Tomonori Kawasaki

Clinicopathological and molecular alterations in early gastric cancers with the microsatellite instability‐high phenotype

The relevance of the clinicopathological and molecular features of early gastric cancers (EGCs) having the microsatellite instability (MSI)‐high phenotype has not been clearly defined in sporadic gastric carcinogenesis. Here, we examined the clinicopathological and molecular characteristics of EGC according to MSI status in 330 patients with EGC (intestinal‐type adenocarcinoma). Tumors were classified as MSI‐high (45 cases), MSI‐low (9 cases), or microsatellite stable (MSS; 276 cases). The specimens were examined using a combination of polymerase chain reaction (PCR)‐microsatellite assays and PCR‐pyrosequencing to detect chromosomal allelic imbalances in multiple cancer‐related chromosomal loci, MSI, gene mutations (KRAS and BRAF) and methylation status [high methylation epigenome (HME), intermediate methylation epigenome and low methylation epigenome]. In addition, the expression levels of various target proteins were examined using immunohistochemistry. Interestingly, EGC with the MSI phenotype showed distinct papillary features. The expression of gastric mucin was more frequent in EGC with the MSI phenotype, while p53 overexpression was common in EGCs, irrespective of MSI status. The frequency of HME was significantly higher in EGCs with the MSI phenotype than in EGCs with the MSS phenotype. Although there was a low frequency of allelic imbalance in EGCs with the MSI phenotype, some markers of allelic imbalance were more frequently detected in EGCs with the MSI‐high phenotype than in EGCs with the MSS phenotype. KRAS and BRAF mutations were rare in EGCs. Thus, the MSI phenotype in EGC is a major precursor lesion in gastric cancer and is characterized by distinct clinicopathological and molecular features.

Well-differentiated neuroendocrine tumor of the breast with recurrence due to needle tract seeding

Dear Editor, Recently, percutaneous imaging-guided core needle biopsy (CNB) ensuring a high degree of diagnostic accuracy and minimal invasiveness has been widely practiced as an alternative to surgical biopsy [1]. However, the procedure itself can, very rarely, contribute to disease recurrence [1, 2]. The WHO classifies mammary carcinomas with neuroendocrine (NE) features as a special tumor entity, representing <1 % of invasive breast carcinomas, and recognizes three subtypes: (i) NE tumor (NET), well-differentiated; (ii) NE carcinoma, poorly differentiated; and (iii) invasive carcinoma with NE differentiation [3, 4]. Herein, we describe the first case of a mammary NE cancer (well-differentiated NET) showing intramammary relapse related to needle implantation. A 60-year-old postmenopausal Japanese woman presented with slight skin retraction in the upper inner portion of the right breast. Ultrasonography revealed an irregular, hypoechoic right breast tumor with an echogenic halo and posterior attenuation. Systemic CT detected no other suspicious lesions. We performed ultrasound-guided 16-G automated CNB of the breast mass after obtaining informed consent, and the histological diagnosis was (ductal) carcinoma. The cut surface of the lumpectomy specimen contained a poorly delimited, grey-whitish and focally brownish-red, solid tumor, measuring 12×10 mm in size. Histologically, this tumor was composed of invasive growing carcinoma cells in solid and/or trabecular clusters with a highly vascular fibrovascular stroma and focal hemorrhage (Fig. 1). Carcinoma cells were polygonal or, occasionally, spindle-shaped with finely granular cytoplasm and ovoid nuclei with a finegranular chromatin pattern (Fig. 1a). Six mitotic figures were counted in 10 high-power fields. In situ carcinoma components were locally observed near invasive cancer nests. Neither lymphatic nor vascular infiltration was detected. Lateral, including nipple-side, margins were negative for cancer. On the other hand, we noted small foci of scarring with fat necrosis and hemosiderin-laden macrophages in the subcutaneous tissues away from the main tumor (Fig. 2a) and identified sporadic epithelial clusters showing cancer cell morphologies within and around the scar tissues (Fig. 2b, c). No metastases were identified in the four excised right axillary lymph nodes. T. Kawasaki (*) :A. Sato :M. Suzuki : R. Sugimoto :Y. Mue : N. Uesugi :K. Ishida : T. Sugai Department of Molecular Diagnostic Pathology, Iwate Medical University School of Medicine, 19-1 Uchimaru, Morioka, Iwate 020-8505, Japan e-mail: tomonori@yamanashi.ac.jp

Well-differentiated neuroendocrine tumour of the breast showing peculiar endovascular spread.

