Mechanisms of methotrexate resistance in osteosarcoma.

High-dose methotrexate is a major component of current protocols for the treatment of osteosarcoma, but some tumors seem to be resistant. Potential mechanisms of resistance include decreased transport through the reduced folate carrier (RFC) and increased expression of dihydrofolate reductase (DHFR). To investigate methotrexate resistance, tumors were obtained from 42 patients with high-grade osteosarcoma. RFC and DHFR mRNA expression were studied by semiquantitative reverse transcription-PCR. The RFC and DHFR genes were studied for deletions and amplification by Southern blot. Thirteen of 20 (65%) osteosarcoma samples were found to have decreased RFC expression at the time of initial biopsy. At definitive surgery and relapse, 10 of 22 (45%) were found to have decreased RFC expression. Seventeen of 26 (65%) samples with a poor response to chemotherapy had decreased RFC expression, whereas 5 of 14 (36%) samples with a good response had a decrease (P = 0.03). None of the samples had an RFC gene deletion. Two of 20 samples (10%) showed increased DHFR expression at initial biopsy. The frequency of increased DHFR expression was significantly higher in metastatic or recurrent tumors (62%, P = 0.014). None of the samples showed evidence of DHFR gene amplification. The high frequency of decreased RFC expression in the biopsy material suggests that impaired transport of methotrexate is a common mechanism of intrinsic resistance in osteosarcoma. Increased DHFR expression in the pulmonary metastases may be a mechanism of acquired methotrexate resistance or a difference between primary and metastatic lesions.

Molecular analysis of the fusion of EWS to an orphan nuclear receptor gene in extraskeletal myxoid chondrosarcoma.

The pathogenesis of myxoid chondrosarcoma (CS) is poorly understood. A recurrent translocation, t(9;22) (q22;q12), has been recognized in CS, specifically in extraskeletal myxoid CS. Recently, this translocation has been shown to represent a rearrangement of the EWS gene at 22q12 with a novel gene at 9q22 designated CHN (or TEC). Sequence analysis suggests that CHN encodes a novel orphan nuclear receptor with a zinc finger DNA-binding domain. The structure of this gene fusion has been characterized in only a limited number of extraskeletal myxoid CSs and its presence in other types of CS has not been extensively examined. We studied 46 cases of CS (8 extraskeletal myxoid, 4 skeletal myxoid, 4 mesenchymal, and 30 other) for the EWS/CHN gene fusion by reverse transcriptase polymerase chain reaction, Southern blotting, and long-range DNA polymerase chain reaction. The EWS/CHN gene fusion was present in 6 of 8 extraskeletal myxoid CSs and was not detected in any of the remaining cases, including the 4 skeletal myxoid CSs. The negative findings in the latter cases suggest that skeletal myxoid CS is pathogenetically distinct from its extraskeletal counterpart. Notably, 2 cases of extraskeletal myxoid CS showed neither an EWS/CHN fusion transcript nor EWS/CHN genomic fusion nor EWS or CHN genomic rearrangement, suggesting genetic heterogeneity within extraskeletal myxoid CS. Finally, we also provide evidence for alternative splicing of the 3' end of the fusion transcript. Extraskeletal myxoid CS thus represents yet another sarcoma type containing a gene fusion involving EWS.

Telangiectatic osteogenic sarcoma. Improved survival with combination chemotherapy.

Since the inception of adjuvant chemotherapy for osteogenic sarcoma (OS), 25 patients were treated for telangiectatic osteogenic sarcoma (TOS) from 1973 through 1980. This represented 12% of all patients with primary OS of an extremity seen during this time period. Tumors that demonstrated only focal areas of TOS with areas of other subtypes were designated not as TOS but as "mixed" subtypes of OS. In the 25 patients with pure TOS, surgery included 18 amputations and seven resections for the primary tumor. Ten patients were treated on the first chemotherapy protocol (T-4) including high-dose methotrexate (HDMTX) with citrovorum factor rescue (CFR), Adriamycin (ADR), and cyclophosphamide (CYC). Of those 10 patients, five have been free of disease for seven to ten years from the time of diagnosis. Nine patients were treated on the second protocol (T-7) including HDMTX with CFR, ADR, and the combination bleomycin, cyclophosphamide, and dactinomycin (BCD). Six of those nine patients are disease-free survivors 63 to 88 months (median, 63 months) from diagnosis. Six were treated on the third chemotherapy protocol (T-10) including HDMTX with CFR, ADR, BCD, and the substitution of cisplatinum for those not having a complete response to preoperative chemotherapy with HDMTX. All six of the latter are disease-free survivors 42 to 56 months (median, 49 months) from the start of treatment. Toxicity included two HDMTX-related drug deaths in patients started on treatment prior to 1977. Of the entire group, 17/25 (68%) have remained free of disease at a mean follow-up time of over five and one-half years. This study demonstrates that TOS is responsive to chemotherapy and is potentially curable. Some prior reports of the uniformly poor prognosis of this variant of OS should not discourage attempts of curative therapy by chemotherapy and surgery.

