INSM1 immunostaining in solid papillary carcinoma of the breast.

Solid papillary carcinoma (SPC) is a histological subtype of breast carcinomas. At least 50% of SPC show neuroendocrine differentiation. Insulinoma-associated protein 1 (INSM1) is a transcription factor now employed as a useful neuroendocrine marker. It is suppressed by the Notch signaling pathway in other neuroendocrine tumors. However, the usefulness of INSM1 as a neuroendocrine marker and the relationships between INSM1 and NOTCH receptors in SPC of the breast currently remain unclear. To clarify the usefulness of INSM1 as a neuroendocrine marker and the relationships between INSM1 and NOTCH receptors in SPC, we performed immunohistochemistry using 19 tissue specimens of SPC of the breast. We complementarily analyzed public RNA sequencing data on breast carcinomas. Immunohistochemical examinations revealed that the staining intensity of INSM1 was significantly higher in the neuroendocrine group than in the non-neuroendocrine group. Positive correlations were observed between INSM1 and synaptophysin (SYP), or chromogranin-A (CHGA). In all cases, NOTCH 2 and 3 were positive, while NOTCH 1 and 4 were negative. According to public RNA data analyses, there were positive correlations between INSM1 and SYP, or CHGA, and negative correlations between INSM1 and NOTCH1-3. INSM1 is useful as a diagnostic marker for SPC with neuroendocrine differentiation in the breast.

T-bet+ lymphocytes infiltration as an independent better prognostic indicator for triple-negative breast cancer.

PURPOSE: T-box transcription factor 21 (T-bet), which is the master regulator of effector T-cell activation, is derived by stimulation of T-cell receptors. In this study, we focused on T-bet and examined the function of activated T cells. METHODS: This study included 242 patients with primary triple-negative breast cancer (TNBC) who underwent resection without neoadjuvant chemotherapy between January 2004 and December 2014. The immunohistochemistry scoring for CD8 and T-bet expression on tumor-infiltrating lymphocytes (TILs) was defined as ≥ 30 per 6.25 × 10-3 mm2. RESULTS: Of the 242 TNBC cases, CD8 was positively expressed in 127 (52.5%) tumors, and T-bet was positively expressed in 67 (27.7%) tumors. T-bet expression was significantly correlated with CD8 expression (p < 0.0001). Patients with T-bet+ tumors had longer overall survival (OS) compared with patients with T-bet- tumors (p = 0.047). The combination of CD8+ and T-bet+ was associated with a better recurrence-free survival (RFS) and OS compared to CD8+/T-bet- tumors (p = 0.037 and p = 0.024, respectively). Adjuvant chemotherapy provided significantly greater benefit to patients with T-bet+ tumors (p = 0.031 for RFS, p = 0.0003 for OS). Multivariate analysis revealed that T-bet expression on TILs was an independent and positive prognostic indicator (HR = 0.36, 95% confidence interval (CI) 0.12-0.94, p = 0.037 for RFS, HR = 0.30, 95% CI 0.07-0.95, p = 0.039 for OS). CONCLUSIONS: OS was significantly improved for patients with high T-bet-expressing TILs in TNBC. Thus, T-bet may be a predictive indicator for survival and various immunotherapy strategies in TNBC.

Usefulness of virtual touch quantification for staging liver fibrosis in patients with hepatitis C, and factors affecting liver stiffness measurement failure compared with liver biopsy.

AIM: The assessment of liver fibrosis in patients with hepatitis C is important to predict carcinogenesis. In this study, we evaluated the usefulness of virtual touch quantification (VTQ) for staging liver fibrosis, and investigated factors causing discrepancies between the estimated fibrosis stage using VTQ and the pathological fibrosis stage. METHODS: Patients with hepatitis C (n = 302) were assessed using VTQ and underwent pathological liver investigation within 1 week before and after VTQ. A receiver operator characteristic (ROC) curve was obtained for VTQ, fibrosis-4 (FIB-4) index, and aspartate aminotransferase-to-platelet ratio index (APRI), and each area under the ROC curve (AUROC) was compared to predict fibrosis stage. We used univariate and multivariate analyses to investigate the factors related to the discrepancy between the estimated fibrosis stage using VTQ and the pathological fibrosis stage. RESULTS: At any stage, VTQ was the most accurate for staging liver fibrosis. The VTQ cut-off values were 1.33 m/s (AUROC = 0.822) for ≥F2, 1.51 m/s (AUROC = 0.836) for ≥F3, and 1.92 m/s (AUROC = 0.890) for F4. Skin liver capsule distance (SCD) was the most relevant factor for the discrepancy between the estimated fibrosis stage using VTQ and the pathological fibrosis stage. The SCD cut-off value was 17.5 mm. CONCLUSIONS: Virtual touch quantification is a non-invasive, simple method that is more accurate for staging liver fibrosis than the FIB-4 index and APRI. However, when the SCD is longer than 17.5 mm, there may be measurement failures.

