoxia induces p53 expression and regarding the absence of p53 mutations, these results suggest that p53 inactivation does not play a major role in the tumorigenesis of hereditary and sporadic paragangliomas.

Clinical importance of molecular determinations in gynecologic patients with multiple tumors.

BACKGROUND: The prognosis and treatment of patients with multiple tumors may depend on the correlation between tumors: multiple primary tumors, or recurrent tumors, and metastatic disease. The authors investigated whether the detection of molecular aberrations in multiple gynecologic tumors in individual patients provided clinically useful information on the correlation between the tumors. METHODS: Between 1999 and 2001, molecular analyses were performed on tissue from 15 gynecologic patients, all with multiple tumors. The molecular analyses included loss of heterozygosity determinations at eight DNA loci and mutation analyses of p53 exons 5-8 using the single-strand conformation polymorphism method. Previously, it was not possible to use routine diagnostic histopathology to determine accurately the correlation between multiple lesions in patients with gynecologic malignancies, information that may have an impact on clinical decision-making and prognosis. RESULTS: Molecular results were obtained from all tumors from each of the 15 patients. The DNA alterations detected provided evidence that two patients had second primary tumors, nine patients had a single tumor with metastases, and four patients had two independent primary tumors as well as metastatic disease. The results provided additional diagnostic information and contributed to clinical decision-making. CONCLUSIONS: The authors demonstrated that, by comparing DNA alterations in multiple tumors within an individual patient, evidence about correlations between the tumors can be obtained. These investigations can be performed on routinely processed tissues, and the results may be of clinical importance in helping to determine the management or prognosis of patients with gynecologic malignancies.

Determination of the molecular relationship between multiple tumors within one patient is of clinical importance.

PURPOSE: To determine the molecular relationship between multiple tumors within one patient and to evaluate the impact of this knowledge on clinical management. PATIENTS AND METHODS: In 25 consecutive patients with multiple tumors, proven by histology and immunohistochemistry to be identical, molecular aberrations were determined. Each patient had at least one lesion in the lung or head and neck region. Loss of heterozygosity (LOH) and p53 aberration analyses were carried out, and similar aberration profiles suggest clonality and metastasis whereas different profiles suggest independent primary tumors. RESULTS: The molecular determinations indicated that 12 patients had a probable second primary tumor and 10 patients had a metastasis of the first lesion. In three patients, both an independent primary tumor and a metastasis were present. The molecular findings determined the course of additional treatment in all 10 patients with metastases, in all three patients with both a second primary tumor and a metastasis, and in seven of 12 patients with a second primary tumor. CONCLUSION: By comparing DNA alterations of multiple tumors within one patient, the relationship between the tumors can be assessed. This study shows that in 20 of 25 patients, knowledge of the nature of both lesions was essential in clinical decision making. Furthermore, after thorough analysis of the five cases where clinical decision making was not influenced, there was in retrospect no clear indication for LOH or p53 analysis. Because these molecular analyses can be performed on routine specimens, they can be applied in almost all patients.