Sir: Spread of an intravascular tumour embolus from a primary lesion can be found in some cancers: intraportal spread of hepatocellular carcinoma, postcaval spread of renal cell carcinoma, and so on. However, to the best of our knowledge, such a finding in breast cancers has not previously been reported in the English literature. The WHO classifies mammary carcinomas with neuroendocrine (NE) features as a special tumour entity, representing <1% of invasive breast carcinomas, and recognises three subtypes: (i) NE tumor (NET), well differentiated; (ii) NE carcinoma, poorly differentiated; and (iii) invasive carcinoma with NE differentiation. Herein, we describe the first case of a well-differentiated mammary NET with extensive intravenous spread. The patient, a 42-year-old premenopausal Thai woman, presented with a palpable mass in the subareolar portion of the left breast. Ultrasonography revealed a sharply marginated, hypoechoic left breast tumour showing heterogeneous internal echoes, with a cranially extending lumen-like structure filled with solid tumour with similar echoic findings (Figure 1A). On MRI, these lesions appeared as an oval-shaped mass and a continuous intravenous occupying lesion, respectively, both of which were strongly enhanced during the early phases of the dynamic study (Figure 1B). These images suggested an invasive cancer with a tumour thrombus. Systemic CT and bone scintigraphy detected no other suspicious lesions. Ultrasound-guided, fine needle aspiration of the breast mass yielded a cytological diagnosis of carcinoma. The cut surface of the lumpectomy specimen contained well-circumscribed, mixed brownish-red and grey-whitish, solid tumour nodules, measuring up to 17 9 15 mm in size. Histopathologically, the tumour was composed of a solid invasive growth of carcinoma cells with a peripheral palisading arrangement and a highly vascular stroma (Figure 2A,B). Haemorrhage was marked in the lesions. Carcinoma cells were polygonal or, occasionally, spindle-shaped with finely granular, slightly eosinophilic cytoplasm (Figure 2B). Their nuclei had ovoid or irregular shapes, a finely granular chromatin pattern, and small nucleoli. Mitotic figures were seen in seven of 10 high-power fields. An in-situ component composed of carcinoma cells with the same histological features was locally observed near the invasive cancer nests (Figure 2C). Massive tumour embolization within prominently dilated veins, spreading from the primary focus, was confirmed by the elastic Van Gieson method (Figure 2A,D). Focal lymphatic permeation

Small-cell carcinoma of the breast with squamous differentiation.