Role of HER2-Related Biomarkers (HER2, p95HER2, HER3, PTEN, and PIK3CA) in the Efficacy of Lapatinib plus Capecitabine in HER2-Positive Advanced Breast Cancer Refractory to Trastuzumab.

OBJECTIVE: The aim of this study was to investigate the correlation between human epidermal growth factor receptor 2 (HER2)-related biomarkers and the treatment outcomes using lapatinib plus capecitabine (LC) and to evaluate the influence of the estrogen receptor (ER) status in trastuzumab-refractory HER2-positive advanced breast cancer. METHOD: Eighty patients were enrolled in this study. Total HER2, p95HER2, and total HER3 expression were quantified using the VeraTag assays. PTEN (phosphatase and tensin homolog) and p95 expression was evaluated using immunohistochemistry and PIK3CA mutation using direct sequencing. RESULTS: The response rate to LC was 30%, clinical benefit rate was 51.3%, and the median progression-free survival (PFS) was 174.5 days. ER negativity significantly correlated with higher HER2 and p95HER2. The lower HER2 and PIK3CA mutations were often observed in the nonresponders. A high p95HER2 expression correlated with longer PFS especially in the high HER2- and ER-positive cases. Patients without the PIK3CA mutation showed longer PFS in the same subset. Overall survival after LC significantly correlated with the number of recurrence organs. CONCLUSION: LC therapy is effective in trastuzumab-refractory HER2-positive breast cancer. Moreover, the biomarker expression differed depending on ER status, and a high p95HER2 expression and wild-type PIK3CA gene correlated with longer PFS especially in the ER-positive cases.

A Comparison of the Hot Spot and the Average Cancer Cell Counting Methods and the Optimal Cutoff Point of the Ki-67 Index for Luminal Type Breast Cancer.

OBJECTIVE: In this case-control study, we investigated the most suitable cell counting area and the optimal cutoff point of the Ki-67 index. METHODS: Thirty recurrent cases were selected among hormone receptor (HR)-positive/HER2-negative breast cancer patients. As controls, 90 nonrecurrent cases were randomly selected by allotting 3 controls to each recurrent case based on the following criteria: age, nodal status, tumor size, and adjuvant endocrine therapy alone. Both the hot spot and the average area of the tumor were evaluated on a Ki-67 immunostaining slide. RESULTS: The median Ki-67 index value at the hot spot and average area were 25.0 and 14.5%, respectively. Irrespective of the area counted, the Ki-67 index value was significantly higher in all of the recurrent cases (p < 0.0001). The multivariate analysis revealed that the Ki-67 index value of 20% at the hot spot was the most suitable cutoff point for predicting recurrence. Moreover, higher x0394;Ki-67 index value (the difference between the hot spot and the average area, ≥10%) and lower progesterone receptor expression (<20%) were significantly correlated with recurrence. CONCLUSION: A higher Ki-67 index value at the hot spot strongly correlated with recurrence, and the optimal cutoff point was found to be 20%.

[Diffuse large B-cell lymphoma developed newly after 9-year remission of a follicular lymphoma].

Follicular lymphoma (FL) occasionally transforms into diffuse large B-cell lymphoma (DLBCL). This is generally associated with a poor prognosis, necessitating more potent chemotherapy as salvage treatment. However, de novo DLBCL, but not DLBCL transformed from FL, can be treated as primary DLBCL. We encountered a 63-year-old woman who developed DLBCL after a 9-year remission following treatment of FL. To differentiate DLBCL transformed from FL and de novo DLBCL, VDJ gene rearrangements in IgH were examined by PCR using biopsy specimens from both lymphomas. The results revealed the two lymphomas to be different clones. Thus, she was diagnosed with primary DLBCL. Therefore, routine chemotherapy and radiation therapy were conducted for the primary DLBCL with a limited stage, achieving complete remission. Treatment based on the clonality assessment of VDJ gene rearrangements is potentially useful for treating late relapse of B-cell lymphoma according to the pathological conditions of patients.

Efficacy of intraoperative entire-circumferential frozen section analysis of lumpectomy margins during breast-conserving surgery for breast cancer.

