Intratumoural immune cell landscape in germinoma reveals multipotent lineages and exhibits prognostic significance.

AIMS: Alterations in microenvironments are a hallmark of cancer, and these alterations in germinomas are of particular significance. Germinoma, the most common subtype of central nervous system germ cell tumours, often exhibits massive immune cell infiltration intermingled with tumour cells. The role of these immune cells in germinoma, however, remains unknown. METHODS: We investigated the cellular constituents of immune microenvironments and their clinical impacts on prognosis in 100 germinoma cases. RESULTS: Patients with germinomas lower in tumour cell content (i.e. higher immune cell infiltration) had a significantly longer progression-free survival time than those with higher tumour cell contents (P = 0.03). Transcriptome analyses and RNA in-situ hybridization indicated that infiltrating immune cells comprised a wide variety of cell types, including lymphocytes and myelocyte-lineage cells. High expression of CD4 was significantly associated with good prognosis, whereas elevated nitric oxide synthase 2 was associated with poor prognosis. PD1 (PDCD1) was expressed by immune cells present in most germinomas (93.8%), and PD-L1 (CD274) expression was found in tumour cells in the majority of germinomas examined (73.5%). CONCLUSIONS: The collective data strongly suggest that infiltrating immune cells play an important role in predicting treatment response. Further investigation should lead to additional categorization of germinoma to safely reduce treatment intensity depending on tumour/immune cell balance and to develop possible future immunotherapies.

Recurrent mutations of CD79B and MYD88 are the hallmark of primary central nervous system lymphomas.

AIMS: Primary central nervous system lymphoma (PCNSL) manifest aggressive clinical behaviour and have poor prognosis. Although constitutive activation of the nuclear factor-κB (NF-κB) pathway has been documented, knowledge about the genetic alterations leading to the impairment of the NF-κB pathway in PCNSLs is still limited. This study was aimed to unravel the underlying genetic profiles of PCNSL. METHODS: We conducted the systematic sequencing of 21 genes relevant to the NF-κB signalling network for 71 PCNSLs as well as the pyrosequencing of CD79B and MYD88 mutation hotspots in a further 35 PCNSLs and 46 glioblastomas (GBMs) for validation. RESULTS: The results showed that 68 out of 71 PCNSLs had mutations in the NF-κB gene network, most commonly affecting CD79B (83%), MYD88 (76%), TBL1XR1 (23%), PRDM1 (20%) and CREBBP1 (20%). These mutations, particularly CD79B and MYD88, frequently coincided within each tumour in various combinations, simultaneously affecting diverse pathways within the network. No GBMs had hotspot mutation of CD79B Y196 and MYD88 L265. CONCLUSIONS: The prevalence of CD79B and MYD88 mutations in PCNSLs was considerably higher than reported in systemic diffuse large B-cell lymphomas. This observation could reflect the paucity of antigen stimuli from the immune system in the central nervous system (CNS) and the necessity to substitute them by the constitutive activation of CD79B and MYD88 that would initiate the signalling cascades. These hotspot mutations may serve as a genetic hallmark for PCNSL serving as a genetic marker for diagnose and potential targets for molecular therapy.

Reirradiation of brain and skull base tumors with fractionated stereotactic radiotherapy.

PURPOSE: We evaluated the feasibility of fractionated stereotactic radiotherapy for small intracranial recurrences after conventional radiotherapy. METHODS AND MATERIALS: Nineteen patients who had initially undergone conventional radiotherapy to intracranial lesions, receiving a median total dose of 50 Gy in 5 weeks, were retreated with stereotactic radiotherapy for their recurrences and received a median total dose of 42 Gy in seven fractions over 2.3 weeks. RESULTS: Of the 19 patients, 15 achieved local control 3-51 months after reirradiation. No patient suffered from acute reaction, but one patient with a history of extensive radiotherapy developed progressive radionecrosis 9 months after reirradiation. CONCLUSIONS: Fractionated stereotactic radiotherapy of intracranial recurrences appears to be effective in achieving in local control with negligible morbidity. We believe it merits further investigation in a prospective study.

Fractionated stereotactic radiotherapy of small intracranial malignancies.