Sir: The WHO classifies mammary carcinomas with neuroendocrine (NE) features as a special tumour entity representing <1% of invasive breast carcinomas. Smallcell carcinoma of the breast is a rare NE subtype that may show aggressive clinical behaviour, whereas the other, more frequent, breast carcinomas with NE differentiation (cellular mucinous carcinoma and solid papillary carcinoma) are usually of low grade. To our knowledge, there is only one brief description concerning metaplastic change in these NE cancers. Herein, we report an exceptionally rare small-cell mammary carcinoma showing squamous differentiation. The patient, a 58-year-old postmenopausal Japanese woman, presented with a palpable mass in the upper outer quadrant of the right breast. Her family history included a sister with ovarian cancer. Ultrasonography revealed a well-defined, focally distorted, hypo-echoic right breast tumour. Systemic CT detected no other suspicious lesions. We performed ultrasound-guided, fine needle aspiration of the breast lesion, and the cytological diagnosis was carcinoma. The cut surface of the mastectomy specimen contained a lobulated grey–whitish tumour, measuring 45 9 40 9 40 mm. Histopathologically, this invasive tumour was composed of solid and/or trabecular growths of densely packed, small to medium-sized carcinoma cells with well-developed vascular stroma (Figure 1A,B). Focal coagulation necrosis and haemorrhage were present. Carcinoma cells were polygonal or occasionally spindle-shaped, with high nuclear/cytoplasmic ratios and ovoid nuclei with finely granular chromatin and absent or inconspicuous nucleoli (Figure 1B). Mitotic figures were numerous [82 per 10 high-power fields (HPFs)]. Squamous differentiation, i.e. formation of nests of polygonal carcinoma cells with abundant, eosinophilic cytoplasm and intercellular bridging with focal keratin pearls, was found (Figure 1C). Mitotic activity was also present in this squamous component, but was lower (12 per 10 HPFs) than that in the small-cell component. Focal lymphatic permeation was detected. An in-situ ductal carcinoma component, predominantly showing cribriform architecture, and with comedo necrosis, was observed within and near the tumour mass (Figure 1A,D). No metastases were identified in the 18 excised right axillary lymph nodes. Immunohistochemically, invasive carcinoma cells were diffusely positive for synaptophysin and CD56 (neural cell adhesion molecule, NCAM) and focally positive for chromogranin A, whereas cells showing squamous metaplasia were negative or weakly positive and in-situ cells were negative for these NE markers (Figure 2A). Carcinoma cells were diffusely positive for cytokeratin 7 and negative for cytokeratin 20. High molecular weight cytokeratin (34bE12) was clearly demonstrated only in carcinoma cells with squamous metaplasia (Figure 2B). Gross cystic disease fluid protein 15 (GCDFP15) was identified in 2% of the invasive carcinoma cells and in 20% of the in-situ carcinoma cells (Figure 2C). The tumour was oestrogen receptor-negative in both invasive and in-situ areas, whereas weak progesterone receptor reactivity was detected in the invasive carcinoma nuclei (positive cell rate: 1.5%), and the in-situ component was progesterone receptor negative. The HER2 score was estimated at 1+, and the MIB-1 labelling index was 51.4%. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis revealed overexpression of chromogranin A mRNA in the invasive cancer tissue. Postoperatively, the patient received doxorubicin (60 mg/m) and cyclophosphamide (600 mg/m) every 4 weeks for four cycles as adjuvant chemotherapy. She remains alive and well, with neither recurrence nor metastasis, 49 months after surgery. The possibility of metastatic small-cell carcinoma from another site should be ruled out. Imaging and clinical history confirmed that our patient had no lesions in other organs, and an in-situ component accompanying the invasive breast cancer was demonstrated histologically. These are regarded as the two most important features for diagnosing primary small-cell mammary carcinoma. In addition, GCDFP15 immunoexpression supports a diagnosis of primary NE carcinoma of the breast, despite oestrogen receptor negativity. Furthermore, mammary NE carcinomas are generally cytokeratin 7-positive and cytokeratin 20-negative, whereas pulmonary smallcell carcinoma is negative for both. It has been suggested that mammary NE carcinomas generally result from a divergent differentiation

Compound heterozygous TYK2 mutations underlie primary immunodeficiency with T-cell lymphopenia

Complete tyrosine kinase 2 (TYK2) deficiency has been previously described in patients with primary immunodeficiency diseases. The patients were infected with various pathogens, including mycobacteria and/or viruses, and one of the patients developed hyper-IgE syndrome. A detailed immunological investigation of these patients revealed impaired responses to type I IFN, IL-10, IL-12 and IL-23, which are associated with increased susceptibility to mycobacterial and/or viral infections. Herein, we report a recessive partial TYK2 deficiency in two siblings who presented with T-cell lymphopenia characterized by low naïve CD4+ T-cell counts and who developed Epstein-Barr virus (EBV)-associated B-cell lymphoma. Targeted exome-sequencing of the siblings’ genomes demonstrated that both patients carried novel compound heterozygous mutations (c.209_212delGCTT/c.691C > T, p.Cys70Serfs*21/p.Arg231Trp) in the TYK2. The TYK2 protein levels were reduced by 35% in the T cells of the patient. Unlike the response under complete TYK2 deficiency, the patient’s T cells responded normally to type I IFN, IL-6, IL-10 and IL-12, whereas the cells displayed an impaired response to IL-23. Furthermore, the level of STAT1 was low in the cells of the patient. These studies reveal a new clinical entity of a primary immunodeficiency with T-cell lymphopenia that is associated with compound heterozygous TYK2 mutations in the patients.

