Overexpression and gene amplification of both ERBB2 and EGFR in an esophageal squamous cell carcinoma revealed by fluorescence in situ hybridization,multiplex ligation‐dependent probe amplification and immunohistochemistry

EGFR and ERBB2 belong to the EGFR gene family. In esophageal squamous cell carcinomas (SCCs), amplification of EGFR or ERBB2 is usually mutually exclusive. EGFR amplification occurs in approximately 15% of SCCs, ERBB2 occurs in less than 5%. Here, we report the co‐amplification of EGFR and ERBB2 in an ulcerative and infiltrating‐type SCC that measured approximately 4.2 × 2.7 × 1.2 cm with a superficial lesion occurring in the thoracic esophagus of a 72‐year‐old man. Multiplex ligation‐dependent probe amplification using representative tumor sections showed gain of CCND1 and coincident amplification of ERBB2 or EGFR or neither. Immunohistochemistry and fluorescence in situ hybridization revealed that the tumor comprised three cancer‐cell populations: well‐differentiated SCC with high‐level ERBB2 amplification and ERBB2 overexpression, more infiltrative poorly‐differentiated SCC with high‐level EGFR amplification and EGFR overexpression, and poorly‐differentiated SCC lacking any ERBB2 or EGFR abnormality. These three populations each had low‐level CCND1 amplification and nuclear cyclin D1 overexpression. This histological topology and gene amplification combinations suggested that genetic instability first produced CCND1 amplification, and then ERBB2 or EGFR gene amplification occurred. It is further speculated that during cancer progression and clonal selection indecisive predominance of either clone caused the rare co‐amplification of ERBB2 and EGFR in a single chimeric tumor.     

Array-based comparative genomic hybridization of ductal carcinoma in situ and synchronous invasive lobular cancer

It has been increasingly recognized that ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS) and invasive cancer of the breast are often closely associated with one another. However, the genomic relationship between these histologically distinct entities has not been well characterized. Refinements in high-resolution comparative genomic hybridization (CGH) techniques allow for a detailed comparison of genomic alterations in synchronously occurring tumors. The following case illustrates how array CGH may be used to better understand whether synchronous neoplasms share a common origin.     

Lobular cancerization: incidence and differential diagnosis with lobular carcinoma in situ of breast

A series of 120 breast biopsies and mastectomy specimens originally diagnosed as carcinoma was reviewed in order to emphasize the differences between lobular carcinoma in situ and cancerization of lobules. Criteria for differential diagnosis between the two types of lobular lesion are proposed. In the reviewed material 15 (12.5%) cases of lobular carcinoma in situ and 64 (53.3%) of lobular cancerization were found. The carcinoma in situ usually co-existed with other types of breast carcinoma, such as intraductal carcinoma, cancerization and invasive carcinoma of different types.     

First‐trimester spontaneous pregnancy loss – molecular analysis using multiplex ligation‐dependent probe amplification

Spontaneous miscarriages are the most frequent complications of pregnancy and, in at least half of cases, are caused by chromosomal abnormalities, mainly aneuploidies. We present the preliminary results of the implementation of multiplex ligation‐dependent probe amplification (MLPA) in the detection of chromosomal aberrations in the tissue derived from first‐trimester miscarriage and evaluate the limitations and requirements of the method. We studied 181 MLPA analyses with subtelomeric and subcentromeric probe kits for all chromosomes (SALSA P070 and SALSA P181) performed on the first‐trimester spontaneous miscarriage products in our Department of Genetics between September 2012 and December 2014. Conclusive MLPA results were obtained in 97.2% of samples. Chromosomal aberrations were detected in 40.3% of samples: 61.8% samples of good quality and 12.6% samples of poor quality (p < 0.001). The normal female karyotype was detected in 14.7% of good quality samples and 84.8% of poor quality samples (p < 0.001). MLPA is a useful tool for the detection of chromosomal aberrations in first‐trimester miscarriage products. However, the tissue has to be well prepared before testing and the results 46,XX should be interpreted with caution.     