PURPOSE: To retrospectively evaluate the effectiveness of fractionated stereotactic radiotherapy (FSRT) in patients with small intracranial malignancies. METHODS AND MATERIALS: From July 1991 to March 1997, 80 patients with a total of 121 brain or skull-base tumors were treated with FSRT alone, and were followed for periods ranging from 3 to 62 months (median 9.8). The majority of patients received 42 Gy in 7 fractions over 2.3 weeks, but in July 1993, protocols using smaller fraction doses were introduced for patients whose radiation-field diameters were larger than 3 cm or whose tumors were close to critical normal tissues. RESULTS: For 64 patients with metastatic brain tumors the overall median survival was 8.3 months and 1-year actuarial survival rate was 33%. Significant prognostic factors were: the presence of extracranial tumors, pre-treatment performance status, and the lung as a primary site. Patients without extracranial tumors prior to FSRT had a median survival of 21.2 months. For seven patients with high-grade glioma, 1-year actuarial local control rate was 75%, with a median survival of 10.3 months. For patients with skull-base tumors the local control was achieved in 6 of 6 patients (100%), with a median survival of 30.7 months. No one suffered from acute complications, but three patients, two of whom had undergone FSRT as the third course of radiotherapy, developed late radiation injuries. CONCLUSION: Overall high local control and low morbidity rates suggest that FSRT is an effective and safe modality, even for those with a history of prior irradiation. However, patients with risk factors should be treated with smaller fraction doses.

Fractionated stereotactic radiotherapy of brain metastases from renal cell carcinoma.

PURPOSE: To retrospectively evaluate the effectiveness of fractionated stereotactic radiotherapy (FSRT) for brain metastases from renal cell carcinoma (RCC). METHODS AND MATERIALS: From May 1983 to September 1998, 35 patients with brain metastases from RCC underwent radiotherapy at the National Cancer Center Hospital, Tokyo; 10 patients treated initially with FSRT (FSRT group); 11 with surgery followed by conventional radiotherapy (S/CR group); and 14 with conventional radiotherapy (CR group). Survival and local control rates were determined for patients who had an ECOG performance status of 0-2. RESULTS: Overall median survival rate was 18 months, and actuarial 1- and 2-year survival rates were 57.6% and 31.0%, respectively. Median survival rates were 25.6 months for the FSRT group, 18.7 months for the S/CR group, and 4.3 months for the CR group. Significant prognostic factors associated with survival were age less than 60 years and good performance status. In patients treated with FSRT, imaging studies revealed that 21 of 24 tumors (88%) were locally controlled during a median follow-up time of 5.2 months (range 0.5-68). Actuarial 1- and 2-year local control rates were 89.6% and 55.2%, respectively. No patient suffered from acute or late complications during and following FSRT. CONCLUSIONS: FSRT offers better tumor control and prolonged survival over the S/CR or CR groups, and should be considered as primary treatment for brain metastases from RCC. Patients under 60-years-old and those with a good performance status at the beginning of radiotherapy had a better prognosis.

Double labeling with iodo- and bromodeoxyuridine for cell kinetics studies.

The rate of progression through the cell cycle was determined in five human glioma cell lines by a new sequential immunohistochemical staining technique. The cells were labeled first with iododeoxyuridine (IdUrd) for 1-3 hr and then with bromodeoxyuridine (BrdUrd) for 30 min. Labeled cells were identified with Br-3, a monoclonal antibody that recognizes only BrdUrd, and with IU-4, an antibody that recognizes both IdUrd and BrdUrd. Each slide was stained sequentially, first with the immunoperoxidase method for Br-3 and then with the alkaline phosphatase-anti-alkaline phosphatase method for IU-4. Cells that were positive only for IU-4 represented the fraction of S-phase cells that passed into the G2 phase during the period of incubation with IdUrd. The rates of progression measured by this method were constant in each cell line and resulted in smaller standard errors than were obtained by measurements from specimens stained singly for IdUrd and BrdUrd in different slides. The duration of the S-phase calculated from this fraction in the five cell lines ranged from 8-13 hr; the estimated potential doubling times were 25-32 hr and were very similar to the actual doubling times.

Linac-based small-field radiotherapy for brain tumors.

