Masahiro Watatani

Mutations of a novel human RAD54 homologue, RAD54B , in primary cancer

Association of breast tumor susceptibility gene products BRCA1 and BRCA2 with the RAD51 recombination protein suggested that cancer could arise through defects in recombination. The identification of NBS1, responsible for Nijmegen breakage syndrome, from the MRE11/RAD50 recombination protein complex also supports this hypothesis. However, our mutation analysis revealed that known members of the RAD52 epistasis group are rarely mutated in human primary cancer. Here we describe the isolation of a novel member of the SNF2 superfamily, characterized with sequence motifs similar to those in DNA and RNA helicases. The gene, designated RAD54B, is significantly homologous to the RAD54 recombination gene. The expression of RAD54B was high in testis and spleen, which are active in meiotic and mitotic recombination. These findings suggest that RAD54B may play an active role in recombination processes in concert with other members of the RAD52 epistasis group. RAD54B maps to human chromosome 8q21.3-q22 in a region associated with cancer-related chromosomal abnormalities. Homozygous mutations at highly conserved positions of RAD54B were observed in human primary lymphoma and colon cancer. These findings suggest that some cancers arise through alterations of the RAD54B function.

Mutations in the RAD54 recombination gene in primary cancers

Association of a recombinational repair protein RAD51 with tumor suppressors BRCA1 and BRCA2 suggests that defects in homologous recombination are responsible for tumor formation. Also recent findings that a protein associated with the MRE11/RAD50 repair complex is mutated in Nijmegen breakage syndrome characterized by increased cancer incidence and ionizing radiation sensitivity strongly support this idea. However, the direct roles of BRCA proteins and the protein responsible for NBS in recombinational repair are not clear though they are associated with the recombinational repair complexes. Since RAD51 forms a complex with other members of the RAD52 epistasis group and with BRCA proteins, it is reasonable to ask if alterations of members of the RAD52 epistasis group lead to tumor development. Here we describe missense mutations at functional regions of RAD54 and the absence of the wild-type RAD54 expression resulting from aberrant splicing in primary cancers. Since RAD54 is a recombinational protein associated with RAD51, this is the first genetic evidence that cancer arises from a defect in repair processes involving homologous recombination.

Nine novel germline mutations of STK11 in ten families with Peutz-Jeghers syndrome

Abstract Peutz-Jeghers Syndrome (PJS) is an autosomal dominant hereditary disease characterized by hamartomatous polyposis involving the entire bowel. Recently STK11, a gene bearing a mutation responsible for PJS, was isolated. We investigated the entire coding region of STK11 in 15 unrelated PJS families by the PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) method and PCR-direct sequence analysis, and found nine different, novel mutations among ten of those families. One nonsense mutation and five different frameshift mutations (two families carried the same mutation), all of which would cause truncation of the gene product, were found in seven families; mutations found in five families were clustered within exon 6. Among these five mutations, three occurred at the mononucleotide-repeat region (CCCCCC) of codons 279–281, suggesting that this region is likely to be a mutational hotspot of this gene. One of the remaining three families carried a 3-bp in-frame deletion that would eliminate an asparagine residue within a kinase domain of the product; the other two carried intronic mutations at or adjacent to the consensus dinucleotide sequences of splice-acceptor or -donor sites, which were likely to lead to aberrant splicing.

Infrequent mutations in the PTEN/MMAC1 gene among primary breast cancers

Recently PTEN/MMAC1, a candidate tumor suppressor gene, was isolated from chromosome 10q23‐24 and somatic mutations of this gene were detected in several malignancies including brain, prostate, and breast tumors. To investigate further the potential role of this gene in mammary carcinogenesis, we examined 69 primary breast cancers for mutations in PTEN/MMAC1 by means of polymerase chain reaction single‐strand conformation polymorphism and sequencing analysis. We detected only one somatic missense mutation, a change from T to C at codon 59 (TCA to CCA) resulting in substitution of Pro for Ser in the predicted protein. This site is located outside of phosphatase or phosphate‐acceptor motifs, but this codon encodes a residue that is conserved in homologous proteins, tensin and auxilin and is likely to be crucial for normal function of PTEN/MMAC1. Among the 69 tumors examined, three low‐frequency polymorphisms were found as well, one in the non‐coding region of exon 1 and one each in introns 2 and 7. Our results suggested that mutation of the PTEN/MMAC1 gene is not a major factor in the development of most primary breast cancers.

Quantitative analysis of the reconstructed breast using a 3-dimensional laser light scanner.

Postoperative cosmesis of the reconstructed breast depends on the interrelation of shape, size (volume), and symmetry. In this study, reconstructed breasts were analyzed with 3-dimensional projections generated by laser light scanning. Fifty-one cases of breast reconstruction following mastectomy (16 cases of rectus abdominis flap, 15 cases of latissimus dorsi flap, and 20 cases of tissue expansion) were evaluated 6 months postoperatively. Shape, volume, and symmetry were quantitatively evaluated. Captured images of the normal breast were mirror-reversed and superimposed on images of the reconstructed breast. Differences in the generated Moire patterns were used to quantitatively compare breasts. The method was rapid, reproducible, and accurate in comparison to thermoplaster casts. It was found that rectus abdominis flaps applied following total mastectomy and latissimus dorsi flaps applied following partial mastectomy gave the best results for their relatively low degree of asymmetry. Application of tissue expansion led to greater asymmetry and poorer overall cosmesis. In conclusion, a 3-dimensional laser light scanning system makes it possible to quantitate the cosmetic outcome following breast reconstruction.