BACKGROUND: Intraoperative frozen section analysis of the surgical margins during breast-conserving surgery (BCS) for breast cancer can reliably achieve clear surgical margins and prevent re-operations. The aim of this study was to assess intraoperative entire-circumferential frozen section analysis (IEFSA) of the lumpectomy margins during BCS. METHODS: A total of 1029 patients who underwent BCS with IEFSA between June 2007 and July 2013 were available for assessment. The inner surfaces of the shaved lumpectomy margins were examined as frozen sections during BCS. The margins were defined as positive when the cancer cells were present within 5 mm from the edge of the outermost margins of the specimens. RESULTS: Out of 1029 patients, 312 patients (30.3 %) had positive margins after the initial lumpectomy and underwent additional resections during BCS. Fourteen patients (1.4 %) underwent mastectomy following the results of additional resections during the first surgery. Of 1015 patients who completed BCS, 60 patients (5.9 %) were found to have positive margins in the final pathology. One patient (0.1 %) underwent re-operation after BCS while the residual diseases of the other 59 patients were judged to be minimal. Of the 312 patients who were judged to have positive margins after the initial lumpectomy with IEFSA, 53 patients (16.9 %) were found to have negative margins in the final pathology. At a median follow-up time of 54.1 months, one patient (0.1 %) had a recurrence of breast cancer in the preserved breast. CONCLUSION: IEFSA is useful for preventing the need for re-operation and local recurrence after BCS.

Breast cancer subtype and distant recurrence after ipsilateral breast tumor recurrence.

BACKGROUND: There is little information about the impact of breast cancer subtype on prognosis after ipsilateral breast tumor recurrence (IBTR). METHODS: One hundred eighty-five patients were classified according to breast cancer subtype, as approximated by estrogen receptor, human epidermal growth factor receptor 2 (HER2), and Ki-67, of IBTR, and we evaluated whether breast cancer subtype was associated with distant recurrence after IBTR. RESULTS: There was a significant difference in distant disease-free survival (DDFS) after IBTR according to breast cancer subtype defined by a cutoff of the Ki-67 index of 20 % (p = 0.0074, log-rank test). The 5-year DDFS rates for patients with luminal A, luminal B, triple-negative, and HER2 types were 86.3, 57.1, 56.6, and 65.9 %, respectively. In addition, breast cancer subtype was significantly associated with distant recurrence after IBTR on adjustment for various clinicopathologic factors (p = 0.0027, Cox proportional hazards model). CONCLUSIONS: Our study suggests that breast cancer subtype based on immunohistochemical staining predicts the outcomes of patients with IBTR. Further analyses are needed (UMIN-CTR number UMIN000008136).

Evaluation of factors related to late recurrence--later than 10 years after the initial treatment--in primary breast cancer.

BACKGROUND: Breast cancer is associated with a relatively good prognosis. Prognostic factors examined to date are related to early recurrence while those related to late recurrence and their countermeasures remain unclear. Therefore, we examined the factors related to late recurrence. PATIENTS AND METHODS: From January 1980 to August 2012, 4,774 patients who underwent primary treatment and estrogen (ER) and progesterone receptor (PgR) assessment were enrolled in this study. The patients were divided into two groups, those with a follow-up period <10 years and those without any recurrence at 10 years but who continued follow-up examinations. Recurrence occurred in 711 patients followed up for <10 years and in 51 patients for ≥10 years. RESULTS: The overall 10-year cumulative disease-free survival rate was 79.5%, and the recurrence rate at ≥10 years was 5.8%. A multivariate analysis revealed that the factors related to late recurrence were PgR positivity and positive nodes. This result differed from that for early recurrence in terms of ER/PgR, Ki-67 index and p53 overexpression. CONCLUSION: PgR positivity and lymph node metastases significantly correlated with late recurrence. Therefore, it is important to evaluate appropriate measures such as treatment period and treatment regimen for hormone-sensitive patients.

Central nervous system lymphoma newly developed 12 years after remission of an ocular adnexal lymphoma.