Small-field radiotherapy based on a 6-MeV linac and a conventional head mold is investigated as an alternative to radiosurgery with stereotactic frames. The system requires no additional device and allows fractionated treatment. The dose distributions obtained are comparable to those reported with a Gamma Unit. Overall positioning errors are within 2 mm. Using this approach, seven patients with brain tumors who could not have been treated otherwise, underwent fractionated radiotherapy with total accumulated doses ranging from 70 to 108 Gy. The treatment was tolerated well with no acute toxicity or adverse effect encountered during the follow-up period of 8-14 months. All of the patients remained free from disease progression in the treated volumes. Although the follow-up is brief, the preliminary results suggest that this is a simple and inexpensive but effective system for the treatment of small intracranial malignancies.

Application of antisense ribonucleic acid complementary to O6-methylguanine-deoxyribonucleic acid methyltransferase messenger ribonucleic acid for therapy of malignant gliomas.

OBJECTIVE: A derivative of chloroethylnitrosoureas, 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), is a drug of choice for the chemotherapy of human malignant brain tumors. However, the cytocidal effect of ACNU is effectively repressed through repair of ACNU-mediated deoxyribonucleic acid lesions by O6-methylguanine-deoxyribonucleic acid methyltransferase (MGMT). Because a variety of human tumors, including brain tumors, contain high levels of MGMT activity, we investigated the effect of antisense ribonucleic acid (RNA) complementary to MGMT messenger RNA on ACNU resistance in tumor cells. METHODS: We established a stable ACNU-resistant clone, C6AR, from the rat glioma cell line C6 exposed to a stepwise increasing concentration of ACNU. We transfected a plasmid deoxyribonucleic acid-encoding antisense MGMT RNA under the control of the human metallothionein promoter into C6AR cells and determined the effect of the antisense RNA on ACNU resistance of tumor cells by a colony-forming efficiency assay. RESULTS: C6AR cells expressed abundant MGMT messenger RNA, although the transcription level of the MGMT gene in parental C6 cells was below the lower limits of detection under the same assay conditions. ACNU resistance of C6AR cells was significantly repressed by transfected gene-dependent antisense MGMT RNA expression that resulted in decreased survival of the tumor cells. CONCLUSION: ACNU resistance resulting from the expression of MGMT in rat glioma cells is significantly overcome by the expression of antisense MGMT RNA. This result suggests that the antisense MGMT RNA system might be a useful strategy for overcoming ACNU resistance in the treatment of intractable malignant gliomas.

Cell kinetics of rat 9L brain tumors determined by double labeling with iodo- and bromodeoxyuridine.

Rats with 9L brain tumors received intraperitoneal injections of iododeoxyuridine (IUdR) and bromodeoxyuridine (BUdR) to estimate the duration of the deoxyribonucleic acid (DNA) synthesis phase (Ts) and the potential doubling time (Tp) of individual tumors. Different sequences and intervals (2 or 3 hours) of IUdR and BUdR administration were evaluated. After denaturation, tumor sections were reacted with Br-3, a monoclonal antibody that identifies only BUdR, and then were stained immunohistochemically by the avidinbiotin complex method. An antibody that recognizes both IUdR and BUdR, IU-4, was applied to the sections and identified by the alkaline phosphatase-antialkaline phosphatase method. Nuclei labeled only with IUdR stained blue, while those labeled with BUdR or with BUdR and IUdR stained brown. The fraction of cells that either left or entered the S-phase during the time between administration of IUdR and BUdR was measured to calculate Ts and Tp, assuming that the labeled cohort completed the DNA synthesis at a constant rate. The Ts was 8.8 hours (coefficient of variation (cv) = 0.05) and the Tp was 64.2 hours (cv = 0.08). The sequence and interval of administration of IUdR and BUdR had a minimal effect on Ts and Tp. In studies of 9L cells in monolayer culture, the Ts was 9.6 hours (cv = 0.08) and the TP was 30.6 hours (cv = 0.06). Double labeling with BUdR and IUdR allows the duration of the S-phase and potential doubling time of individual brain tumors to be estimated in situ from a single biopsy specimen.

Malignant intracerebral nerve sheath tumor with intratumoral calcification. Case report.

The authors present the clinical, radiological, and pathological features of a malignant intracerebral nerve sheath tumor that occurred in the right parietooccipital lobe of a 4-year-old girl. Computerized tomography scanning and magnetic resonance imaging demonstrated a 5x5x4-cm multiloculated mass with considerable enhancement of the irregularly shaped septa and clearly calcified areas within the mass. Among five cases reported in the literature, this patient is the youngest and represents the first case in which there is radiological evidence of intratumoral calcification.