Imaging with ultra-small-angle X-ray scattering using a Laue-case analyzer and its application to human breast tumors

PURPOSE In this study, we demonstrate a novel imaging technique, based on ultra-small-angle X-ray scattering (USAXS) that uses a Laue-case Si wafer as the angle analyzer. METHODS We utilized the (1 1 1) diffraction plane of a 356 μm thick, symmetrically cut Si wafer as the angle analyzer, denoted by A[L]. With this device, we performed USAXS imaging experiments using 19.8 keV synchrotron X-rays. The objects we imaged were formalin-fixed, paraffin-embedded breast tumors (an invasive carcinoma and an intraductal papilloma). During image acquisition by a charge-coupled device (CCD) camera, we varied the rotation angle of the analyzer in 0.02″ steps from -2.40″ to +2.40″ around the Bragg angle. The exposure time for each image was 2 s. We determined the amount of ultra-small-angle X-ray scattering from the width of the intensity curve obtained for each local pixel during the rotation of the analyzer. RESULTS We acquired USAXS images of malignant and benign breast tumor specimens using the A[L] analyzer; regions with larger USAXS form brighter areas in the image. We varied the sensitivity of the USAXS image by changing the threshold level of the object rocking curve. CONCLUSIONS The USAXS images can provide information about the internal distribution of closely packed scattering bodies in a sample with reasonable sensitivity. This information differs from that obtainable through refraction-contrast imaging. Although further validation studies will be necessary, we conclude that USAXS imaging using a Laue-case analyzer may have significant potential as a new diagnosis technique.

Molecular analysis of isolated tumor glands from endometrial endometrioid adenocarcinomas.

We studied the extensive molecular alterations of endometrial endometrioid adenocarcinoma (EEA) using a crypt isolation method. We analyzed copy number variation (CNV) using a single nucleotide polymorphism (SNP) array, genetic mutations (KRAS, BRAF, p53, PIK3CA), DNA methylation and microsatellite instability (MSI) status. In addition, loss of PTEN protein expression was examined. Increased chromosome copy numbers of 1q21.2–44 (22%) and 10q11.21–23.31 (28%) were seen relatively frequently in EEA, and copy‐neutral loss of heterozygosity (LOH) was also observed in 10q22.1–26.3 (22%). The CNV patterns of EEA were classified into four groups through hierarchical cluster analysis. Cluster 1 had many CNVs of 10q, and cluster 2 was characterized by MSI status. In cluster 3, increased CNVs of 1q were often seen. In cluster 4, p53 mutations were detected. KRAS and PIK3CA mutations and reduced PTEN protein expression were common to all groups. On the other hand, CpG island methylator phenotype (CIMP) was rare in all groups. The data indicated an association with chromosomal gain of 1q and 10q or 10q copy‐neutral LOH in some cases. We suggest that EEA consists of four groups that are characterized with molecular alterations.

Proposal for novel histological findings of colorectal liver metastases with preoperative chemotherapy