Recurrence of non-Hodgkin's lymphoma more than 5 years after the initial diagnosis is rare. When late relapse occurs, it is difficult to determine whether it is a true recurrence or a new lesion. We experienced a case of an 81-year-old woman who developed central nervous system (CNS) lymphoma 12 years after remission of ocular adnexal lymphoma. Both showed the histology of diffuse large B-cell lymphoma. To elucidate whether the CNS lymphoma was clonally related to the first lymphoma, rearrangement of the immunoglobulin heavy chain genes of each lymphoma was studied using a polymerase chain reaction-based method. The results revealed that the sizes of the amplified products of the rearranged regions from the two lymphomas were different. This suggested different clonal origins of the lymphomas. It is clinically important to determine the origin of a second neoplasm because patients with a clonally related second lymphoma are usually treated with more intensive regimens, while those with a clonally unrelated lymphoma receive standard first-line therapy. The present case shows that, in the case of recurrent non-Hodgkin's lymphoma, not only histological confirmation but also genetic assessment is important to clarify the origin of the second lymphoma.

Granzyme B Expression in the Tumor Microenvironment as a Prognostic Biomarker for Patients with Triple-Negative Breast Cancer.

Tumor-infiltrating lymphocytes in the tumor microenvironment are important in the treatment of triple-negative breast cancer (TNBC). Cytotoxic T cells produce cytokines and cytotoxic factors, such as perforin and granzyme, which induce apoptosis by damaging target cells. To identify biomarkers of these cells, we investigated granzyme B (GZMB) in the tumor microenvironment as a biomarker of treatment response and prognosis in 230 patients with primary TNBC who underwent surgery without preoperative chemotherapy between January 2004 and December 2014. Programmed cell death ligand 1 (PD-L1) positivity was defined as a composite positive score ≥10 based on the PD-L1 immunostaining of tumor cells and immune cells. GZMB-high was defined as positivity in ≥1% of tumor-infiltrating lymphocytes (TILs). Among the 230 TNBC patients, 117 (50.9%) had CD8-positive infiltrating tumors. In the PD-L1-positive group, a Kaplan-Meier analysis showed that GZMB-high TNBC patients had better recurrence-free survival (RFS) and overall survival (OS) than GZMB-low patients and that OS was significantly longer (RFS: p = 0.0220, OS: p = 0.0254). A multivariate analysis also showed significantly better OS in PD-L1- and GZMB-high patients (hazard ratio: 0.25 (95% IC: 0.07-0.88), p = 0.03). Our findings indicate that GZMB is a useful prognostic biomarker in PD-L1-positive TNBC patients.

Mucocele-like tumor associated with ductal carcinoma in situ diagnosed as mucinous carcinoma by fine-needle aspiration cytology: report of a case.

Mucocele-like tumors (MLTs) of the breast are rare, with only 11 cases reported from Japan and 35 cases from other countries. MLTs of the breast were first described by Rosen in 1986. They are believed to be related to atypical ductal hyperplasia, ductal carcinoma, or mucinous carcinoma. It is difficult to diagnose this tumor preoperatively, and especially difficult to differentiate between benign and malignant forms. We report a case of MLT associated with ductal carcinoma in situ, which was initially diagnosed as fibroadenoma by mammography and ultrasonography, and as mucinous carcinoma by fine-needle aspiration cytology. We discuss the characteristic findings of imaging and the appropriate clinical treatment of this tumor. The characteristic image first signals the possibility of this tumor, following which the diagnosis can be confirmed by pathological examination of a fully excised tumor specimen. Breast-conserving surgery is recommended because of the low risk of high-grade malignancy, even when malignancy is confirmed, and lymph node dissection may be avoided.

Triple Negative Breast Cancer: An Analysis of the Subtypes and the Effects of Menopausal Status on Invasive Breast Cancer.

BACKGROUND: Triple negative breast cancer (TNBC) is a subtype of breast cancer which lacks hormone receptor (HR) expression and HER2 gene amplification and is the most aggressive subtype, with a heterogeneous genetic profile. The aim of this retrospective study was to evaluate the clinical significance of menopausal status in breast cancer cases with TNBC. METHODS: Primary breast cancer patients who underwent curative surgery were enrolled in this retrospective study. A total of 5153 invasive breast cancer cases with Stage I-III were analyzed. The distribution of cases according to the menopausal status and subtypes was investigated and the clinicopathological characteristics and prognosis were compared between pre- and postmenopausal TNBC patients. RESULTS: TNBC was frequently seen in postmenopausal patients and Luminal B and Luminal/HER2 subtypes were more common in premenopausal patients. There was no difference in DFS in the Luminal A/B and HER2 subtypes, but a significant difference was seen in the TNBC patients. Premenopausal patients with TNBC frequently had an overexpression of the p53 protein, a significantly higher Ki-67 index value, and a higher nuclear grade. A multivariate analysis revealed that menopausal status, nodal status, and tumor size were significant factors for DFS in TNBC cases. CONCLUSION: Menopausal status significantly correlates with breast cancer subtypes. TNBC was often seen in postmenopausal patients and these patients tend to have more favorable factors and a better DFS than premenopausal patients. These findings suggest that menopausal status is an important factor for evaluating biology and prognosis in TNBC cases.