Metastatic brain tumor. A clinical and pathologic analysis of 101 cases with biopsy.

A clinical and pathologic analysis was made of 101 patients with surgically treated metastatic brain tumors. The most common primary site was the lung (58.4%), followed by th breast (11.9%), gastrointestinal tract (6.9%), and uterus (5.)%). In 14 patients, the disease began with cerebral symptoms. The overall average survival after craniotomy was 8.0 months, the longest survival being 8l/2 years; patients with one-year survivals composed 18.8% of the cases. The histologic features were almost the same in primary and metastatic tumors. The undifferentiated tumor metastasized most frequently to the brain, and adenocarcinoma of the papillary type also seemed to find the brain fertile ground for metastatic growth.

Seven cases of breast cancer recurrence limited to the central nervous system without other visceral metastases.

We report 7 rare cases of recurrent breast cancers who presented with central nervous system (CNS) metastases as the initial relapse site without any other organ metastases. The average age of the patients at surgery was 42.6 years old of age (median 45:range 32-60), and 6 of the 7 cases (86%) were premenopausal. The mean disease-free period was 25.7 months (median 22, range 2-60 months). The primary tumors were all invasive ductal carcinomas. The estrogen receptor and progesterone receptor status of the 3 tumors available for study were all negative. The metastatic CNS lesions included the cerebrum (4 cases), cerebellum, cervical spinal cord, and meninges. In 6 out of these 7 cases (86%), the CNS metastasis was the initial recurrent lesion. Multidisciplinary treatments including surgery, radiotherapy and systemic or intrathecal chemotherapy were given. Although the mean survival time from clinical manifestations of the metastases of the 4 deceased patients was 20 months (median 20.5; range 6-33), one patient treated with surgery and radiotherapy is been still alive18 years later. These cases were also notable for the fact that the only metastatic site was in the CNS only during the entire clinical course, except for 2 cases, one with ocular adnexa metastasis, and the other with cervical lymph node metastasis. Premenopausal patients with negative hormone receptor status are more likely to develop this type of recurrence, regardless of the histological type. It is necessary to pay attention to neurological symptoms and signs during follow-up of breast cancer patients.

Developmental process of chronic subdural collections of fluid based on CT scan findings.

One hundred twenty-four follow-up scans were performed on 24 patients who had had posttraumatic subdural collections of low density. Of the 24 patients, 6 developed chronic subdural hematomas. Six other subdural collections showed a temporary increase in attenuation but eventually resolved. The remaining 12 subdural collections resolved without apparent increase in density. Illustrative cases are presented with computed tomographic scans. The identity of these posttraumatic subdural lesions of low density is discussed. They seem to be posttraumatic subdural hygromas.

Ossified and calcified epidural hematoma incidentally found 40 years after head injury: case report.

The authors report a 57-year-old man with an ossified and calcified epidural hematoma found incidentally 40 years after incurring a severe head injury. Since the introduction of computed tomography (CT) scan, few such cases have been described in the literature. A characteristic intracranial "double-outlined" contour on plain skull x-ray films and CT scans represented bone formation and calcification of the hematoma capsule adjacent to the dura.

Investigation of chemoresistance-related genes mRNA expression for selecting anticancer agents in successful adjuvant chemotherapy for a case of recurrent glioblastoma.