Histopathological and clonal study of combined lobular and ductal carcinoma of the breast

Lobular carcinoma in situ (LCIS) clinically constitutes a risk factor for the subsequent development of either invasive lobular carcinoma (ILC) or invasive ductal carcinoma (IDC). In order to approach the possibility of this common precursor of both ILC and IDC, we investigated combined lobular and ductal carcinomas. Thirty‐two cases of lobular carcinoma were picked up out of 773 cases of operated breast carcinomas. The histopathological detailed re‐examination using immunostain of E‐cadherin and β‐catenin revealed a rather high frequency of combined lobular carcinomas than previous reports. Clinicopathologically, combined lobular carcinomas were younger and smaller than pure lobular carcinomas, and the cytological atypia was relatively low. These results suggested that combined lobular carcinomas could be detected in the earlier stage of breast cancer. Furthermore, the lobular and ductal components of combined carcinomas coexisted in the neighborhood and were distributed contiguously. The immunohistochemical phenotypes of both components were accorded in most combined cases. A genetic analysis using methylation‐specific PCR on the HUMARA gene demonstrated that the same allele was inactivated in both lobular and ductal components in all detectable cases of combined carcinoma. Therefore, it is reasonable to assume that both lobular and ductal components of combined carcinomas are clonal and derived from the LCIS as the common precursor lesion, which may contradict the conventional concept that the lobular and ductal carcinomas arise from distinct differentiation pathways.     

乳腺浸润性导管癌与小叶癌的超声诊断分析

目的探讨乳腺浸润性导管癌和浸润性小叶癌的超声声像图特征。方法对2006年1月至2008年6月经手术、病理证实为乳腺浸润性导管癌（IDC）136例和浸润性小叶癌61例的超声声像图、彩色多普勒表现进行对比分析。结果两种病理类型的肿瘤的超声声像图在肿块形状、内部与后方回声以及血流图上差异有统计学意义（P〈0．05）。在侧方回声与内部钙化情况上差异无统计学意义（P〉0．05）。结论乳腺浸润性导管癌和浸润性小叶癌可以根据各自超声声像的特点进行诊断，组织学的不同影响乳腺癌的超声影像表现。     

Expression of autophagy related proteins in invasive lobular carcinoma: comparison to invasive ductal carcinoma

The aim of this study is to compare the expression of autophagy related proteins in invasive lobular carcinoma (ILC) with that of autophagy related proteins in invasive ductal carcinoma (IDC), and to determinate its implication. Tissue microarray containing 114 ILC and 692 IDC was constructed, and immunohistochemistry was performed for autophagy related protein (beclin-1, LC3A, LC3B, p62) and Ki-67. No significant difference in expression of autophagy-related proteins between pleomorphic type (n = 12) and classic type (n = 102) of ILC was observed, whereas ILC and IDC showed distinguished features that tumoral beclin-1, stromal LC3A, tumoral LC3B, tumoral p62 were highly expressed in IDC and tumoral BNIP3 was highly expressed in ILC (P < 0.001). Beclin-1 expression was correlated with ER negativity (P = 0.016) and TNBC type (P = 0.024). BNIP3 expression was correlated with ER positivity (p = 0.040). Using multivariate Cox analysis, shorter overall survival was associated with tumoral beclin-1 positivity (hazard ratio: 21.19, 95% CI: 1.098-409.1, P = 0.043). In conclusion, ILC and IDC showed different expression pattern of autophagy-related proteins in tumor and stroma that demonstrated by higher expression of tumoral beclin-1, stromal LC3A, tumoral LC3B, tumoral p62 in IDC, and higher expression of tumoral BNIP3 in ILC.     

Infiltrating lobular carcinoma of the breast

On review of the histology of 1050 primary breast carcinomas, 103 cases of infiltrating lobular carcinoma were identified and clinical and survival data collected in each case. The tumours were then separated into four groups on the basis of histological pattern and these groups shown to have significantly different survival times. Assessment of the presence of in situ carcinoma, elastosis and intracyto-plasmic lumina was performed in each case and the effect of these features on survival investigated. In addition to stage of disease at presentation, the major significant factor in predicting survival of patients with this type of invasive carcinoma is histological type.     

Mixed ductal‐lobular carcinomas: evidence for progression from ductal to lobular morphology