An evaluation of lymphovascular invasion in relation to biology and prognosis according to subtypes in invasive breast cancer.

Lymphovascular invasion (LVI) is associated with a poor outcome in breast cancer. The purpose of the present study was to evaluate the clinical significance of LVI in primary breast cancer and to investigate disease-free survival as a prognostic marker according to the breast cancer subtypes. This study examined 4,652 consecutive cases of invasive breast cancer excluding the patients with non-invasive cancer, stage IV and those who underwent neo-adjuvant therapy from February 2002 to February 2021. The clinicopathological characteristics and prognosis of LVI-positive and -negative tumors were compared. LVI was evaluated in H&E staining specimens from surgically resected samples. The LVI expression rates were 29.2% (low, 19.7%; high, 9.5%) in all primary cases. The LVI-positive rate was significantly associated with specimens with the following characteristics: ER/PgR-negative, HER2-positive, p53 overexpression, higher Ki-67 index values, higher nuclear grade, positive nodes and larger tumors. Moreover, the subtypes were significantly associated with LVI positivity; 20% in Luminal A, 34.6% in Luminal B, 40.9% in Lumina/HER2, 38.1% in HER2-enriched and 29.8% in triple negative (TN). There were significant differences in disease-free survival between LVI status in Luminal A, Luminal B and TN subtypes, but there was no difference in the Luminal/HER2 and HER2-enriched subtypes. A multivariate analysis revealed that LVI was a significant factor in Luminal B and TN subtypes. Overall, LVI was significantly associated with the advanced and aggressive characteristics in breast cancer. Luminal A type had a lower LVI rate, and HER2 type had a higher LVI rate. Moreover, LVI was a significant prognostic factor in Luminal B and TN subtypes. These data suggested that the LVI status was useful in predicting the prognosis in HER2 negative breast cancer cases.

Time to first tumor progression as a predictor of efficacy of continued treatment with trastuzumab beyond progression in human epidermal growth factor receptor 2-positive metastatic breast cancer.

BACKGROUND: Trastuzumab demonstrates significant clinical benefits in HER2-positive metastatic breast cancer (MBC), and recent clinical trials suggest that trastuzumab should be continued in combination with other chemotherapy beyond progression. There is an urgent need to assess if patients could substantially benefit from continuing trastuzumab-based therapy. METHODS: We reviewed 91 patients with HER2-positive MBC treated with trastuzumab and investigated correlations between survival and clinical response to first trastuzumab-based therapy and biological markers, time to first tumor progression (1st TTP), response rate (RR), estrogen receptor (ER), Ki-67, and p53 overexpression. RESULTS: With a median follow-up of 33 months, 76 patients had received two or more lines of consecutive trastuzumab-based therapy. Median 1st TTP was 8.6 months; patients who received trastuzumab with chemotherapy had a longer 1st TTP and better RR than those without chemotherapy. In terms of survival after first progression, patients with a longer 1st TTP (≥ 8.6 months) had significantly better survival compared with those who had a shorter 1st TTP (24.3 months vs. 15.4 months, P = 0.024), and multivariate analysis revealed that 1st TTP was a significant prognostic factor (HR 0.44, 95% CI 0.23-0.82, P = 0.01). There were no correlations between survival and ER or Ki-67; however, there was a correlation with p53 overexpression (HR 1.92, 95% CI 1.01-3.64, P = 0.045). CONCLUSIONS: 1st TTP is a significant prognostic factor for patients who receive subsequent trastuzumab-based therapy. This factor should be considered when determining the efficacy of continuing trastuzumab or switching to another anti-HER2 therapy beyond progression.

Changes in the ER, PgR, HER2, p53 and Ki-67 biological markers between primary and recurrent breast cancer: discordance rates and prognosis.