Primary osteogenic sarcoma of the femur: a model for the use of preoperative chemotherapy in high risk malignant tumors.

The value of adjuvant chemotherapy in primary osteogenic sarcoma (OSA) is still considered controversial by some. One reason may be that various reported series include patients with widely varying prognostic variables. To address this, the effect of chemotherapy on the continuous disease-free (CNED) survival was analyzed in 100 patients aged 21 yr or less with OSA of the femur. This classically poor prognostic group of patients represented 51% of all primary OSA seen at the Memorial Sloan-Kettering Cancer Center during the study interval. This study includes all patients aged 21 yr or less with fully malignant (Grade III-IV/IV) OSA of the femur and no metastases treated from November 1973 through November 1981. The first (T-4) protocol (31 patients) consisted of high dose methotrexate (HDMTX) with leucovorin rescue, cyclophosphamide (Cyc), and adriamycin. In the second (T-7) protocol (23 patients) the dose of HDMTX was increased to 12 g/m2 for prepubescent patients, and bleomycin, Cyc, and dactinomycin replaced Cyc. The current (T-10) protocol (46 patients) uses the same CT as T-7, but patients not having a complete response of the primary tumor to preoperative CT receive additional cisplatinum (120 mg/m2) with adriamycin (30 mg/m2/day for two consecutive days). In 31 patients treated with T-4 the CNED survival was 32% with a minimum follow up of over 7 yr. On T-7, 15/23 patients with femur primaries had a CNED survival of 65% with all of the surviving patients followed for more than 5 yr. The addition of cisplatinum in T-10 has resulted in CNED survival rate of 77% in 34/44 patients (excluding two patients that died CNED during and after treatment); the median follow-up patients who are alive CNED is 33 months, with a minimum of 2 yr follow up on the last patient entered.

Chemotherapy of malignant fibrous histiocytoma of bone. A report of five cases.

Five patients with evaluable malignant fibrous histiocytoma (MFH) of bone (three with primary tumor and two with primary tumor and metastatic disease) were treated with preoperative chemotherapy including high dose methotrexate (HDMTX) with citrovorum factor rescue (CFR) as is used for patients with osteogenic sarcoma. All five patients demonstrated a clinical response to chemotherapy. Three of four patients who underwent surgery had complete responses and one patient had greater than 90% tumor necrosis as documented by histologic examination of the resected primary tumor. All four patients who underwent surgery following preoperative chemotherapy are surviving free of disease from one to six years from the start of treatment; chemotherapy was discontinued after six to 11 months in these patients. The median disease-free survival time is 31.5 months. This study demonstrates the effectiveness of chemotherapy in MFH of bone, and in particular the effectiveness of HDMTX with CFR which caused measurable responses in all patients while receiving this therapy as a single agent.

Primary osteogenic sarcoma: eight-year experience with adjuvant chemotherapy.

Since October 1973, 185 patients 21 years of age or younger with primary osteogenic sarcoma of an extremity were treated with adjuvant chemotherapy. Twenty-five of the first fifty-two patients (48%) have remained free of disease for a median of 7 years. In the next chemotherapy protocol most patients had chemotherapy prior to amputation or resection, during which time the dose of high-dose methotrexate was escalated in many patients to that needed to shrink the primary tumor. For a median of 4 years 43 of 54 patients (80%) have remained free of disease. In the current protocol, the response of the primary tumor to chemotherapy with high-dose methotrexate was used to select postoperative adjuvant chemotherapy for the patient. With the latter approach 73 of 79 patients (92%) have remained continuously free of disease for a median of 2 years. This experience demonstrates the value of chemotherapy in increasing the cure rate in osteogenic sarcoma and that the response to preoperative chemotherapy can help select postoperative chemotherapy to produce an even higher potential cure rate for osteogenic sarcoma.

Preoperative chemotherapy for osteogenic sarcoma: selection of postoperative adjuvant chemotherapy based on the response of the primary tumor to preoperative chemotherapy.