Mixed ductal–lobular carcinomas (MDLs) show both ductal and lobular morphology, and constitute an archetypal example of intratumoural morphological heterogeneity. The mechanisms underlying the coexistence of these different morphological entities are poorly understood, although theories include that these components either represent ‘collision’ of independent tumours or evolve from a common ancestor. We performed comprehensive clinicopathological analysis of a cohort of 82 MDLs, and found that: (1) MDLs more frequently coexist with ductal carcinoma in situ (DCIS) than with lobular carcinoma in situ (LCIS); (2) the E‐cadherin–catenin complex was normal in the ductal component in 77.6% of tumours; and (3) in the lobular component, E‐cadherin was almost always aberrantly located in the cytoplasm, in contrast to invasive lobular carcinoma (ILC), where E‐cadherin is typically absent. Comparative genomic hybridization and multiregion whole exome sequencing of four representative cases revealed that all morphologically distinct components within an individual case were clonally related. The mutations identified varied between cases; those associated with a common clonal ancestry included BRCA2, TBX3, and TP53, whereas those associated with clonal divergence included CDH1 and ESR1. Together, these data support a model in which separate morphological components of MDLs arise from a common ancestor, and lobular morphology can arise via a ductal pathway of tumour progression. In MDLs that present with LCIS and DCIS, the clonal divergence probably occurs early, and is frequently associated with complete loss of E‐cadherin expression, as in ILC, whereas, in the majority of MDLs, which present with DCIS but not LCIS, direct clonal divergence from the ductal to the lobular phenotype occurs late in tumour evolution, and is associated with aberrant expression of E‐cadherin. The mechanisms driving the phenotypic change may involve E‐cadherin–catenin complex deregulation, but are yet to be fully elucidated, as there is significant intertumoural heterogeneity, and each case may have a unique molecular mechanism. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Molecular profiling pleomorphic lobular carcinomas of the breast: evidence for a common molecular genetic pathway with classic lobular carcinomas

Pleomorphic lobular carcinomas (PLC) of the breast display histological features associated with classic invasive lobular carcinoma (ILC), yet they also exhibit more conspicuous nuclear atypia and pleomorphism, and an aggressive clinical behaviour. From a breast cancer progression perspective, it is unclear whether PLC is a variant of ILC or is a high-grade invasive ductal carcinoma (IDC) that has lost E-cadherin. The molecular features of 26 PLC were studied using immunohistochemistry [oestrogen receptor (ER), progesterone receptor (PR), HER2, p53 and E-cadherin], 0.9 Mb resolution, microarray-based comparative genomic hybridization (aCGH), fluorescent (FISH) and chromogenic (CISH) in situ hybridization and loss of heterozygosity. Comparative analysis was performed with aCGH data from PLC with classic ILC (16 cases) and high grade IDC (35 cases). PLCs were frequently ER- and PR-positive, E-cadherin-negative and occasionally HER2- and p53-positive. Recurrent copy number changes identified by aCGH included gains on 1q, 8q, 11q, 12q, 16p and 17q and losses on 8p, 11q, 13q, 16q and Xq. Highly recurrent 1q+ (100% of cases), 16p+ (93%), 11q- (53%) and 16q- (93%) and evidence of the der(1;16)/der(16)t(1;16) rearrangement, as detected by FISH, suggested that PLC had a 'lobular genotype'. Focal amplifications were evident at 8p12-p11, 8q24, 11q13.1-q14.1, 12q14, 17q12 and 20q13. Loss of BRCA2 was detected in 40% of PLC by LOH. Comparative analysis of aCGH data suggested the molecular features of PLC (ER/PR-positive, E-cadherin-negative, 1q+, 11q(-), 16p+ and 16q(-)) were more closely related to those of ILC than IDC, implicating an overlapping developmental pathway for these lobular tumour types. Molecular alterations found in PLC that are more typical of high-grade IDC than ILC (p53 and HER2 positivity, 8q+, 17q24-q25+, 13q(-) and amplification of 8q24, 12q14, 17q12 and 20q13) are likely to drive the high-grade and more aggressive biology of PLC.     

Multiplex ligation‐dependent probe amplification and fluorescence in situ hybridization are complementary techniques to detect cytogenetic abnormalities in multiple myeloma