BACKGROUND: In breast cancer, ER/PgR, HER2, and Ki-67 are important biological markers for predicting prognosis and making effective treatment decisions. In addition, changes in markers due to relapse are also clinically experienced; however, the frequency and clinical significance are still not fully understood. Thus, changes in markers and their correlations with prognosis were investigated. PATIENTS AND METHODS: Out of the patients with relapse from 1997 to March 2011, there were 97 consecutive patients from whom the lesion was resected and evaluated by immunostaining. The biopsy sites were chest wall, lymph node, ipsilateral breast tumor recurrence, lungs, bones, ovaries and brain. The markers sought were ER, PgR, HER2, p53 and Ki-67. RESULTS: The hormone receptor positive rate from the primary tumor to recurrence decreased from 63.9% to 57.7% and from 56.7% to 43.3% for ER and PgR, respectively. Changes in the positive/negative evaluation were seen at the rate of 10.3% and 25.8% for ER and PgR, respectively. The Ki-67 index increased significantly from a mean of 29.1% at primary tumor to 36.3% at relapse. When divided into 2 groups (< 50% and ≥50%), changes were seen in 24.7%. On the other hand, the rates of changes in HER2 and p53 positivity were 14.4% and 12.4%. The changes in subtypes were seen in 25%, however, the lowest rate of change was seen in the triple negative cases. Although there was no notable difference in the rate of change between disease-free interval (DFI) and PgR, Ki-67, p53 and HER2, there was a significant difference in the change rates in the ER. A multivariate analysis revealed that the status of distant metastasis and PgR level at relapse, and Ki-67 levels at primary tumor were all significant factors. CONCLUSION: Estrogen receptor and PgR decreased while Ki-67 increased due to relapse; however, the rate of change was high for PgR and Ki-67. Change in the subtypes was seen in 25%. In addition, PgR at relapse and Ki-67 at primary tumor were significant factors for post-relapse prognosis while PgR becoming negative was a poor prognostic factor. These findings are important for making effective treatment decisions.

[A resected case of gastric regional lymph node metastasis from ascending colon cancer].

A 67-year-old man underwent right hemi-colectomy for ascending colon cancer in 2007. One year later, he was diagnosed as early gastric cancer, and endoscopic submucosal dissection was performed. Pathologically, cancer cells were detected on the vertical margin, so we conducted distal gastrectomy. A dissected lymph node around the hepatic artery was histologically proved to contain adenocarcinoma cells. The cancer cells were positive for CK20. Colon cancer cells were also positive for CK20 but gastric cancer cells were focally positive for CK20. This pattern of CK staining suggested the ascending colon cancer metastasized to a gastric regional lymph node.

[A resected case of locally invasive rectal cancer successfully treated with neoadjuvant capecitabine, oxaliplatin, bevacizumab and radiation therapy].

A 57-year-old man was admitted to our hospital with a complaint of perineal pain. He was diagnosed as advanced rectal cancer with an invasion of prostate, and we conducted neoadjuvant capecitabine, oxaliplatin, bevacizumab and radiation therapy. After chemoradiation therapy, the tumor regressed to an ulcerative lesion without invasion of the prostate. Abdominoperineal resection was then performed without radical resection. The tumor regressed to an ulcer and only a few cancer cells were detected in the submucosal layer pathologically.

Incidence, predictive factors and severity of methotrexate-related liver injury in rheumatoid arthritis: a longitudinal cohort study.

OBJECTIVES: The aims were to determine the incidence rate, predictive factors and severity of liver injury that develops during MTX treatment for RA and to evaluate the role of pretreatment hepatic fat deposition. METHODS: We used an ongoing real-life registry containing RA patients who had started MTX between August 2007 and April 2018 at participating institutions. The liver-to-spleen attenuation ratio on CT scans at enrolment was used to evaluate pretreatment fat deposition quantitatively. Patients were followed until persistent transaminitis developed or until the end of the study. Liver biopsy was performed for patients who presented with persistent transaminitis. RESULTS: We followed 289 new MTX users without pretreatment elevations of transaminases (mean follow-up time, 58.3 months). Hepatic fat deposition was detected in half of the patients at enrolment. During follow-up, persistent transaminitis occurred at a crude incidence rate of 3.13 per 100 person-years, and the cumulative incidence at 5 years was estimated to be 13%. A multivariate Fine-Gray regression analysis showed that the most important predictive factors were pre-existing moderate to severe fat deposition (adjusted hazard ratio, 7.69; 95% CI: 3.10, 19.10) and obesity (adjusted hazard ratio, 2.68; 95% CI: 1.37, 5.25). Non-alcoholic steatohepatitis (NASH) was the most predominant pattern in liver biopsy samples. Hepatic fibrosis was found in 90% of samples, but most cases were not advanced. CONCLUSION: Aggravation of underlying fatty liver to NASH with fibrosis seems to be an important mechanism of liver injury that occurs in MTX-treated RA patients.