Allelic loss of chromosome 17p, mutation of the p53 gene, and microsatellite instability in right‐ and left‐sided colorectal cancer

Epidemiologic and genetic studies suggest that cancer of the right and left sides of the bowel arise through different mechanisms. To investigate the molecular mechanisms, allelic loss of chromosome 17p, p53 mutations, and microsatellite instability were analyzed in colorectal cancer according to tumor site.

Switching addictions between HER2 and FGFR2 in HER2-positive breast tumor cells: FGFR2 as a potential target for salvage after lapatinib failure.

Agents that target HER2 have improved the prognosis of patients with HER2-amplified breast cancers. However, patients who initially respond to such targeted therapy eventually develop resistance to the treatment. We have established a line of lapatinib-resistant breast cancer cells (UACC812/LR) by chronic exposure of HER2-amplified and lapatinib-sensitive UACC812 cells to the drug. The mechanism by which UACC812/LR acquired resistance to lapatinib was explored using comprehensive gene hybridization. The FGFR2 gene in UACC812/LR was highly amplified, accompanied by overexpression of FGFR2 and reduced expression of HER2, and a cell proliferation assay showed that the IC(50) of PD173074, a small-molecule inhibitor of FGFR tyrosine kinase, was 10,000 times lower in UACC812/LR than in the parent cells. PD173074 decreased the phosphorylation of FGFR2 and substantially induced apoptosis in UACC812/LR, but not in the parent cells. FGFR2 appeared to be a pivotal molecule for the survival of UACC812/LR as they became independent of the HER2 pathway, suggesting that a switch of addiction from the HER2 to the FGFR2 pathway enabled cancer cells to become resistant to HER2-targeted therapy. The present study is the first to implicate FGFR in the development of resistance to lapatinib in cancer, and suggests that FGFR-targeted therapy might become a promising salvage strategy after lapatinib failure in patients with HER2-positive breast cancer.

Adrenal metastasis from carcinoma of the colon and rectum: a report of three cases.

We report herein three cases of patients with adrenal metastases from colorectal carcinoma. Recurrent disease was suspected following markedly elevated levels of serum carcinoembryonic antigen (CEA), and adrenal metastases were confirmed by computed tomography (CT) scanning in all three patients. The adrenal metastasis was solitary in one patient and this patient is still alive and free from disease 1 year after undergoing complete removal of the adrenal metastasis. On the other hand, metastatic disease was not limited to the adrenal gland in the other two patients and both died of recurrent disease, 33 months and 4 months after undergoing removal of the adrenal metastases, respectively. Thus, although the prognosis of adrenal metastasis from colorectal cancer is usually poor, we believe that patients with a solitary adrenal metastasis will benefit from complete removal of the metastasis.

Second-look operation for recurrent colorectal cancer based on carcinoembryonic antigen and imaging techniques

PURPOSE: The usefulness of postoperative carcinoembryonic antigen (CEA) monitoring and improvements in imaging techniques have renewed enthusiasm for second-look operations (SLO) as the most effective treatment for recurrent colorectal cancer by reresection following early detection. The aim of our study is to evaluate the role of CEA and imaging techniques-directed SLO. METHODS: Seven hundred fifty-six patients with Dukes Stages B and C, who had undergone curative resection, were monitored postoperatively using CEA and imaging techniques. An SLO was performed on any potentially resectable recurrence, and in addition, an SLO was done when a persistently rising CEA value was detected. RESULTS: Recurrence developed in 18.8 percent (142/756) of patients, and 90.8 percent (129/ 142) of the recurrences were detected within the first three years following curative resection. When comparing carcinomas of the colon with that of the rectum, the former were associated with significantly more hepatic and intra-abdominal recurrences, whereas the latter had significantly more locoregional and pulmonary recurrences. Seventy-two patients underwent SLO. Of these patients, 54.2 percent (39/72) had all of their disease resected, and 1.4 percent (1/72) had no detectable disease at the SLO. Among the 142 patients with recurrence, 71 (50 percent) patients underwent SLO. The resectable group at SLO carried a significantly better survival than the unresectable recurrence group (41.3vs.5.2 percent;P<0.01). CONCLUSIONS: Complete removal of colorectal cancer recurrences by SLO, on the basis of postoperative, follow-up CEA and imaging technique findings, results in improved survival.

Proliferating cell nuclear antigen in breast lesions: Correlation of c-erbB-2 oncoprotein and EGF receptor and its clinicopathological significance in breast cancer

Monoclonal anti-proliferating cell nuclear antigen (PCNA PC10), which is directed against a 36 kDa auxiliary protein for DNA polymerase delta specific for the S-phase of cell cycle, was used to measure tumour cell proliferation in 4 lactating breasts and 98 benign and malignant breast tumours. The percentage of PCNA-positive cells determined by point counting was significantly lower in the lactating breast [mean 3.6%, standard deviation (SD) 0.67,n=5] than in fibroadenoma and mastopathy (mean 23.7, SD 5.0,n=2). Primary breast carcinoma showed a PCNA index ranging from 2% to 36% (mean 12.3, SD 9.3,n=50), whereas in recurrent carcinoma the index was mean 28.5, SD 4.0. A high index was correlated with c-erbB-2 and epidermal growth factor (EGF) receptor membrane reactivity, worsening histological grade, poor survival and disease-free survival. The expression of c-erbB-2 and EGF receptor was associated with poor survival and disease-free survival in primary breast cancer patients.