Multiple myeloma (MM) is a genetically heterogeneous disease with diverse clinical outcomes. Interphase fluorescence in situ hybridization (i‐FISH) is the most commonly used approach to detect recurrent cytogenetic abnormalities in this malignancy. We aimed to assess the performance of multiplex ligation‐dependent probe amplification (MLPA) to reveal copy number abnormalities (CNAs) in MM. Diagnostic bone marrow samples from 81 patients were analyzed using 42 MLPA probes for the following regions: 1p32‐31, 1p21, 1q21.3, 1q23.3, 5q31.3, 12p13.31, 13q14, 16q12, 16q23, and 17p13. All samples were also screened by i‐FISH for the presence of hyperdiploidy, deletion/monosomy of chromosome 13, deletion of TP53, disruption of the immunoglobulin heavy‐chain gene, t(4;14), t(11;14), t(14;16), t(8;14), gain of 5q and abnormalities of chromosome 1. A total of 245 alterations were detected in 79 cases (98%). Investigating the same aberrations, the two methods showed a congruency of higher than 90%. A low proportion of cells with the relevant abnormality, focal CNAs and unmatched probes were responsible for the discrepancies. MLPA revealed 95 CNAs not detected by i‐FISH providing additional information in 53 cases (65%). Scrutiny of CNAs on chromosome 1, using more than 20 probes, revealed significant heterogeneity in size and location, and variable intra‐chromosomal and intra‐clonal rates of loss or gain. Our results suggest that MLPA is a reliable high‐throughput technique to detect CNAs in MM. Since balanced aberrations are key to prognostic classification of this disease, MLPA and i‐FISH should be applied as complementary techniques in diagnostic pathology. © 2013 Wiley Periodicals, Inc.     

HER2 status in pure ductal carcinoma in situ and in the intraductal and invasive components of invasive ductal carcinoma determined by fluorescence in situ hybridization and immunohistochemistry

AIM: To determine the HER2 status of ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) of the breast. The increased prevalence of HER2 amplification and overexpression in DCIS is considered to be maintained in the intraductal component of IDC; however, HER2 amplification and overexpression are detected much less in IDC. METHODS AND RESULTS: Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) were performed to detect HER2 in 270 IDCs with an intraductal component and in 50 pure DCIS samples; IHC was also performed in 116 metastatic nodes. HER2 was found to be amplified in 77 cases (28.5%) and overexpressed in 79 (29.3%) of the 270 IDCs. HER2 amplification was similar between intraductal and invasive components of the same tumour. The concordance for HER2 status between invasive and intraductal components of individual tumours was 98.5% and 99.3% by FISH and IHC, respectively. HER2 was amplified in 25 (50%) of the 50 pure DCIS samples. HER2 overexpression in metastatic nodes resembled the HER2 status in the primary tumour for 108 (93.1%) of 116 cases (kappa =0.831). CONCLUSION: Our study indicates that the intraductal component of IDC may differ biologically when compared with pure DCIS. HER2 appears to lack a critical role in the progression from DCIS to IDC and HER2 status is maintained in metastatic lesions.     

Fine-needle aspiration cytology of lobular carcinoma in situ

Fine-needle aspiration cytology (FNAC) plays a key role in the preoperative diagnosis of breast carcinoma but is less reliable in the diagnosis of in situ lesions. The objective of the present study was to investigate the cytological features of lobular carcinoma in situ (LCIS), regarding which little data is available to date. Cytological features of FNAC of the breast from 21 patients with histology-proven LCIS were described and compared with surgical specimens. Aspirates from 8/21 cases had cell groups diagnostic for or compatible with LCIS. Aspirates from an additional two cases demonstrated hypercellular, dissociated, and more pleomorphic tumor cells, which were originally diagnosed as invasive lobular carcinoma (ILC). The remaining 11 aspirates were diagnosed as benign or nondiagnostic. FNAC from the eight diagnostic specimens were characterized by loosely cohesive cell groups composed of uniform cells with occasional intracytoplasmic lumina, slightly irregular and eccentric nuclei. We conclude that the main difficulty in diagnosing LCIS by FNAC is sampling rather than recognition of the lesions. However, one should be aware of the cytological features of LCIS in order to reach a correct diagnosis. There are no reliable cytological criteria that help in differentiating pleomorphic and dissociated LCIS from ILC.     

DNA profiling of primary serous ovarian and fallopian tube carcinomas with array comparative genomic hybridization and multiplex ligation-dependent probe amplification