Since June 1978, 57 patients with primary osteogenic sarcoma of an extremity were treated with high-dose methotrexate (HDMTX) and citrovorum factor rescue (CFR), Adriamycin, and the combination of bleomycin, cyclophosphamide and dactinomycin (BCD) given for 4-16 weeks prior to definitive surgery. Histologic examination of the resected primary tumor determined the effect of preoperative chemotherapy with many primary tumors showing greater than 90% tumor necrosis attributable to preoperative chemotherapy. All patients having this favorable effect of chemotherapy on the primary tumor were continued on the same chemotherapy regimen postoperatively (regimen B). However, in those patients not having a good effect of preoperative chemotherapy on the primary tumor, HDMTX with CFR was subsequently deleted from their postoperative chemotherapy and they were placed on a regimen containing cisplatinum at the dose of 120mg/M2 with mannitol diuresis combined with Adriamycin in addition to BCD (regimen A). In the current study, 35 of the 57 patients did not demonstrate a good effect of chemotherapy on the primary tumor and were assigned to regimen A postoperatively. Of these 35 patients, 32 (91%) have remained continuously free of recurrent or metastatic disease from 6-34 months following the start of therapy. Among the 22 remaining patients having a good histologic response and treated with regimen B postoperatively, there has been only one relapse in a patient who had a local recurrence in the area of an inadequately resected primary tumor three months after the cessation of chemotherapy. Thus, 53 of 57 patients (93%) are continuously with no evidence of recurrent or metastatic disease from 6-35 months (median, 20 months) from the start of treatment. This study demonstrates the value of thorough histologic examination in predicting survival in responding patients and in helping identify patients whose disease-free survival rate can be substantially increased if they are given alternative postoperative adjuvant chemotherapy after failing to have a good response to preoperative chemotherapy. This individualized chemotherapeutic strategy has yielded the highest disease-free survival rate reported to date for osteogenic sarcoma.

Quotient imaging with N-13 L-glutamate in osteogenic sarcoma: correlation with tumor viability.

An investigation was performed to correlate the regional uptake of N-13 L-glutamate with histologic changes in tumor tissue in patients undergoing adjuvant chemotherapy for osteogenic sarcoma. A parametric image was produced by calculating the ratio of N-13 uptake in the tumor in a pixel-by-pixel fashion, using the presurgical scan as the numerator and the pretherapy scan as the denominator. The change in N-13 uptake in 2 x 2-cm regions of the tumor was compared with residual cell viability as determined by microscopic examination of multiple thin sections obtained from the surgical specimens. Regions that showed decreases in N-13 uptake of more than 30% were frequently associated with areas of highly necrotic tumor, and regions that showed increasing uptake were associated with high residual cell viability and incomplete response to chemotherapy.

Prognostic factors in the response of primary osteogenic sarcoma to preoperative chemotherapy (high-dose methotrexate with citrovorum factor).

Forty-three patients, ranging in age from 7 to 30 years (median age, 17 yr), with primary osteogenic sarcoma (OS), confirmed by biopsies and with no evidence of metastatic disease at the time of diagnosis, received T-7 chemotherapy for an average of 4 months before surgery, including high-dose methotrexate (HDMTX) and citrovorum factor rescue (CFR) (median, 7 courses), and 1 course each of bleomycin, cyclophosphamide, and dactinomycin, and adriamycin. At the time of definitive surgery, the surgical specimen showed a good histologic response to chemotherapy (grade III or IV response) in 29 (67%) of 43 patients and a poor histologic response (grade I or II response) in 14 (33%) of 43 patients. Among those who responded well, no patient relapsed, as all received a complete course of preoperative and postoperative chemotherapy for more than 5 to over 28 months after the initiation of treatment (medium, 13 mo). Among those who responded poorly, 6 of 14 patients relapsed with pulmonary metastases (a thoracotomy was beneficial to 1), 4 of 6 patients are alive with disease, and 1 patient died of progressive disease. On retrospective analysis, we observed that good and poor responders did not differ in the distribution of sex, age, race, primary site of disease, or histologic subtype of OS. An elevated alkaline phosphatase level that returned to normal under preoperative chemotherapy indicated a good response. Neither the 24-, 48-, and 72-hour serum MTX levels nor the fluid intake and urinary output during 3 days that followed HDMTX with CFR correlated significantly with tumor response. Based on our studies with this form of therapy, we concluded that the response of OS to preoperative chemotherapy is of prognostic value.

Primary osteogenic sarcoma: the rationale for preoperative chemotherapy and delayed surgery.