This study aimed to clarify the histological characteristics related to preoperative chemotherapy for colorectal liver metastases (CRLM). Sixty‐three patients with CRLM were divided into two groups: CRLM with chemotherapy (41 cases, group A) and CRLM without chemotherapy (22 cases; surgical treatment alone, group S) to identify the histological differences associated with chemotherapy. In addition, we investigated the effects of combination chemotherapy on the histology of metastatic lesions. Infarct‐like necrosis (ILN), three‐zonal changes, and cholesterol clefts were more frequent in group A than in group S (P < 0.05). ILN and three‐zonal changes were more common in the 5‐FU with leucovorin and oxaliplatin (FOLFOX), or 5‐FU with leucovorin and irinotecan (FOLFIRI) with or without additional bevacizumab groups than in group S (P < 0.05). Cholesterol clefts in the FOLFOX or FOLFIRI with bevacizumab group and foamy macrophages in the FOLFOX or FOLFIRI group were more common than in group S (P < 0.05). Cases with more than three of the four histological findings—i.e. ILN, three‐zonal changes, cholesterol clefts, and foamy macrophages—were more frequent in the FOLFOX or FOLFIRI with or without additional bevacizumab groups than in group S (P < 0.05). We showed histological findings for every representative chemotherapy regimen for CRLM to clarify the effects of preoperative chemotherapy.

A rare case of breast cancer showing distinct TTF‐1 nuclear expression: small‐cell carcinoma or not?

Sir: We read with great interest the article entitled ‘TTF-1 expression in breast carcinoma: an unusual but real phenomenon’, as presented in Histopathology. Ni et al. evaluated thyroid transcription factor-1 (TTF-1) expression in 1132 primary invasive breast carcinomas (IBCs) and 208 primary pulmonary carcinomas using tissue microarray sections. They detected moderate to strong TTF-1 nuclear expression in only one IBC (0.09%) and 149 pulmonary carcinomas (71.6%) using clone 8G7G3/1, and in no IBC (0%) and 147 (70.6%) pulmonary carcinomas using clone SPT24. The single IBC case with 8G7G3/1 positivity was histologically assessed as of no special type, with histological grade 2, and showed oestrogen receptor positivity, progesterone receptor positivity and HER2 negativity. The authors concluded that both TTF-1 antibodies were useful in differentiating breast from pulmonary carcinomas. TTF-1, a 38-kDa homeodomain-containing nuclear protein encoded by the NKX2-1 gene, regulates the transcription of genes, particularly in the thyroid, lungs and diencephalon, and immunostaining for TTF-1 is used accordingly in routine surgical pathology practice to facilitate identifying the sites of origin of carcinomas. However, it has been demonstrated that TTF-1 is commonly detectable not only in pulmonary, but also in extra-pulmonary small-cell carcinomas. In a study of the breast, Shin et al. described two of 10 cases (20%) with primary small-cell carcinomas as showing moderate to strong, diffuse nuclear staining for TTF-1. Similar TTF-1 immunoreactivity in such cancers was subsequently reported by other investigators. In addition, in an extremely rare mammary carcinoma with both small-cell neuroendocrine and metaplastic features, which we reported very recently in this journal, only the former component was selectively TTF-1 (clone 8G7G3/1) positive (Figure 1). Ni et al. described the IBC positive for TTF-1 (8G7G3/1) as being of no special type with histological grade 2 and luminal immunoexpressions. However, the figure showing TTF-1 nuclear expression could prompt readers to consider the possibility of a small-cell carcinoma. Unfortunately, immunohistochemical analyses of this cancer for neuroendocrine markers, such as chromogranin A, synaptophysin and CD56 (NCAM), were not included in their study. In our opinion, morphological review as well as further immunohistochemical examination should be implemented for this specific case. Indeed, the results for neuroendocrine markers, whether positive or negative, would add to the interest of their investigation. In addition, we hope that if the authors accumulate additional cases of small-cell mammary carcinoma, TTF-1 expression in these cancers, including the differences between antibody clones 8G7G3/1 and SPT24, will be examined and comprehensively discussed.

Cytological features of lymphoepithelioma-like carcinoma of the breast.

dated forms of varying size with narrow-based budding, whereas a mucicarmine stain highlighted their thick capsule (Figure 1). Subsequent serum Cryptococcal antigen was positive with a quantitative titre of 1:1024. Blood and ascitic fluid cultures produced creamy white colonies which were later identified as C. neoformans. Despite aggressive treatment with high-dose fluconazole, the patient continued to decline. He developed multiorgan failure with an impending need for dialysis. After consultation with the family, the patient was transitioned to palliative care and died within a few days.