Primary serous ovarian carcinoma (OVCA) and serous Fallopian tube carcinoma (FTC), both belonging to the BRCA-linked tumour spectrum, share many properties and are treated similarly. However, a detailed molecular comparison has been lacking. We hypothesized that comparative genomic studies of serous OVCAs and FTCs should point to gene regions critically involved in their tumorigenesis. Array comparative genomic hybridization (array CGH) analysis indicated that serous OVCAs and serous FTCs displayed common but also more distinctive patterns of recurrent changes. Targeted gene identification using a dedicated multiplex ligation-dependent probe amplification (MLPA) probe set directly identified EIF2C2 on 8q as a potentially important driver gene. Other previously unappreciated gained/amplified genes included PSMB4 on 1q, MTSS1 on 8q, TEAD4 and TSPAN9 on 12p, and BCAS4 on 20q. SPINT2 and ACTN4 on 19q were predominantly found in FTCs. Gains/amplifications of CCNE1 and MYC, often in conjunction with changes in genes of the AKT pathway, EVI1 and PTK2, seemed to be involved at earlier stages, whereas changes of ERBB2 were associated with advanced stages. The only BRCA1-mutated FTC shared common denominators with the sporadic tumours. In conclusion, the data suggest that serous OVCAs and FTCs, although related, exhibit differences in genomic profiles. In addition to known pathways, new genes/pathways are likely to be involved, with changes in an miRNA-associated gene, EIF2C2, as one important new feature. Dedicated MLPA sets constitute potentially important tools for differential diagnosis and may provide footholds for tailored therapy.     

Recognizing breast ductal carcinoma in situ on fine‐needle aspiration: A diagnostic dilemma

In this study, we evaluated cytomorphologic features of different subgroups of ductal carcinoma in situ (DCIS); we compared seven cytologic features between DCIS and invasive ductal carcinoma (IDC) aspirates to determine whether diagnosis of stromal invasion can be made based on fine‐needle aspiration (FNA) findings. There were 142 cases of DCIS and 1,978 cases of IDC enrolled in our study. FNA analysis revealed 80.3% sensitivity for DCIS and 94.7% sensitivity for IDC. High and intermediate grade DCIS exhibited marked nuclear abnormality (92.1% vs. 35.7%, 30.0%; P1 < 0.001, P2 < 0.001) and necrosis (69.7% vs. 0%, 10.0%; P1 < 0.001, P2 = 0.001) in a higher percentage of cases compared to low grade DCIS and intraductal/intracystic papillary carcinoma. The rates of background macrophages (71.3% for DCIS and 21.9% for IDC, P < 0.001) and extensive necrosis (54.0% for DCIS and 16.7% for IDC, P < 0.001) were significantly higher in DCIS compared to IDC. Lymphocytes were observed in conjunction with tumor cells more frequently in IDC (81.3%) compared to DCIS (36.8%, P < 0.001). Stromal fragments associated with tumor cells were only observed in invasive lesions (11.9% micro‐invasive DCIS and 52.1% IDC). Tubular structures were found exclusively in IDC (11.5%). Cytologic criteria for diagnosis of high and low grade DCIS are different. The suspicion of DCIS is raised when background macrophages and extensive necrosis are observed. Stromal invasion is suggested by FNA if lymphocytes are entwined around tumor cells or if stromal fragments associated with tumor cells or tubular structures are observed. Diagn. Cytopathol. 2013;41:710–715. © 2013 Wiley Periodicals, Inc.     

Screen‐detected pleomorphic lobular carcinoma in situ (PLCIS): risk of concurrent invasive malignancy following a core biopsy diagnosis

Carder PJ, Shaaban A, Alizadeh Y, Kumarasuwamy V, Liston JC & Sharma N
(2010) Histopathology 57, 472–478
Screen‐detected pleomorphic lobular carcinoma in situ (PLCIS): risk of concurrent invasive malignancy following a core biopsy diagnosis Aims: Pleomorphic lobular carcinoma in situ (PLCIS) is an uncommon, recently recognized variant of lobular carcinoma in situ (LCIS). Its natural history, biological behaviour and clinical characteristics are uncertain. The aim was to review the radiological and pathological findings in a series of screen‐detected PLCIS diagnosed on needle core biopsy with a view to determining the diagnostic features, immunohistological profile and risk of concurrent invasive malignancy. Methods and results: Ten cases of core biopsy‐diagnosed, screen‐detected PLCIS were identified. Core biopsy findings were compared with pathological findings at subsequent surgery. Two cases were associated with possible microinvasion on the core. Two of 10 had invasive lobular carcinoma and one had microinvasive lobular carcinoma on subsequent surgical excision (positive predictive value for malignancy = 30%). There was associated conventional LCIS on either core or excision biopsy in all cases except one. All three cases of oestrogen receptor (ER)‐negative PLCIS arose in the context of ER+ conventional LCIS. Conclusions: PLCIS is a potentially more aggressive lesion than conventional LCIS and may present as mammographic calcification through a breast screening programme. Diagnosis may be problematic and immunohistochemical markers including ER may prove a useful diagnostic adjunct. There is a significant risk of concurrent invasive carcinoma following a core biopsy diagnosis.