BACKGROUND: Glioblastoma multiforme represents one of the most malignant forms of primary intracranial tumors, often intractable to multimodality of treatment including chemotherapy. The unsatisfactory results of chemotherapy are chiefly attributed to chemoresistance. Since various molecules that could confer drug resistance have been elucidated, screening of the amount of such molecules in the tumor cells could provide possibilities for predicting their chemoresistance beforehand and help select more effective drugs. METHODS: We present a 45-year-old woman with recurrent glioblastoma multiforme in the cerebellum and invading the brain stem, treated successfully by postoperative chemotherapy. In this patient, anticancer drugs were determined by measurements of mRNA expression of chemoresistance-related genes, such as O6-methylguanine-DNA methyltransferase (MGMT), mdr1, glutathione S-transferase (GST)-pi, and metallothionein (MT) in the resected tumor. RESULTS: Northern blot analysis demonstrated the moderate mRNA level of MGMT, a major molecule causing ACNU (1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitroso ure a hydrochloride) resistance. On the other hand, expression levels of mdr1 which codes the P-glycoprotein responsible for multidrug resistance, and GST-pi, a detoxification enzyme, were low. Transcript of MT, another thiol containing molecule for cellular detoxification possibly associated with cisdiamminedichloroplatinum(II) (CDDP) resistance, was only faintly detectable. Postoperatively, the patient was treated initially with intravenous administration of ACNU and etoposide (VP16), resulting in a minor response of tumor regression. For maintenance therapy, we changed ACNU to CDDP according to the findings of the Northern blot analysis. Consequently, the residual tumor showed a marked response and almost disappeared after two courses of systemic chemotherapy with CDDP and VP16. CONCLUSIONS: The successful tumor regression in this case suggests that Northern blot analysis on expression of these chemoresistance-related genes in tumor tissues could provide beneficial information for determination of optimal anticancer agents to improve the efficacy of chemotherapy.

Triple primary malignant neoplasms including a malignant brain tumor: report of two cases and review of the literature.

BACKGROUND: Two rare cases of triple primary malignant neoplasms (PMN), including malignant brain tumors, which were glioblastoma multiformes, are described. METHODS: The clinical characteristics and underlying genetic alterations in triple or more PMN, including malignant brain tumors are discussed with intensive review of the literature. RESULTS: The first patient, a 77-year-old male, suffered metachronously from tubular adenocarcinoma of the stomach, transitional cell carcinoma of the bladder, and glioblastoma in the brain. This glioblastoma had loss of heterozygosity in exons 7-8 in p53 gene. The second patient, a 68-year-old male, developed papillary adenocarcinoma of the lung, adenocarcinoma of the rectum, and glioblastoma in the brain during a period of 7 years. In 42 such cases described in the literature, age distribution demonstrated two characteristic peaks, one in the third decade and the other over 50 years of age. The younger group consisted mainly of Turcot's syndrome, and of a case of Li-Fraumeni familial cancer syndrome. On the other hand, neither of these hereditary cancer syndromes were contained in the elder group. Regarding the site of PMN, colorectal cancers were associated most frequently with malignant brain tumors, followed by stomach cancers, and thyroid cancers. Malignant brain tumors, mostly glioblastoma multiforme, tend to occur as the last tumor of triple or more PMN. CONCLUSIONS: These results suggest that genetic background might play an important role in tumorigenesis of PMN in the younger group, whereas epigenetic factors would be more important in the older group. Characteristic organ association and factors influencing carcinogenesis, such as aging, environmental carcinogens, and underlying genetic alterations in these tumors are further discussed.

Combined chemotherapy and radiation therapy for central nervous system germ cell tumors: preliminary results of a Phase II study of the Japanese Pediatric Brain Tumor Study Group.

The authors conducted a multiinstitutional phase II study to establish a postsurgical combined chemotherapy and radiation therapy regimen for patients with primary germ cell tumors of the brain. After surgical debulking of the tumor and histological verification, patients were divided into three therapeutic groups: good prognosis, intermediate prognosis, and poor prognosis. Patients received two kinds of chemotherapy (three courses) prior to receiving radiation therapy: carboplatin-etoposide combination ([CARB-VP]: carboplatin 450 mg/m(2) on Day 1, etoposide 150 mg/m(2) on Days 1-3) or ifosphamide-cisplatin-etoposide combination ([ICE]: ifosphamide 900 mg/m(2), cisplatin 20 mg/m(2), and etoposide 60 mg/m(2) on Days 1-5). Patients in the good prognosis group (those with germinomas) were treated with CARB-VP followed by local radiation therapy (24 Gy). Patients in the intermediate prognosis group received CARB-VP followed by local radiation therapy (50 Gy); they received five additional chemotherapy treatments. Patients in the poor prognosis group received ICE followed by whole craniospinal radiation therapy; they also received five additional chemotherapy treatments. Eighty-two patients were evaluated. For the 56 patients with germinomas, a 93% rate of complete remission after treatment was achieved. The remission rate was 76% for 21 patients in the intermediate prognosis group, and no recurrence was detected during a median follow-up period of 2.6 years. In the group of five patients with poor prognosis, the disease in three patients progressed during chemotherapy or radiation therapy and they died within 6 months. There were no serious complications in the surviving patients. The authors found their treatment protocols to be currently effective for patients with germinomas and those with an intermediate prognosis.