From 1973--1975, 31 patients with biopsied primary osteogenic sarcoma were treated with preoperative chemotherapy followed by surgical ablation of the primary tumor. Surgery was delayed in order to obtain a custom-fitted prosthetic bone implant in an attempt to avoid amputation. Preoperative chemotherapy included high dose methotrexate (HDMTX) with citrovorum factor rescue (CFR) and adriamycin (T-5 protocol) and was administered for 3 months preoperatively and continued with the inclusion of cyclophosphamide for approximately 5 months postoperatively. At a follow-up period of 30--52 months, 23 of 31 patients (75%) are surviving (21 of 23 with no evidence of disease). Histologic examination of primary tumor removed at surgery revealed varying degrees of tumor destruction (from very little effect to no evidence of viable tumor) attributable to the effect of chemotherapy. The 21 patients that are disease-free survivors had a more complete effect of preoperative chemotherapy on the primary tumor. Some patients achieving favorable effects upon the primary tumor did so only after the dose of HDMTX was escalated to greater than the starting dose of 8 g/m2. Preoperative chemotherapy for all patients with osteogenic sarcoma would seem to offer the following advantages: 1) Evaluation of the effect of HDMTX with CFR on the primary tumor with escalation of the dose of HDMTX until a clinical response is observed, thus defining the dose of HDMTX effective in that patient, to be continued postoperatively as adjuvant therapy; 2) The early use of systemic therapy to eradicate distant microfoci of disease that will eventually kill the patient if not adequately treated by effective chemotherapy; 3) Allow more time for postoperative healing without the need to start adjuvant chemotherapy immediately; and 4) Provide the surgeon time to plan resection surgery. To date, 20 additional patients with biopsy proven osteogenic sarcoma have been treated with more aggressive preoperative chemotherapy (T-7) for approximately 2 1/2 months prior to definitive surgery (resection or amputation). Doses of HDMTX were escalated where necessary and good clinical responses were obtained in 19 of 20 patients. In the majority of patients, no evidence of viable tumor was found on histologic examination of the surgically removed primary tumor. All 20 patients are surviving free of active disease at this brief follow-up period of 4--20 months.

Pathologic aspects of chemotherapy response in the treatment of osteogenic sarcoma. An analysis of two cases.

Two patients with osteogenic sarcoma of the proximal tibia were treated pre-operatively with intensive chemotherapy with high dose methotrexate (HDMTX) with citrovorum factor rescue (CFR) and adriamycin (ADR). One patient died before surgery. The other underwent en bloc resection of the primary tumor with prosthetic replacement of the involved tibia, following by adjuvant chemotherapy consisting of HDMTX with CFR and ADR, but died of metastases. Complete control of the primary or metastatic tumor was not achieved. It is emphasised that intensive multiple drug chemotherapy should be administered with extreme caution. The histologic findings are carefully analysed in relation to objective tumor response and toxic chemotherapy effects.

Chemotherapy, en bloc resection, and prosthetic bone replacement in the treatment of osteogenic sarcoma.

In an attempt to shrink primary osteogenic sarcoma and allow complete surgical removal of the primary tumor, without amputating the involved limb, intensive preoperative chemotherapy with high dose methotrexate (HDMTX) with citrovorum factor rescue (CFR) and adriamycin (ADR) was initiated in 20 patients with biopsy-proven primary osteogenic sarcoma of the distal femur (15 patients) and proximal tibia (five patients). Following intensive chemotherapy, en bloc resection of the primary tumor with prosthetic replacement of the involved bone was planned. After surgery, adjuvant chemotherapy, consisting of HDMTX with CFR, ADR, and high dose cyclophosphamide was given sequentially for 1 year. Of 20 patients with primary osteogenic sarcoma (two with evidence of pulmonary metastases), 18 had primary tumors that could be clinically measured. Of these 18, 17 demonstrated a decrease in the size of primary tumor prior to surgery, while on chemotherapy. To date, 12 of these patients with osteogenic sarcoma of the distal femur have had total femur and knee joint replacement, and three patients with osteogenic sarcoma of the proximal tibia have had total knee replacement. In all 15 patients, surgical margins were grossly and microscopically free of tumor. There has been no evidence of soft tissue recurrence in any of the 15 patients who have undergone surgery for from 2 to 15 months postoperatively. These preliminary results indicate that with the use of aggressive chemotherapy, it is possible to demonstrate objective tumor regression in primary osteogenic sarcoma, allowing the surgeon to perform en bloc resection of tumor and prosthetic replacement of the involved bone. Although the limb is preserved, it is important to stress that extensive surgery yielding tumor-free margins is performed. The ultimate evaluation of this approach to the treatment of primary osteogenic sarcoma awaits longer observation, to determine limb function and the continued disease-free status, once adjuvant chemotherapy is discontinued.