Early stage detection of chemotherapeutic effect on 203 GL glioma in mice as studied by P-31 NMR and flow cytometry.

The effect of chemotherapy against glioma in mouse was evaluated by 31P NMR spectroscopy and flow cytometry. We found that administration of ACNU or tegafur at a dose less than LD50 resulted in the partial suppression of the ratio of inorganic phosphate (Pi)/phosphocreatine (PCr) and phosphomonoester (PME)/creatine phosphate (PCr) after 24 or 48 hr, although these ratios are usually increased together with growth of tumors. Flow cytometric analysis of glioma in vivo showed an accumulation in cells containing tetraploid DNA by G2M block 24-48 hr after treatment. However, the change occurred at a period slightly later than that of the Pi/PCr ratio. In contrast, histological change was noted at eight days after administration. Hence, it is concluded that in vivo 31P NMR spectroscopy can detect a change in metabolic pathways in tumors as early as 24-48 hr after the administration of chemotherapeutic agents.

[Treatment of metastatic brain tumor from renal cell carcinoma].

Twenty-eight patients with metastatic brain tumor from renal cell carcinoma were treated at the National Cancer Hospital, Tokyo, between 1962 and March 1989. In 13 patients, the median time interval between the initial diagnosis and pulmonary metastasis was 18 months, and the interval between pulmonary metastasis and brain metastasis was 13 months. In 10 patients, whose initial diagnosis was pulmonary metastasis, the median interval between pulmonary metastasis and brain metastasis was also 13 months. There were 2 patients who presented brain metastasis initially. The median survival time from the diagnosis of brain metastasis was 17 months for the patients whose brain tumors were surgically resected, but only 4 months for the patients who didn't receive surgery. The median survival time of the patients who received postoperative radiation was 20 months, while it was 10.5 months for the patients who received radiation therapy alone. Repeated serial CT scans of 7 patients with measurable brain metastases revealed partial response (PR) to radiotherapy in 2 patients (28.6%), no change (NC) in 4 patients (57.2%), and progressive disease (PD) in one patient (14.3%). BrdU labeling indices of resected brain metastases were about 2%, and the doubling time calculated on repeated serial CT scans was about 20 days. As these lesions are rather resistant to radiotherapy and grow relatively slowly they should be resected as much as possible.

[Primary intracranial epidermoid carcinoma accompanied with epidermoid cyst in the cerebellopontine angle--a case report].

A case of epidermoid carcinoma arising in an epidermoid cyst in the cerebellopontine angle is presented. Clinical features and CT appearance are discussed in comparison with those of benign epidermoid cyst. A 43-year-old man was admitted to the Department of Neurosurgery, University of Tokyo Hospital on April 14, 1983, with complaints of right facial numbness and weakness of six months' history. On neurological examination, sensation of the right half of the face was decreased in all modalities. Marked atrophy of the ipsilateral temporal muscle was also noted. Right facial paresis of peripheral type was evident. Gag reflex was decreased on the same side. Except for a slightly increased left deep tendon reflexes, there were no pyramidal tract signs. A CT without contrast material failed to show any abnormalities. A postcontrast CT demonstrated an irregular enhancement in the right cerebellopontine angle. The finding of asymmetry of the ambient cistern indicated minimum mass effect on the metrizamide CT cisternography. Suboccipital exploration of the right cerebellopontine angle was carried out on April 28, 1983. Leaving a part of the capsule indenting the pons between the roots of the fifth and the seventh nerve, we removed a white pearly tumor. Histological diagnosis was typical epidermoid cyst. He left the hospital one month later with signs of the right seventh and the eighth nerve. His postoperative course, however, was beyond our expectation. Over a few months following his discharge, left hemiparesis as well as horizontal and vertical nystagmus gradually developed. He was readmitted on November 10, 1983. A postcontrast CT revealed enlargement of the enhanced lesion filling the right ambient cistern.(ABSTRACT TRUNCATED AT 250 WORDS)